Effects of Increasing Intravenous Glucose Infusions on ... thesis...Effects of Increasing Intravenous Glucose Infusions on Lactation Performance, Metabolic Profiles, ... BHBA . β-Hydroxybutyrate

From the Institute of Physiology, Faculty of Veterinary Medicine, University of Leipzig

Effects of Increasing Intravenous Glucose Infusions on Lactation Performance, Metabolic Profiles, and Metabolic

Gene Expression in Dairy Cows

Inaugural-Dissertation to obtain the degree of a

Doctor medicinae veterinariae (Dr. med. vet.) from the Faculty of Veterinary Medicine

University of Leipzig

Presented by Bahaa Al-Trad

From Irbid/Jordan

Leipzig, 2010

Supported by Pfizer Animal Health

[I]

Mit Genehmigung der Veterinärmedizinischen Fakultät der Universität Leipzig

Dekan: Prof. Dr. Arwid Daugschies

Betreuer: Prof. Dr. Gotthold Gäbel

Gutachter: Prof. Dr. Gotthold Gäbel,

Veterinär-Physiologisches Institut,

Veterinärmedizinische Fakultät,

Universität Leipzig

Prof. Dr. Holger Martens,

Veterinär-Physiologisches Institut,

Veterinärmedizinische Fakultät

Freie Universität Berlin

Tag der Verteidigung: 30.03.2010

[II]

DEDICATION

TO MY FATHER’S SOUL

and always

TO MY MOTHER

[III]

CONTENTS

1 Introduction 1

2 Literature Review 3

2.1 Glucose Requirement and Partitioning in Dairy Cows 3

2.2 Glucose Metabolism in Ruminants 4

2.2.1 Hormonal Control of Glucose Metabolism 4

2.2.2 Hepatic Glucose Metabolism 5

2.2.2.1 Hepatic Gluconeogenesis 5

2.2.2.2 Regulation of Hepatic Gluconeogenesis 7

2.3 Metabolic Disorders and Shortage in Glucose Supply 8

2.4 Postruminal Starch Digestion and Glucose Absorption 9

2.5 Metabolic Consequences of Increased Glucose Supply 10

2.5.1 Lactational Performance 10

2.5.2 Feed Intake 12

2.5.3 Glucose Metabolism 12

2.5.3.1 Hepatic Glucose Production 12

2.5.3.2 Peripheral Glucose Metabolism 13

2.5.4 Protein Metabolism 14

2.5.5 Lipid Metabolism 14

2.5.5.1 Lipogenesis and Lipolysis 14

2.5.5.2 Mitochondrial Fatty Acid β-Oxidation 15

2.6 Main Objectives 16

3 Manuscript I. Increasing Intravenous Infusions of Glucose Improve Body Condition

but Not Lactation Performance in Mid-Lactation Dairy Cows

18

4 Manuscript II. Expression and Activity of Key Hepatic Gluconeogenesis Enzymes in

Response to Increasing Intravenous Infusions of Glucose in Dairy Cows

32

5 Manuscript III. Activity of Hepatic but Not Skeletal Muscle Carnitine

Palmitoyltransferase Enzyme Is Depressed by Intravenous Glucose Infusions in

Lactating Dairy Cows

55

6 General Discussion 74

6.1 Glucose Supply Is Not a Limiting Factor for Lactation Performance in Midlactating

Dairy Cows

74

[IV]

6.2 Dysregulation of Blood Glucose Occurs Postparandially at High Glucose Loads 76

6.3 Gradual Increases in Glucose Supply Cause Insulin Resistance in a Dose-Dependent

Fashion

77

6.4 Nutritionally Relevant Increases of Glucose Supply Have No Negative Effect on the

Enzymatic Capacity for Gluconeogenesis

78

6.5 Excessive Glucose Has a Protein-Sparing Effect 80

6.6 Surplus Glucose Is Used Mainly by Glycogen and Fat Synthesis Pathways 81

6.7 Surplus Glucose Diverts Lipid from Energy-Generating Pathways into Anabolic

Pathways

81

6.8 Hepatic But Not Skeletal Muscle CPT Activity Is Sensitive to the Changes in Energy

Balance Status

82

6.9 Adaptations are Rapidly Reversible after Ceasing of Glucose Infusions Except for

Those of Lipid Metabolism

83

7 Summary 86

8 Zusammenfassung 88

9 References 90

Acknowledgements 100

[V]

ABBREVIATIONS

AA Amino acids AUC Area under the curve AST Aspartate transaminase ATP Adenosine triphosphate ATPase Adenosine Triphosphatase B2M β2-Microglobulin BFT Back fat thickness BHBA β-Hydroxybutyrate BUN Blood urea nitrogen BW Body weight cAMP Cyclic adenosine monophosphate CACT Carnitine-acylcarnitine translocase CBC Complete blood count CPT Carnitine palmitoyltransferase DMI Dry matter intake DTNB 5,5'-dithiobis-2-nitrobenzoic acid EB Energy balance EDTA Ethylenediaminetetraacetic acid FBPase Fructose 1,6-bisphosphatase Fig. Figure G6-Pase Glucose 6-phosphatase GI Glucose infusion GLUT Glucose transporter GGT γ-glutamyl transferase HEPES 4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid HOT Hepatic oxidation hypothesis LCFA Long chain fatty acids MEO Milk energy output NADPH Nicotinamide adenine dinucleotide phosphate NEB Negative energy balance NEL Net energy for lactation NEFA Non-esterified fatty acids OAA Oxaloacetate PC Pyruvate carboxylase PCC Propionyl-CoA carboxylase PDV Portal-drained viscera PEP Phosphoenolpyruvate PEPCK Phosphoenolpyruvate carboxykinase qRT-PCR Quantitative Reverse Transcription-Polymerase Chain Reaction RQUICKI Revised quantitative insulin sensitivity check index SCFA Short chain fatty acids SI Saline infusion TAG Triacylglycerides TCA Tricarboxylic acid cycle VLDL Very low density lipoprotein

CHAPTER‐1: INTRODUCTION

[1]

1 Introduction Lactating dairy cows have a high demand for glucose to synthesize milk lactose

(BICKERSTAFF et al. 1974; RIGOUT et al. 2002b). However, little glucose is available for

absorption from normal diets since the majority of ingested carbohydrates are converted to

short chain fatty acids (SCFA) in the rumen (e.g., to acetate, propionate and butyrate;

REYNOLDS et al. 1994). As an alternative source, endogenous glucose production through

hepatic gluconeogenesis is considered to supply most of the glucose in dairy cows (YOUNG

1977). It has been estimated that 90% of the circulating glucose (~3kg/d) originates from this

process (YOUNG 1977; REYNOLDS et al. 1988), and 60-80% from this amount are utilized

for milk lactose synthesis (BICKERSTAFF et al. 1974). Given the high glucose requirements,

limitations in glucose availability play a crucial role in the development of metabolic (BOBE

et al. 2004) and reproductive disorders (BUTLER 2000; MARTENS 2007) in high-yielding

cows. Therefore, much effort has focussed recently on measures to increase the direct

absorption of glucose from the intestine via postruminally digestible starch, which is

energetically more efficient than hepatic glucose synthesis (OWENS et al. 1986).

With the intention to address the consequences of additional glucose supply, numerous

studies investigated the effects of different levels of intravenous (AMARAL et al. 1990; KIM

et al. 2000) or postruminal glucose infusions (RIGOUT et al. 2002a,b) on lactation

performance and/or on tissues intermediary metabolism (e.g. glucose and lipid metabolism).

Although the effects of glucose supply on milk yield have been studied thoroughly, the

beneficial effect of surplus glucose on lactation performance is still a subject of continuing

research and debate. Some studies have demonstrated that milk yield increased with glucose

infusion (FROBISH and DAVIS 1977; RIGOUT et al. 2002b), whereas others have shown no

change (AMARAL et al. 1990; HURTAUD et al. 1998). Reasons for the inconsistent effect of

postruminal glucose supply on milk yield may lie in the infusion dose as suggested by

RIGOUT et al. (2002b). In their study, moderate doses of duodenal glucose infusion

improved net mammary balance of glucose and, in turn, increased lactose synthesis and milk

output. In contrast, excessive glucose supply impaired the intracellular glucose metabolism

which negatively affected milk lactose synthesis and milk yield. However, it is still an open

question whether the dose effects on lactation performance observed by RIGOUT et al.

(2002b) after postruminal application of glucose were primarily caused by increased glucose

availability for the mammary gland. An alternative explanation could be that postruminal

application of glucose induced the release of gastrointestinal hormones (e.g. glucagon-like

CHAPTER‐1: INTRODUCTION

[2]

peptide-1) that affected intermediary glucose metabolism and feed intake (HOLST 2007).

Therefore, the first goal of the present study was to test whether intravenously applied glucose

elicits dose effects on lactation performance that are similar to those observed previously after

gastrointestinal glucose supply (see chapter 3).

Amongst the metabolic adaptations that underlie the effects of glucose supply on

performance, hepatic gluconeogenesis is of particular interest. The observed decreases in the

rate of hepatic glucose production during surplus glucose supply (BARTLEY and BLACK

1966; THOMPSON et al. 1975) might explain the limited or missing effects of surplus

glucose on milk yield in certain studies. However, only a few experiments investigated

whether the decrease in hepatic gluconeogenesis might be caused by changes in hepatic

gluconeogenesis enzymes catalytic capacity. One previous study showed a decrease in the

activities of five gluconeogenic enzymes by extreme overfeeding prepartum (MURONDOTI

et al. 2004) while another study showed a decrease in the activity of hepatic glucose 6-

phospatase (G6-Pase, EC 3.1.3.9) by high doses of glucose infused intraduodenally

(BARTLEY et al. 1966). So far, no data are available about the dose-dependent adaptive

responses of hepatic gluconeogenesis enzymes activity and gene expression levels to glucose

supply in dairy cows. Accordingly, the second goal of the present study was to examine the

dose effects of surplus glucose on the activities and gene expression levels of four rate-

limiting gluconeogenesis enzymes (see chapter 4).

In non-ruminants, one adverse consequence of prolonged increases in systemic glucose

availability is the development of insulin resistance (DOBBINS et al. 2001; CAHOVÁ et al.

2007). It has been shown that decreases in the skeletal muscle activity of carnitine

palmitoyltransferases, (CPT, EC 2.3.1.21), the rate limiting enzyme in the β-oxidation of fatty

acids, play a crucial role in this process (i.e. lipotoxicity theory, KREBS and RODEN 2004;

CAHOVÁ et al. 2007). HOLTENIUS et al. (2003) reported that overconsumption of energy

during the dry period was associated with the development of peripheral insulin resistance in

dairy cows, too. In dairy cows, this peripheral insulin resistance could be related to

adaptations of hepatic CPT activity to changing energy supply (AIELLO et al. 1984; DANN

and DRACKLEY 2005) but a similar responsiveness of CPT in muscle tissue has never been

thoroughly investigated. Therefore, the last goal of the present study was to test whether the

development of peripheral insulin resistance in dairy cows may be linked to inadequate

responses of skeletal muscle CPT activity to increasing energy supply (see chapter 5).

CHAPTER‐2: LITERATURE REVIEW

[3]

2 Literature Review 2.1 Glucose Requirement and Partitioning in Dairy Cows

Dairy cows have a requirement for glucose as a primary metabolic fuel for many tissues. In

the non-pregnant, non-lactating cow, about 200 g/day of glucose is estimated for non-

mammary tissues maintenance (REYNOLDS 2005), while during late pregnancy, the gravid

uterus consumes approximately half (~650 g/d) of the maternal glucose blood supply for fetal

oxidative metabolism (BELL 1995). Nonetheless, previous studies have shown that 60 to 85%

of the total glucose used in lactating dairy cows is utilized by the mammary gland

(BICKERSTAFF et al. 1974). Using the following formula which calculated the daily

turnover of glucose in lactating dairy cows: Y = 1.64 + 0.396x (where Y = daily glucose

turnover Mol/d; x = daily milk production kg/d; DANFÆR 1994), MARTENS (2007)

estimated that ~3.1 kg/d of glucose is required for a cow to produce 40 litres of milk per day.

The dependence of mammary secretion on glucose supply is almost certainly due to the role

of glucose as a precursor of milk lactose synthesis which accounts for 50 to 85% of the total

glucose extracted by the mammary gland (ANNISON et al. 1974; BICKERSTAFF et al.

1974).

In addition to the mammary gland, the portal-drained viscera (PDV) are also net

consumers of glucose in ruminants, even in situations when postruminal starch delivers

moderate amounts of additional glucose for direct absorption from the intestine (REYNOLDS

et al. 1994; FREETLY and KLINDT 1996). Glucose further constitutes an essential

metabolite for the brain with approximately 10 – 15% of whole-body glucose utilization

(LINDSAY 1979; ORTIGUES-MARTY et al. 2003). Glucose consumption by skeletal

muscle depends on both glucose availability and physical activity. Glucose may be oxidized

in the muscle cells to CO2 which accounts for ~30 - 60 % of total CO2 produced by the

muscle or it may be stored as glycogen (HOCQUETTE et al. 1998). The oxidization of

glucose in muscle cells and many other cells of the body generates adenosine triphosphate

(ATP) as an universal fuel for energy-dependent processes, while some tissues additionally

use intermediates of glycolysis for synthesis pathways (e.g. glycerol for the production of

esterified fatty acids in fat cells; ORTIGUES-MARTY et al. 2003; HUNTINGTON et al.

2006).


[4]

2.2 Glucose Metabolism in Ruminants

As in monogastric animals, glucose is the main energy source for several metabolic processes

in ruminants; however, there are great differences regarding to the source and metabolism of

glucose between the two groups of species. Absorption of glucose in the small intestine

constitutes the major source for glucose supply in monogastric animals (GRAY 1992). In

contrast, only little glucose is available for absorption in the small intestine of ruminants

under many dietary regimes as most of the ingested carbohydrates are microbially fermented

to SCFA in the rumen (YOUNG 1977; REYNOLDS et al. 1994). These SCFA may supply

70-80% of the daily energy requirements to the animal (BERGMAN 1990). Ruminants have

adapted to the limited availability of glucose by high rates of hepatic gluconeogenesis, by the

limited use of glucose for oxidation, and by the primary use of acetate instead of glucose for

fatty acid synthesis (BROCKMAN and LAARVELD 1986; DANFÆR et al. 1995). To ensure

that the liver fulfils its role as an organ of net glucose production, ruminant hepatocytes lack

measurable glucokinase activity to counteract glucose uptake and utilization by the liver

(BALLARD et al. 1969).

2.2.1 Hormonal Control of Glucose Metabolism

Insulin and glucagon are the major hormones regulating glucose metabolism in ruminants,

just as they are in monogastric species (MCDOWELL 1983). Insulin controls a variety of

cellular glucose homeostasis processes, the most important being the stimulation of glucose

uptake into certain cell types. Most mammalian cells take up glucose by facilitated diffusion

via tissue-specific glucose transporter (GLUT) proteins (BELL et al. 1990). These

transporters are divided into insulin-sensitive (e.g., GLUT-4) and non insulin-sensitive

glucose transporters (e.g., GLUT-1, 2, 3 and 5) in both monogastric animals (BELL et al.

1990) and ruminants (HOCQUETTE and ABE 2000). The insulin-sensitive transporter,

GLUT-4, is found mainly in adipose and skeletal muscle tissues both in ruminants (ZHAO et

al. 1993; ABE et al. 1997) and in monogastric species (BELL et al. 1990).

While insulin is a central hormone in the regulation of glucose metabolism in all

mammalian species, insulin sensitivity of whole body glucose utilization differs greatly

between ruminants and monogastric species. Application of the hyperinsulinemic, euglycemic

clamp technique has demonstrated that ruminants are generally less sensitive to insulin

compared with monogastric animals (KASEK et al. 2001; SASAKI 2002). The lower insulin-

mediated glucose utilization rates are to a significant part caused by lower insulin-mediated


[5]

glucose uptake rates in ruminants (JANES et al. 1985b; ROSE et al. 1998). In lactating dairy

cows, it has been estimated that about 80 % of the cellular glucose uptake occurs independent

of insulin (ROSE et al. 1998). One major GLUT mediating insulin-independent glucose

uptake is GLUT-1 which is expressed in many tissues including the brain, kidney and

mammary gland (ZHAO et al. 1993; KOMATSU et al. 2005). In contrast to monogastric

species, GLUT-1 is also highly prevalent in adult bovine glycolytic muscles and adipose

tissue, which can explain their capability and high degree of insulin-independent glucose

uptake (ABE et al. 2001; DUHLMEIER et al. 2005). Since GLUT-1 and GLUT-4 have

comparable affinity for glucose (Km = 5 mM for both transporter; BELL et al.1993), the ratio

of their membrane expression relates directly to the insulin-sensitivity of glucose uptake.

Another important difference between ruminant and monogastric animals is the low

correlation between plasma glucose and insulin concentrations (MCATEE and TRENKLE

1971). Although intravenous (THOMPOSON et al. 1975; AMARAL et al. 1990;

GRÜNBERG et al. 2006) and duodenal (LEMOSQUET et al. 1997) infusions of glucose can

increase blood insulin concentrations in dairy cows, some studies showed little effect of

glucose infusion on circulating insulin concentrations, especially, in lactating cows

(FROBISH and DAVIS 1976; LOMAX et al. 1979). Moreover, higher plasma levels of

insulin were observed after the intravenous infusion of propionate compared to glucose,

suggesting that ruminants are more sensitive to propionate as a stimulator of insulin release

than to glucose (MCATEE and TRENKLE 1971; ROSS and KITTS 1973; FUHRMANN et

al. 1989). The action of insulin is mainly antagonized by pancreatic glucagon which acts

primarily to increase hepatic glucose output by promoting hepatic gluconeogenesis and

glycogenolysis (MCDOWELL 1983).

2.2.2 Hepatic Glucose Metabolism

2.2.2.1 Hepatic Gluconeogenesis

Since the absorption of glucose from the gut is low, there is no general need for the

ruminant’s liver to remove excessive glucose from the portal vein. Therefore, ruminants have

little or not detectable hepatic glucokinase activity; the rate-limiting enzyme in the glycolytic

pathway (BALLARD et al. 1969). In turn, gluconeogenesis is very prominent in the bovine

liver (YOUNG 1977). Approximately 90% of circulating glucose needed by the animal is

provided through hepatic gluconeogenesis (YOUNG 1977; BELL 1995). The remaining

glucose supplies come from gluconeogenesis by the kidneys (BERGMAN et al. 1974) and


[6]

from the postruminal glucose supply from the diets. It has been shown that the net hepatic

glucose production (~3.1 kg/d) in early lactating dairy cows was more than adequate to meet

the mammary glucose demands (~2.3 kg/d; REYNOLDS et al. 1988). According to an

estimation by DANFÆR et al. (1995), 2 - 2.5 kg are needed daily for a cow at six month of

pregnancy, which produced 25 kg of milk/day and gained 0.5 kg of body weight/day; ~90 -

95% of that quantity were provided by endogenous hepatic glucose production through

gluconeogenesis.

Precursors for glucose synthesis include propionate, lactate, glycerol, and glucogenic

amino acids (AA; HUNTINGTON et al. 2006). In fed animals, almost all of PDV-absorbed

propionate is removed by the liver and used for glucose synthesis, generating ~50 - 60% of

the total glucose entry. The remaining carbons are supplied by lactate and gluconeogenic AA

(AMARAL et al. 1990; HUNTINGTON et al. 2006). The initial step of hepatic propionate

metabolism is the activation of propionate to its coenzyme A derivative (propionyl-CoA) by

the mitochondrial enzyme propionyl-CoA synthase (E.C. 6.2.1.17). Propionyl-CoA is

carboxylated by the biotin-dependent enzyme, propionyl-CoA carboxylase (PCC; E.C.

6.4.1.3), to form methylmalonyl-CoA which is metabolized to succinyl-CoA through the

tricarboxylic acid cycle (TCA). Succinyl-CoA is converted to oxaloacetate (OAA) and then

to phosphoenolpyruvate (PEP) by the action of phosphoenolpyruvate carboxykinase

(PEPCK, EC 4.1.1.32). In rats, cattle, and several other species, the PEPCK enzyme has two

isoforms which are compartmentalized to the mitochondria (PEPCK-M) and cytosol

(PEPCK-C) with approximately equal activities (HOD et al. 1986; AGCA et al. 2002).

However it has been shown that the mRNA abundance of hepatic PEPCK-C is greater than

that of PEPCK-M in dairy cows; with a close relationship existing between total PEPCK

enzyme activity and PEPCK-C mRNA level (AGCA et al. 2002). In addition, hepatic

PEPCK-C activity and mRNA are regulated by nutritional and hormonal stimuli while

PEPCK-M activity and mRNA levels appear to be constitutive (AGCA et al. 2002).

Lactate and some of the glucogenic AA (e.g. alanine, cysteine,) are metabolized first to

pyruvate and then directly to OAA through an ATP-dependent reaction catalyzed by pyruvate

carboxylase (PC, EC 6.4.1.1). Then, OAA is shuttled to the cytosol as malate, from which

NADH and OAA are regenerated, followed by PEP generation that is catalyzed by the

PEPCK-C (ENGELKING 2004).


[7]

Phosphoenolpyruvate is converted via several 3-carbon intermediates to fructose 1,6-

bisphosphate, which is hydrolyzed by fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) to

fructose 6-bisphosphate and inorganic phosphate (ENGELKING 2004). FBPase has been

identified as the rate-limiting enzyme in hepatic glucose production from plasma glycerol

which originated from adipose tissue as a result of lipolysis (RUKKWAMSUK et al. 1999;

ENGELKING 2004). Glycerol enters the gluconeogenic pathway after conversion to

dihydroxyacetone phosphate, the latter being a direct precursor of fructose 1,6-bisphosphate.

After fructose 6-phosphate has been isomerised to glucose 6-phosphate, the final step of

the gluconeogenic pathway is the dephosphorylation of glucose 6-phosphate by G6-Pase. G6-

Pase is found primarily in the liver and kidney, where it produces free glucose. Because this

terminal step of glucose release is shared between both the gluconeogenic and the

glycogenolytic pathways, G6-Pase activity is a key determinant in the output of glucose from

the liver (VAN SCHAFTINGEN and GERIN 2002).

2.2.2.2 Regulation of Hepatic Gluconeogenesis

In general, the gluconeogenic rate in ruminants is mainly controlled by substrate availability

from the diet (BBROCKMAN and LAARVELD 1986). In steers, increasing propionate

supply by feeding sodium propionate increased the relative contribution of propionate to the

carbon for gluconeogenesis from 43 % to 67 % (VEENHUIZEN et al. 1988). In a similar

way, the contribution of AA to glucose production was positively correlated with dietary

protein intake and hepatic AA entry rate (DANFÆR et al. 1995; WARY-CAHEN et al. 1997).

When glucogenic precursor supply becomes excessive, increased hepatic glucoseneogenesis

is balanced by anabolism or upregulation of glucose oxidation as it has been shown in steers

(VEENHUIZEN et al. 1988) and sheep (JUDSON and LENG 1973a).

Glucagon and insulin play important roles in the hormonal regulating of hepatic glucose

metabolism in ruminants as in other species (MCDOWELL 1983). Glucagon acts primarily

on the liver to increase hepatic glucose output by promoting gluconeogenesis and

glycogenolysis in states of starvation and energy mobilization. It has been found that

glucagon stimulates conversion of propionate to glucose in cultured hepatocytes from calves

(DONKIN and ARMENTANO 1995), and from propionate and lactate in ovine hepatocytes

(FAULKNER and POLLOCK 1990). The action of glucagon is mediated mainly through

increases in the intracellular concentration of cyclic adenosine monophosphate (cAMP;


[8]

PILKIS et al. 1988). Elevation of intracellular cAMP leads to activation of cAMP-dependent

protein kinases that catalyze the phosphorylation of a number of protein substrates. The end

result of this cascade of events is a stimulation of gluconeogenesis and an inhibition of

glycolysis (PILKIS et al. 1988). Insulin, on the other hand, opposes the action of glucagon to

stimulate gluconeogenesis. One mechanism probably involves the ability of insulin to activate

cAMP phosphodiesterase, which results in lower cAMP levels (PILKIS et al. 1988). In sheep,

intravenous insulin infusion caused a marked decrease in the rate of hepatic glucose

production and in net hepatic utilization of the gluconeogenic substrates alanin, glutamine,

lactate and glycerol (BROCKMAN 1985; BROCKMAN 1990). Conversely, insulin has no

effect on the rate of conversion of propionate to glucose (BROCKMAN 1990). Similarly,

insulin has no effect on the conversion of propionate to glucose in monolayer cultures of

hepatocytes isolated from ruminating calves (DONKIN and ARMENTANO 1995), but there

was a decrease in glucose synthesis from lactate (DONKIN et al 1997). Using an

hyperinsulinemic, euglycemic clamp, EISEMANN and HUNTINGTON (1994) showed that

increased insulin supply in beef steers will reduce liver glucose production to the point where

all glucose production could be derived from propionate.

In non-ruminants, the most extensively studied gluconeogenic enzyme with regard to the

hormonal regulation at the gene expression level is PEPCK (PILKIS et al. 1988). PEPCK

gene expression was stimulated in the fasted state by glucagon, acting through cAMP, and

was inhibited in the fed state by high levels of insulin (PILKIS et al. 1988; LEMAIGRE and

ROUSSEAU 1994; CHRIST et al. 1988). In contrast, hepatic PEPCK mRNA levels of

lactating dairy cows were decreased (SHE et al. 1999) or remained unchanged (WILLIAMS

et al. 2006) when infused with glucagon. Although it is not well understood how insulin

regulates the transcription rates of glucneogenesis enzymes mRNA expression in ruminants,

SHE et al. (1999) suggested that PEPCK gene expression was down-regulated by an increase

in the endogenous insulin secretion during infusions of glucagon. In monogastric animals,

insulin has a depressive effect on hepatic glucose production by modulating the gene

expression of PC (JITRAPAKDEE and WALLACE 1999), PEPCK (PILKIS et al. 1988),

FBPase (EL-MAGHRABI et al. 1991) and G-6Pase (NORDLIE et al. 1999).

2.3 Metabolic Disorders and Shortage in Glucose Supply

The rapid increase in milk production after parturition greatly increases the demand for

glucose for milk lactose synthesis (BELL 1995; DRACKLEY 1999). BELL (1995) calculated


[9]

that for a milk yield of 30 kg/d, mammary requirements for glucose, AA and fatty acids at 4

days postpartum are approximately 2.7, 2.0 and 4.5 times those of the gravid uterus during

late pregnancy. Consequently, transition from pregnancy to lactation is a huge challenge to

the metabolism of high-yielding lactating dairy cows.

Limited feed intake during the early lactation; likely because of physiological stress and

immune challenges, means that the supply of energy, espically the supply of propionate for

glucose synthesis, is not sufficient to meet the energetic requirements of maintenance and the

abundant milk secretion. Thus, the animal experiences a period of negative energy balance

(NEB; BELL 1995; MARTENS 2007). Alteration of metabolism to sustain high levels of

milk production during NEB results in mobilization of large amounts of body fat and the

release of non-esterified fatty acids (NEFA) into the blood. The ability of the liver to utilize

NEFA as fuel or export it as very low density lipoprotein (VLDL) is limited during periods of

elevated plasma NEFA (e.g. postpartum NEB). When a limit is reached, excess fatty acids

accumulate in the liver in the form of triacylglycerides (TAG), a condition referred to as fatty

liver (GRUMMER 1993). As a consequence to the incomplete metabolism of mobilized

NEFA, the acetyl-CoA that is not incorporated into the TCA is converted to ketone bodies

(acetoacetate and β-hydroxybutyrate; BHBA). Appearance of these metabolites in blood,

milk, and urine as a result of the excessive rate of ketogenesis is diagnostic of the metabolic

disorder known as ketosis (GOFF and HORST 1997). Ketosis/fatty liver complex is

associated with loss of appetite, central nervous system dysfunction, longer calving intervals,

decrease in milk production and with increased veterinary costs (BOBE et al. 2004; GOFF

2006). Therefore, improving the nutritional strategies to meet mammary gland glucose

demands and also to minimizing such adverse consequences of glucose shortage is an area of

current research in dairy nutrition. To alleviate glucose shortage in such periods, recent

feeding strategies aim to increase glucose availability via postruminally digestible starch.

2.4 Postruminal Starch Digestion and Glucose Absorption

In ruminants, glucose availability in the small intestine is highly dependent on the type of the

fed diet. For example, in ruminants fed on forage-based diets which contain essentially no

starch carbohydrates, insignificant amounts of glucose would be available for absorption in

the lumen of the small intestine, as documented in previous studies (JANSE et al. 1985a;

HARMON and MCLEOD 2001). In contrast, many studies with lactating dairy cows and

sheep indicated that considerable amounts of starch might disappear from the lower gut of


[10]

animals fed on a high starch diet (such as wheat, corn, sorghum, barley, and oats; JANSE et

al. 1985b; REYNOLDS et al. 1994). Depending on a variety of conditions (e.g. grain type,

processing); rumen escape starch can exceed 40% of the dietary starch intake in dairy cattle

(ØRSKOV 1986; OWENS et al. 1986). The maximum amount of postruminal digestible

starch has been estimated with ~1.5 - 2 kg/d in dairy cows (GÄBEL and ASCHENBACH

2004). If this starch was digested and absorbed by the small intestine as glucose, it could

contribute significantly to the total entry rate of glucose.

There is evidence that the efficiency of conversion of dietary starch energy to tissue energy

is greater in ruminants when glucose is absorbed directly as a result of postruminal starch

digestion, rather than when dietary carbon is absorbed as SCFA, AA, and other glucose

precursors (REYNOLDS et al. 1994; HARMON and MCLEOD 2001). Taken together, when

starch that is resistant to ruminal degradation is fed, glucose absorption balances glucose

utilization by PDV tissues, sparing endogenously synthesized glucose for tissue metabolism

and, consequently, increasing total glucose availability to the mammary gland and to the rest

of the body (NOCEK and TAMMINGA 1991; REYNOLDS et al. 1994).

2.5 Metabolic Consequences of Increased Glucose Supply

2.5.1 Lactation Performance

The availability of glucose should be relevant for milk yield in view of the fact that lactose is

the major osmoregulator for the transfer of water from the plasma to the milk (LINZELL and

PEAKER 1971). In a number of studies conducted in lactating dairy cows fed on a grass

silage-based diet, duodenal glucose infusion increased milk yield (HURTAUD et al. 2000;

RIGOUT et al. 2002a,b; LEMOSQUET et al. 2004). However, with corn silage-based diet,

postruminal glucose infusion had no effect on milk production (LEMOSQUET et al. 1997;

HURTAUD et al. 1998). Since grass silage diet has a limited capacity to provide postruminal

glucose for absorption compared with corn silage, this might indicate that the total availability

of glucose in the doudenoum from postruminal starch and infused glucose might play role in

the ability of infused glucose to increase milk yield (i.e. dose-dependent effect). In line with

that, RIGOUT et al. (2002b) suggested that the differing responses of dairy cows to surplus

glucose supply might be linked to such dose effects. Using duodenal glucose infusion in their

study, moderate amounts of duodenal glucose were identified to increase the whole body

appearance rate of glucose and mammary blood flow, leading to increased lactose synthesis

and milk output. In contrast, excessive provision of glucose caused a decrease in milk yield


[11]

which was explained by a decreased mammary conversion of glucose to lactose based on a

decreased conversion of glucose 6-phosphate to glucose 1-phosphate in mammary tissue

(RIGOUT et al. 2002b). In this regard, no study tested the dose effects of glucose on lactation

performance using the intravenous administration route. The intravenous infusion route

differs from the postruminal infusion route in that it avoids the release of gastrointestinal

hormones that affect glucose metabolism and feed intake (HOLST 2007). Such hormonal

effects could largely bias the true effect of glucose on lactation performance. The intravenous

infusion route also avoids glucose utilization within the PDV (REYNOLDS et al. 1994) and,

therefore, allows a more precise determination of dose effects of infused glucose with regard

to the mammary function.

Apart from milk yield, the effect of glucose on milk composition is also of interest.

Excessive glucose has repeatedly been shown to modifiy milk composition. Most consistent

results were decreases in milk fat yield (HURTAUD et al. 2000; RIGOUT et al. 2002a) and

increases in milk protein yield (AMARAL et al. 1990; HURTAUD et al. 2000). Several

theories have been proposed to explain the depression in milk fat which occurs during

increases of glucose supply. The most widely held theory is the glucogenic-insulin theory

which explains the depression in milk fat by a shortage in the supply of lipogenic precursors

to the mammary gland (MCCLYMONT and VALLANCE 1962; BAUMAN and GRIINARI

2003). According to this theory, increases in glucose availability would stimulate insulin

secretion which, in turn, increases the rate of adipose tissues lipogenesis and inhibits lipolysis.

These changes in the rates of lipid synthesis and lipolysis are proposed to preferentially direct

nutrients to adipose tissue rather than mammary gland, thus causing a shortage of lipogenic

precursors for mammary synthesis of milk fat (MCCLYMONT and VALLANCE 1962;

BAUMAN and GRIINARI 2003). Another theory attributes milk fat depression during

glucose infusion to a direct inhibition of mammary gland synthesis of milk fatty acids

(LEMOSQUET et al. 1997; RIGOUT et al. 2002a,b). Numerous hypotheses have also been

proposed to explain the increase in mammary gland protein synthesis by glucose infusion.

Increasing glucogenic substrate (i.e., propionate) supply or glucose infusion may reduce the

utilization of some glucogenic AA for gluconeogenesis and subsequently increase the supply

of AA to the mammary gland (REYNOLDS et al. 1994). However, this kind of AA-sparing

effect of glucose could not be prooved in a study by RULQUIN et al. 2004 where isoenergetic

duodenal infusion of glucose did not increase arterial mammary glucogenic AA fluxes.

Alternatively, an increased insulin concentration during glucose infusion could enhance


[12]

mammary gland AA uptake, leading to increased mammary availability of AA for milk

protein synthesis (MACKLE et al. 2000).

2.5.2 Feed Intake

Surplus glucose has had somewhat inconsistent effects on feed intake of dairy cows. Some

studies have demonstrated that feed intake decrease with abomasal (FROBISH and DAVIS

1976; CLARK et al. 1977) or intravenous (AMARAL et al. 1990) glucose infusions in dairy

cows, whereas others have shown no change ( DHIMAN et al. 1993; KNOWLTON et al.

1998). In non-ruminants, the hepatic branches of the vagus nerve carry signals from the liver

to the brain which regulates many aspects of food intake and metabolism (LANGHANS et al.

1985). Vagally mediated hepatic satiety signals are generated from extensive hepatic

metabolism of glucose and other metabolic fuels (NIIJIMA 1983; LANGHANS et al. 1985).

Since hepatic hexokinase activity is low in ruminants compared with non-ruminants

(BALLARD et al. 1969), hepatic removal and oxidation of glucose appears to be

insignificant. Therefore, part of the differences in the hypophagic effects of glucose supply

observed between ruminants and non-ruminants could be related to the differences in hepatic

oxidation of glucose according to the hepatic oxidation hypothesis (HOT; ALLEN 2000;

ALLEN and BRADFORD 2007). In accordance with HOT, depression of feed intake by

propionate infusions has been well documented in ruminants (ALLEN 2000). Increased

hepatic oxidation of propionate during a meal would increase the energy state of hepatocytes,

generating a satiety signal to terminate the meal (ALLEN et al. 2009). It had also been

suggested that the hypophagic effects of propionate may be mediated by plasma insulin

because high plasma insulin concentrations were correlated with a greater depression in DMI

during high-grain feeding (BRADFORD and ALLEN 2007). However, the hypophagic effect

of propionate infusions has been observed without an increase in the plasma insulin level in

other studies (ALLEN et al. 2009).

2.5.3 Glucose Metabolism

2.5.3.1 Hepatic Glucose Production

It has been suggested that hepatic glucose synthesis decreases in ruminants when the

absorption of glucose from the gut increases (HUNTINGTON 1997). Intravenous, abomasal,

or duodenal glucose infusion decreased the hepatic endogenous glucose production in dairy

cows (BARTLEY and BLACK 1966; THOMPSON et al. 1975; CLARK et al. 1977;

RIGOUT et al. 2002b) and sheep (JUDSON and LENG 1973b; FREETLY and KLINDT


[13]

1996). These decreases were probably due to simultaneously observed increases in the blood

insulin concentrations (THOMPSON et al. 1975; EISEMANN and HUNTINGTON 1994).

However, some longer term infusions studies (>11d) showed no significant effect on

endogenous glucose production (AMARAL et al. 1990; LARSEN and KRISTENSEN 2009).

In monogastric animals, it has been shown that PEPCK activity is reduced with high-

carbohydrate diet and increased during fasting (LEMAIGRE and ROUSSEAU 1994).

Exogenous glucose decreased PEPCK mRNA levels in the liver which resulted from an

accelerated rate of mRNA degradation and from a decrease in the transcription rate

(LEMAIGRE and ROUSSEAU 1994). G6-Pase gene expression may also be regulated by

glucose via an insulin-independent pathway (NORDLIE et al. 1999). In ruminants, a limited

number of studies showed that the pattern of liver gluconeogenesis enzymes changed during

periods of increased glucose availability. Comparative studies showed that hormonal and

nutritional regulations of PEPCK in ruminants are different from those in non-ruminants

species (BALLARD et al. 1969; FILSELL et al. 1969). It has been shown that the activity and

mRNA expression levels of hepatic PEPCK lacks appreciable sensitivity to dietary and

hormonal changes in ruminants (FILSELL et al. 1969; GREENFIELD et al. 2000). However,

one study by RUKKWAMSUK et al. (1999) observed a decrease in hepatic PEPCK activity

in cows fed on a high-energy diet during the dry period. Intraduodenal infusion of glucose

decreased the activity of liver G6-Pase in dairy cows (BARTLEY et al. 1966) and sheep

(PEARCE and UNWORTH 1982). Also, the specific activity of FBPase was decreased in

sheep fed on a concentrate diet (PEARCE and UNWORTH 1976). To present, no study

addressed the dose effects of surplus glucose on gene expression and catalytic activity of

hepatic gluconeogenesis enzymes in dairy cows.

2.5.3.2 Peripheral Glucose Metabolism

It has been shown in ruminants, that glucose became the first choice for oxidation by many

tissues in the body when glucose supplies increased (KNOWLTON et al. 1998). Postruminal

infusion of starch or glucose in lactating dairy cows increased the glucose irreversible loss

rate (i.e. the rate at which glucose is utilized by, e.g., oxidation or milk lactose synthesis;

AMARAL et al. 1990; KNOWLTON et al. 1998; RIGOUT et al. 2002b). Duodenal glucose

infusions in an amount of 1.5 kg/day for 21 d doubled the rate of glucose oxidation as

measured by the fraction of respiratory CO2 derived from plasma glucose (BARTLEY and

BLACK 1966). In another study, the fraction of respiratory CO2 supplied by the oxidation of


[14]

plasma glucose increased from 4.1 to 6.8% with intravenous glucose infusion (737g/d for 11d;

AMARAL et al. 1990) and from 5.4 to 7% by abomasal infusion of starch (1500g /d for 14d;

KNOWLTON et al. 1998). In line with that, increased utilization of glucose in gut tissues has

been observed with interavenous glucose supply in sheep (1-2 mg/kg BW/min for 8 h;

BALCELLS et al. 1995).

2.5.4 Protein Metabolism

When exogenous glucose supply to ruminants was increased by intravenous or postruminal

glucose infusion, the urinary nitrogen excretion was reduced and the whole-body nitrogen

retention increased (MATRAS and PRESTON 1989; OBITSU et al. 2000). In line with that,

decreases in blood urea nitrogen (BUN) concentrations which indicate a decrease in AA

catabolism have been described earlier during intravenous (AMARAL et al. 1990) or

abomasal (OBITSU et al. 2000) glucose infusions. This protein-sparing effect of exogenous

glucose may arise mainly through three mechanisms. Firstly, hepatic gluconeogenesis may be

inhibited when sufficient exogenous glucose is supplied (BARTLEY and BLACK 1966;

JUDSON and LENG 1973b) and hence sparing AA from hepatic utilization (REYNOLDS et

al. 1994; VANHATALO et al. 2003). Secondly, an elevation of plasma insulin concentrations

due to the glucose infusion can increase AA uptake by peripheral tissues (AHMED et al.

1983). This stimulation of the peripheral uptake of AA may reduce the hepatic inflow of AA

for urea synthesis (OBITSU et al. 2000). Finally, JAHOOR and WOLFE (1987) suggested

that glucose may directly inhibit the hepatic capacity for urea synthesis.

2.5.5 Lipid Metabolism

2.5.5.1 Lipogenesis and Lipolysis

It has been shown that acetate but not glucose is the major substrate for lipid synthesis in

ruminant’s adipose tissue and liver. The mammary gland additionally uses appreciable

amounts of BHBA for milk fat synthesis (INGLE et al. 1972; VERNON 1980). There is no

doubt, however, that glucose is required to generate nicotinamide adenine dinucleotide

phosphate (NADPH) needed for de novo fatty acids synthesis and is also the major precursor

of the glycerol 3-phosphate needed for esterifying the fatty acids to TAG (VERNON 1980;

NAFIKOV and BEITZ 2007). Surplus glucose thus increased the conversion of acetate into

lipid in the ruminant’s liver (BALLARD et al. 1972). A similar effect occurred in adipose

tissue but, in this tissue, glucose itself became a quantitatively important precursor of TAG

synthesis in times of excess glucose supply (BALLARD et al. 1972; PRIOR and SCOTT


[15]

1980; PEARCE and PIPEROVA 1984). Glucose administration to ruminants also reduced the

plasma NEFA levels, mainly through an increase in insulin concentrations (VERNON 1980).

Insulin is the key anabolic hormone of adipose tissue. It suppresses lipolysis and stimulates

lipogenesis, resulting in a decrease of plasma NEFA concentrations (VERNON 1980).

2.5.5.2 Mitochondrial Fatty Acid β-Oxidation

Mitochondrial fatty acid β-oxidation involves three key steps: 1) uptake and activation of fatty

acids to fatty acyl-CoA, 2) translocation of the activated fatty acyl-CoA into the mitochondria,

3) β-oxidation of fatty acyl-CoA (BAUCHART et al. 1996). If β-oxidation becomes

excessive, a fourth step becomes prominent, i.e. ketogenesis (DRACKLEY et al. 2001). The

CPT system allows fatty acids to be translocated from the cystol to the mitochondrial matrix.

The CPT system is composed of 3 enzymes: CPT-1, carnitine-acylcarnitine translocase

(CACT), and CPT-2 (KERNER and HOPPEL 2000). CPT-1 is believed to be a key

regulatory enzyme of cell NEFA metabolism by controlling the entry of NEFA into

mitochondria for β-oxidation (KERNER and HOPPEL 2000). CPT-1 exists in three isoforms

in mammalian tissues, CPT-1A, B and C. CPT-1A has been demonstrated in liver, kidney,

brain, and pancreas; whereas, CPT-1B was highly expressed in muscle, heart and adipose

tissue. CPT-1C appeared to be limited to the central neural tissues (RAMSAY et al. 2001).

In monogastric animals, it has been shown that the concentration of malonyl-CoA, an

intermediate precursor for de novo synthesis of fatty acids, is a critical factor regulating entry

of fatty acid into the mitochondria for oxidation. In the carbohydrate-fed state, the insulin-to-

glucagon ratio increased, the rates of fatty acid synthesis were high, and hepatic malonyl-CoA

formation was high. Under these conditions, the authors observed decreases in hepatic CPT-1

activity and gene expression levels and an increase in CPT-1 sensitivity to inhibition by

malonyl-CoA (KERNER and HOPPEL 2000). Vice versa, under conditions of increased fatty

acid oxidation, e.g. starvation, hepatic CPT-1 becomes more active and less sensitive to

inhibition by malonyl-CoA, resulting in an increase in hepatic fatty acids oxidation (KERNER

and HOPPEL 2000).

Inefficient hepatic β-oxidation of NEFA could be one of the predisposing factors for fatty

liver in lactating dairy cows (DRACKLEY 1999). Therefore, it is of great importance to

identify the factors that regulate the disposition of NEFA between oxidation and esterification

in the liver. In ruminants, the rate of hepatic β-oxidation of NEFA is also controlled by CPT


[16]

activity through controlling the entry of NEFA into the mitochondria (JESSE et al. 1986a).

Despite the fact that hepatic lipogenesis rate is low in ruminants, malonyl-CoA is also

considered to be important in the partitioning of fatty acids between esterification and

oxidation in ruminants, similar to what has been observed in non-ruminants species (JESSE et

al. 1986a,b). Previous studies reported that insulin decreased hepatic CPT activity and the

capacity for total hepatic NEFA oxidation in ruminants, presumably through increased

malonyl-CoA concentration and increased sensitivity of CPT to malonyl-CoA inhibition

(CHOW and JESSE 1992; ANDERSON et al. 2002). In dairy cows, the influence of

nutritional status on CPT is evident from the observations that CPT activity increased or

tended to increase during periods of NEB (DANN and DRACKLEY 2005; DOUGLAS et al.

2006) and decreased when feeding a high-concentrate diet (AIELLO et al. 1984).

In man, an increase in muscle glucose oxidation was linked to a rise of the intracellular

concentration of malonyl-CoA, which is a potent inhibitor of CPT, and accordingly prevents

the entry of fatty acids to mitochondrial oxidation (CAHOVÁ et al. 2007). Accumulation of

intracellular lipids due to insufficient fatty acid oxidation, in turn, has been shown to suppress

insulin signalling (CAHOVÁ et al. 2007), leading to insulin resistance (KREBS and RODEN

2004). Therefore, impaired muscle LCFA oxidation and CPT activity might be involved in the

mechanisms by which peripheral tissue insulin resistance developed by overconsumption of

energy during the dry period in dairy cows (HOLTENIUS et al. 2003). Consequently, studies

investigating the changes in skeletal muscle CPT activity relative to the energy balance are

warranted in dairy cows.

2.6 Main Objectives

Given the considerable efforts to identify the metabolic responses to surplus glucose supply in

dairy cows, there is still scarce information available on how intermediary metabolism adapts

at different levels of glucose availability over a long time. The overall objectives of the

present study were to investigate the effects of increasing rates of continuous glucose supply

in mid-lactating dairy cows on lactation performance, body condition, and on some aspects of

liver and skeletal muscle glucose and lipid intermediary metabolism. The present study was

carried out to address precisely these questions:

Do lactating dairy cows use excessive glucose to improve lactational performance or

not?


[17]

To what upper limit can cows adapt to surplus glucose without dysregulation of

glucose homeostasis?. Further, if dysregulation of glucose homeostasis occured, might

it be linked to compromised insulin sensitivity of peripheral organs?

What is the dose effect of surplus glucose on gene expression and catalytic activity of

key hepatic gluconeogenesis enzymes in dairy cows?

What is the dose effect of surplus glucose on body protein metabolism?

What is the possible fate of infused glucose?

What is the dose effect of glucose supply on lipid metabolism?

Do the catalytic activities of CPT in liver and skeletal muscle respond in a co-ordinate

manner to positive energy balance or not?

How quick can possible adaptations to surplus glucose be reversible after ceasing of

glucose infusion?

CHAPTER‐3: MANUSCRIPT‐I

[18]


[19]


[20]


[21]


[22]


[23]


[24]


[25]


[26]


[27]


[28]


[29]


[30]


[31]

CHAPTER‐4: MANUSCRIPT‐II

[32]

4. Manuscript-II: Expression and activity of key hepatic gluconeogenesis enzymes in

response to increasing intravenous infusions of glucose in dairy cows1 B. Al-Trad,* T. Wittek,† G. B. Penner,‡, K. Reisberg,§ G. Gäbel,§ M. Fürll,# and J. R.

Aschenbach*,2

*Institute of Physiology and Pathophysiology, University of Veterinary Medicine Vienna,

1210 Vienna, Austria †Division of Animal Production and Public Health, University of Glasgow, G61 1QH, UK. ‡Animal and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan,

Canada, S7N 5A8 §Institute of Veterinary Physiology, University of Leipzig, D-04103 Leipzig, Germany #Clinic for Large Animal Internal Medicine, University of Leipzig, D-04103 Leipzig,

Germany

Address for correspondence:

Prof. Dr. Jörg R. Aschenbach, Institute of Physiology and Pathophysiology, University of

Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria

Phone: +43-1-25077 4500

Fax: +49-1-25077 4590

e-mail: [email protected]

Journal of Animal Science (2010); doi:10.2527/jas.2009-2463

1 Experiments were performed at the University of Leipzig 2 Corresponding author: [email protected]


[33]

ABSTRACT

The present study aimed at investigating whether increasing levels of glucose supply have a

depressive effect on the mRNA abundance and activity of key gluconeogenesis enzymes in

dairy cows. Twelve Holstein-Friesian dairy cows in mid-lactation were intravenously infused

with either saline (SI; n = 6) or a 40% glucose solution (GI; n = 6). For GI cows, the infusion

dose increased by 1.25%/d relative to the initial NEL requirement until a maximum dose

equating to surplus 30% NEL was reached on d 24. Cows receiving SI received an equivalent

volume of 0.9% saline solution. Blood samples were taken every 2 d and liver biopsies were

collected every 8 d. A treatment × quadratic dose interaction was observed for both the

concentration of plasma glucose and serum insulin. The interactions were due to positive

quadratic responses of the concentrations of glucose and insulin for GI cows whereas; the

concentrations of glucose and insulin did not change over time for SI cows. The concentration

of β-hydroxybutyric acid (BHBA) and serum urea nitrogen (BUN) responded in a treatment ×

quadratic dose manner such that greater decreases in BHBA and BUN concentrations were

observed for cows receiving GI than SI as the dose level increased. Serum NEFA

concentration tended to follow a similar pattern as serum BHBA and BUN, however, the

interaction was not significant. The mRNA abundance of gluconeogenesis enzymes followed

a linear treatment x dose interaction for only pyruvate carboxylase (PC), which was paralleled

by a trend for a linear treatment x dose interaction for PC enzyme activity. The lowest PC

expression and activity were observed at the highest glucose dose. The activity, but not

mRNA abundance, of fructose 1,6-bisphosphatase (FBPase) showed treatment x quadratic

dose interactions with decreasing activity at increasing glucose dose. Activities and

expression levels of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase were not

affected by treatment. In conclusion, hepatic gluconeogenesis enzymes are only moderately

affected by slowly increasing glucose supply, including a translational or posttranslational

downregulation of FBPase activity and a decrease in the mRNA abundance of PC with

possible consequences for PC enzyme activity.

Key Words: enzyme activity, gene expression, glucose infusion, hepatic gluconeogenesis,

dairy cows


[34]

INTRODUCTION

The majority of ingested carbohydrates are converted to short chain fatty acids in the

forestomach of ruminants (Young, 1977; Seal and Reynolds, 1993). Under many dietary

settings, this implies low availability of glucose for absorption (Baird et al., 1980; Reynolds et

al., 1988); thereby, generating a need for hepatic gluconeogenesis (Young, 1977). Efficient

gluconeogenesis is especially important in dairy cows because it is the major pathway for

maintaining adequate glucose supply for the mammary gland (Reynolds et al, 1988;

Huntington et al., 2006). When cows approach peak lactation, insufficient gluconeogenesis

might contribute to the occurrence of metabolic disorders like ketosis and fatty liver (Mills et

al., 1986; Rukkwamsuk et al., 1999; Murondoti et al., 2004). Consequently, knowledge on the

regulation of hepatic gluconeogenesis has implications for both the performance and the

health status of dairy cows.

While surplus supply of glucose has often been shown to downregulate hepatic glucose

production (Bartley and Black, 1966; Thompson et al., 1975; Lomax et al., 1977; Rigout et

al., 2002), it is a crucial question whether this also involves a down-regulation of the involved

enzymes. A down-regulation of gluconeogenic enzymes could have consequences extending

beyond the period of surplus glucose or glucogenic substrate supply. For example, a down-

regulation of gluconeogenic enzymes in cows overfed prepartum could exacerbate metabolic

disturbances during the post-partum phase of the transition period (Rukkwamsuk et al., 1999;

Murondoti et al., 2004). Therefore, the objective of this study was to investigate the dose effect

of intravenous glucose infusion on the mRNA abundance and activity of key enzymes of

hepatic gluconeogenesis. The hypothesis was that mRNA abundance and activity of hepatic

gluconeogenic enzymes would decrease with increasing dose of glucose and that, at least, the

decreases of enzyme activity would persist beyond the actual period of surplus glucose

supply.


[35]

MATERIALS AND METHODS

Animals and Experimental Design

Experimental procedures were pre-approved by the local authorities, Regierungspräsidium

Leipzig (reference 24-9168.11, TVV 49/06). The use of animals, the general experimental

design and the production traits during the experiment have been described in detail in a

previous report (Al-Trad et al., 2009). In brief, experiments were carried out on 12 Holstein-

Friesian cows from the dairy herd of the University of Leipzig. Cows were confirmed to be in

the 2nd to 4th month of gestation (193 ± 14 DIM) at the start of the experiment and had an

average BW and energy-corrected milk yield of 632 ± 33 kg and 30.5 ± 2.3 kg/d, respectively.

Cows were housed in individual tie stalls with straw bedding. Twice daily (0600 and 1500 h),

cows received a diet based on grass haylage and supplements containing a commercial energy

concentrate for lactating dairy cows (Multilac, Leikra GmbH, Leipzig, Germany) and soybean

meal. The ingredient and chemical composition of the diet has previously been reported (Al-

Trad et al., 2009). The diet was low in starch (11.3% DM) and sugar content (4.4% DM) in

order to minimize the direct entry of glucose from the digestive tract. Water was available for

ad libitum intake. Cows were milked at 0630 and 1600 h.

Cows were assigned to either a glucose infusion (GI; n = 6) or saline infusion (SI; n = 6)

treatment balanced for actual lactation performance and DIM. Infusions were made via a 14-

ga, 20-cm jugular catheter (Cavafix Certo Splittocan 338, Melsungen, Germany) which was

replaced every 8 d. Glucose infusion treatment consisted of continuous jugular infusions of

40% glucose solution (Serumwerk Bernburg, Bernburg, Germany) over a period of 28 d. The

infusion dose was calculated for each animal individually as a percentage of their daily NEL

requirement at the start of the study (see Calculations and Statistical Analysis). Dose level

was 0% of the NEL requirement on d 0 and increased linearly by 1.25% each day until a

maximum dose of surplus 30% of the NEL requirement was reached at d 24. This was

equivalent to 2.65 ± 0.19 kg glucose per cow per day. After maintaining the infusion dose at

30% of the NEL requirement between d 24 and 28, responses to glucose withdrawal were

assessed by withholding infusions between d 29 and 32. Infusion canisters were loaded at

1500 h each day, starting in the afternoon of d 0. This assured that cows had been on the

assigned dose level for at least 19 h prior to the collection of biopsies (see below). The

infusion protocol and the individual calculation of infusion volumes were identical for the SI

treatment as the GI treatment except that the cows on the SI treatment received a volume-


[36]

equivalent dose of 0.9% saline (Serumwerk Bernburg, Bernburg, Germany) as a control

treatment.

Sampling and Measurements

Liver biopsies were obtained between 1000 h and 1200 h on d 0, 8, 16, 24, and 32 with a 2.5-

mm wide, 250-mm long biopsy needle (Model Berlin, Walter Veterinär-Instrumente,

Rietzneuendorf, Germany) under ultrasonography control (Pie Medical Scanner 100 LC, Pie

Medical, Maastricht, The Netherlands) as described by Gröhn and Lindberg (1982).

Following collection, liver samples were washed immediately in ice-cold 0.9% saline

solution. Samples for enzyme activity determinations (~ 200 mg) were snap frozen in liquid

nitrogen and stored at -80 °C. Samples (~300 mg) for real-time reverse transcription-

polymerase chain reaction (rt-PCR) were transferred into tubes with 3 mL RNAlater (Qiagen,

Germantown, MD), placed in a refrigerator for 24 h, and stored at -20 °C thereafter.

Blood samples were taken from a coccygeal vein every 2 d at 1000 h and processed as

described previously. Monovette tubes with heparin (16 IU lithium heparin/ml blood;

Sarstedt, Nümbrecht, Germany) were used to obtain plasma for glucose analysis. Kaolin-

coated Monovette tubes (Sarstedt) were used for serum preparation to determine insulin,

serum urea nitrogen (BUN), non-esterified fatty acids (NEFA), and β-hydroxybutyric acid

(BHBA) concentrations. All plasma and serum samples were stored at –20 °C.

Blood Analyses

An automatic analyzer (Hitachi 912, Boehringer Mannheim, Mannheim, Germany) was used

to analyze the concentrations of plasma glucose (glucose/HK kit; Roche Diagnostics GmbH.,

Mannheim, Germany; Peterson and Young, 1965), serum BUN (urea/BUN kit; Roche; Talke

and Schubert, 1965), serum NEFA (NEFA kit; Randox Laboratories Ltd., Crumlin, UK;

Matsubara et al., 1983) and serum BHBA (Ranbut/kit; Randox; McMurray et al., 1984). A

radiometric immunoassay was used for analysis of serum insulin (INS-IRMA Kit; BioSource

Europe SA, Nivelles, Belgium) which has a high specificity for insulin and no significant

cross-reactivity to proinsulin (Temple et al., 1990). The kit originally comes with a human

calibration standard. However, we routinely prepare calibration standards by diluting bovine

insulin in the human insulin-free serum provided with the test to account for the partial

differences in the amino acid sequence between human and bovine insulin.


[37]

Gluconeogenic Enzymes Activity

The activities of the following key hepatic gluconeogenic enzymes was measured: pyruvate

carboxylase (PC, EC 6.4.1.1), phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32),

fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), and glucose 6-phospatase (G6-Pase, EC

3.1.3.9). For PC and PEPCK assays, liver tissues were homogenized with an Ultra-Turrax T25

homogenizer (IKA, Staufen, Germany). Thereafter, mitochondria were disrupted by

sonication (Bandelin Sonoplus HD 2070; Bandelin Electronic, Germany) according to the

protocol of Agca et al. (2002). Pyruvate carboxylase activity was assayed

spectrophotometrically at room temperature using the previously published method of

Crabtree et al. (1972). This procedure measures the reduction of 5,5'-dithiobis-2-nitrobenzoic

acid by CoA. The latter is released when citrate synthase couples acetyl-CoA to oxaloacetate

emerging from the PC reaction. Activity of PEPCK was measured at 37 °C based on the 14CO2 incorporation assay of Ballard and Hanson (1967) with the modifications described by

Agca et al. (2002). To avoid the release of 14CO2 into the environment, the 14CO2 not

incorporated into oxaloacetate was trapped in barium hydroxide (0.2 M) at the end of the

procedure.

For FBPase and G6-Pase assays, tissues were homogenized (1:10, w/v) in ice-cold

homogenization buffer containing 20 mM HEPES, 100 mM sucrose and 0.25 mM EDTA (pH

7.4). The homogenate was centrifuged for 5 min at 1,000 × g to remove cell debris. The

supernatant was recentrifuged at 10,000 × g for 10 min. The resulting supernatant was used to

determine FBPase and G6-Pase activities by the methods of Marcus et al. (1973) and

Swanson (1950), respectively. The latter methods spectrophotometrically measure the release

of inorganic phosphate from either fructose 1,6-bisphosphate or glucose 6-phosphate.

RNA Isolation and Quantitative rt-PCR

Ribonucleic acid was extracted from liver tissues stored in RNAlater using Trizol reagent

(Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The resulting RNA

pellets were dissolved in RNase-free water and the quantity and quality of the isolated RNA

were determined by absorbance at 260 and 280 nm. Total RNA (1 µg) was reverse transcribed

using oligo-(dT)15 primer in a 20-µl reaction according to the manufacturer's instructions

(AMV-RT kit, Roche Diagnostics, Mannheim, Germany). Reverse transcription reactions

were carried out at 25 °C for 10 min followed by 42 °C for 60 min and 95 °C for 5 min. The

resulting first-strand cDNA was stored at −20 °C until use for rt-PCR.


[38]

Quantitative rt-PCR was carried out on a Rotor-Gene 6000 (Corbett Research, Australia),

using β2-microglobulin (B2M) as non-regulated reference gene. Primers and dual-labelled

fluorescent probes (Table 1) for quantitative rt-PCR were designed using the web-based

quantitative rt-PCR probe design software provided by MWG Biotech AG (Ebersberg,

Germany, http://www.eurofinsdna.com/de/home.html) and synthesized by the same company.

To ensure the specificity of the primers and probes, a gel was run, in which a single band of

expected size was obtained. Primer and probe concentrations were optimized to the

concentration that provided the lowest fluorescence threshold cycle (CT) values and the

highest increase of fluorescence compared to the background. For each sample, the target

gene and the control gene were run under duplex reaction conditions in duplicate. The

following reagents were used for amplification in 20 μl final volume: 1 μl of sample cDNA,

2 μl Mg-free 10X buffer, 0.75 U Dynazyme II DNA polymerase (Finnzymes, Espoo,

Finland), 5.5 mM MgCl2, 0.3 mM dNTP, 900 nM of each primer and 150 nM of each probe

for the genes of interests, and 200 nM of each primer and 50 nM of the probe for the B2M.

Amplification conditions for quantification were: 95 °C for 2 min and 45 cycles of 95 °C for

30 s, 55 °C for 30 s and 72 °C for 30 s. After the amplification efficiency of each target and

reference gene was validated, the relative gene expression levels were determined by the 2-

ΔΔCT method as described by Livak and Schmittgen (2001). Level of gene expression was

expressed as the normalized ratio of gene expression relative to B2M mRNA level using one

sample from the SI group as an interplate calibrator. The suitability of B2M as a reference

gene was checked by demonstrating that B2M CT values at the beginning of the experiment

(i.e. d 0) were not different from those obtained with later samples (d 8, 16, 24, 32) in both

groups (P > 0.05; data not shown).

.


[39]

1B2M, β2 microglobulin; PEPCK-C, cytosolic phosphoenolpyruvate carboxykinase; PEPCK-M, mitochondrial phosphoenolpyruvate carboxykinase;

Table 1. Sequences of primers and probes used for quantitative real time RT-PCR Gene1 GenBank accession Nucleotide range Primers and probes sequences2

B2M BC118352.1 102-123 138-168 170-192

Forward: 5´-AGCGTCCTCCAAAGATTCAAGT-3´ Probe: 5´-FAM-CACCAGAAGATGGAAAGCCAAATTACCTGAA- BHQ1-3´ Reverse: 5´-GGATGGAACCCATACACATAGCA-3´

PC NM_177946.3 2,042-2,058 2,066-2,095 2,102-2,122

Forward: 5´-CAACGCCGTGGGCTACA-3´ Probe: 5´-ROX-CCCCGACAATGTGGTCTTCAAGTTCTGCGA-BHQ2-3´ Reverse: 5´-ATGTCCATGCCATTCTCCTTG-3´

PEPCK-C NM_174737.2 2,184-2,203 2,234-2,260 2,268-2,288

Forward: 5´-TTGGGGAGGGAAATAGCAGG-3´ Probe: 5´-JOE-ATGCACCTTTGTTCAACTTAGGGACAC-TAMRA-3´ Reverse: 5´-AGGGGGAAAGACAAAGAAGAC-3´

PEPCK -M XM_583200.3 2,000-2,019 2,069-2,096 2,101-2,120

Forward: 5´-ACACCACCCAGTTGTTCTCC-3´ Probe: 5´-JOE-TGACAGAACAGGTCAACCAGGATCTGCC-TAMRA-3´ Reverse: 5´-TCCAGTTCAGCCAGCACTTC-3´

FBPase NM_001034447.1 975-994

997-1,023 1,055-1,073

Forward: 5´-AGAAGGCAGGAGGAATGGCT-3´ Probe: 5´-ROX-CACCGGGAAGGAAACTGTGCTGGACAT-BHQ2-3´ Reverse: 5´-CAG GAGACCCCAAGA TGAT-3´

G6-Pase NM_001076124.1 458-478 495-517 529-547

Forward: 5´-GTCACATCCACCCTCTCTATC-3´ Probe: 5´-JOE-AGCCAACCTACAGATTTCGGTGC-TAMRA-3´ Reverse: 5´-CCAGAATCCCAACCACAAC-3´

PC, pyruvate carboxylase; FBPase, fructose 1,6-bisphosphatase; G6-Pase, glucose 6-phosphatase, catalytic subunit. 2FAM, 6-carboxy-fluorescein; TAMRA, 6-carboxy-tetramethylrhodamine; JOE, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein; ROX, 6-carboxy-X-rhodamine; BHQ1,2, blackhole quencher-1,2.

http://www.ncbi.nlm.nih.gov/sites/?Db=gene&Cmd=retrieve&dopt=full_report&list_uids=538710&log$=databasead&logdbfrom=nuccore

http://www.ncbi.nlm.nih.gov/sites/?Db=gene&Cmd=retrieve&dopt=full_report&list_uids=538710&log$=databasead&logdbfrom=nuccore


[40]

Calculations and Statistical Analysis

Net energy required for maintenance and lactation (MJ/d) was calculated as 4.184 × ([BW0.75

× 0.08] + milk yield (kg) × [(0.0929 × fat %) + (0.0563 × protein %) + (0.0395 × lactose %)])

according to the NRC (2001). Based on the energy requirement of the individual cow,

infusion dose (kg glucose/d) was calculated as follows; designated dose level × NEL/(15.6

MJ/kg).

All data were analyzed using the PROC MIXED procedure of SAS (version 9.1.3; SAS

Institute Inc., Cary, NC; ref. SAS, 2002) accounting for repeated measures. The model to test

for global effects of treatment, dose and the dose x treatment interaction included the fixed

effects of treatment (GI vs. SI), dose (representing dose levels of 0, 10, 20, and 30% NEL

requirement) and their interaction. The NEL calculated prior to the start of the experiment was

used as a covariate. In addition, because cows were gradually exposed to increasing dose

levels; dose was included in the model as a repeated measure. The covariate error structure

which yielded the lowest Akaike’s and Bayesian information criterion values for each

dependent variable was used. Because the first part of the statistical model could not account

for the steady increase in dose over time, we continued to assess the slopes of the responses of

the infusion dose and their interaction with treatment (i.e. linear vs. quadratic effects) using

the same model except that dose was considered to be a continuous variable. The biological

significance of the statistical tests was finally assessed based on the treatment effects derived

from the first model, and the linear and quadratic responses of dose and treatment × dose

derived from the second model (Table 2). Priority was given to treatment × dose interactions

because dose effects can be partially confounded by time and infusion volume effects given

the long duration of the experiment and the constant increase in infusion volume..

Comparisons of post-infusion samples (d 32) to pre-infusion samples (d 0) were performed

using a Student’s paired t-test. Differences were considered significant when P < 0.05 and

tendencies are discussed when P < 0.15.

RESULTS

Production data

Production data from this study have been reported previously (Al-Trad et al., 2009). Briefly,

dry matter intake was not different between the GI and SI groups over the period from d 0 to

d 24 (period mean ± pooled SEM for GI vs. SI cows: 17.5 vs. 17.7 ± 0.9 kg/d).

Correspondingly, surplus glucose linearly improved the energy balance of GI cows between d


[41]

0 and d 24 (from -14.6 to 35.4 MJ/d); while it was unchanged in the SI group during the same

period (from -10.4 to -1.8 MJ/d; pooled SEM: 6.3 MJ/d). The improved energy balance was

coupled to a net BW gain of 32 kg in GI cows (from 651 to 670 kg) relative to the SI cows

(from 614 to 601 kg; pooled SEM: 28 kg), while the energy-corrected milk yield was not

affected by the treatment (period mean between d 0 and d 24 ± pooled SEM: 27.8 and 29.7 ±

1.9 kg/d for GI and SI cows, respectively) (Al-Trad et al., 2009).

Plasma and Serum Metabolites and Hormones

Plasma glucose and serum insulin concentrations showed predominantly quadratic dose

effects and treatment × quadratic dose interactions (P < 0.05; Figure 1A and 1B,

respectively). Additionally, there was a treatment effect on serum insulin concentration (P =

0.005) and a trend for a treatment effect on plasma glucose concentrations (P = 0.06). These

effects were due to increases in plasma glucose and serum insulin concentrations in the GI

group occurring mainly when infusion doses exceeded 20% NEL requirement; whereas, cows

receiving the SI treatment had no response to increasing dose level (Figure 1).

Treatment did not affect the concentrations of BUN, BHBA, and NEFA; however, BUN,

BHBA and NEFA decreased in a quadratic manner in response to increasing dose level (P =

0.001; Figure 2A-C). Additionally, treatment × quadratic dose interactions were detected for

BUN and BHBA (P = 0.001), and tended to be present for NEFA (P = 0.07), indicating that

the concentrations of these metabolites decreased specifically with increasing dose of glucose.

In contrast to glucose and insulin concentrations, however, the major part of the decrease in

BUN, BHBA, and NEFA concentrations occurred at low infusion levels < 20% NEL

requirement.

Post-infusion values (d 32) were lower in the GI group for NEFA compared to pre-

treatment values (d 0). However, glucose, insulin, BHBA, and BUN values were not different

between d 0 and d 32 in both groups, indicating a quick reversal of the GI effect on these

other blood metabolites.


[42]

Figure 1. Plasma glucose (A) and

serum insulin concentrations (B) of

cows treated with glucose (■) or

saline (●).

Open symbols depict pre- and post-

infusion values that were not

included in statistical trend

modeling. Data are expressed as

least square means; pooled SEM:

0.31 mmol/L (A), 120 pmol/L (B).

Probability levels for statistical

contrasts were as follows; treatment:

0.06 (A), 0.005 (B); dose linear:

0.03 (A), 0.10 (B); dose quadratic:

0.02 (A), 0.001 (B); treatment ×

dose linear: 0.61 (A), 0.44 (B);

treatment × dose quadratic: 0.001

(A), 0.006 (B). Post-infusion values

(d 32) were not different from pre-

infusion values (d 0; P > 0.05).


[43]

Figure 2. Serum urea nitrogen (BUN;

panel A), beta-hydroxybutyric acid

(BHBA; panel B) and nonesterified

fatty acid (NEFA; panel C)

concentrations for blood samples taken

every 2 d for cows treated with glucose

(■) or saline (●).

Open symbols depict pre- and post-

infusion values that were not included

in statistical trend modeling. Data are

expressed as least square means; pooled

SEM, 1.6 mg/dL (A), 0.15 mmol/L (B),

29 µmol/L (C). Probability levels for

statistical contrasts were as follows;

treatment: 0.22 (A), 0.98 (B), 0.56 (C);

dose linear: 0.001 (A), 0.06 (B), 0.001

(C); dose quadratic: 0.001 (A), 0.001

(B), 0.001 (C); treatment × dose linear:

0.12 (A), 0.76 (B), 0.19 (C); treatment ×

dose quadratic: 0.001 (A), 0.001 (B),

0.07 (C). Post-infusion values (d 32)

were different to pre-infusion values (d

0) for only the GI group in panel C (P =

0.01).

Hepatic mRNA Abundance and Activity of Gluconeogenic Enzymes

Hepatic gluconeogenic enzyme activity and relative mRNA abundance levels are listed in

Table 2. Treatment did not affect the mRNA levels or enzyme activity measured in this study.

However, the relative mRNA abundance of PC showed a linear treatment × dose interaction

(P = 0.048) and a tendency for a quadratic dose effect (P = 0.06), indicating decreased

abundance of PC mRNA with increasing glucose dose. The decreased abundance of PC


[44]

mRNA coincided with a numerical decrease in PC activity only at the highest dose of glucose

(linear treatment × dose interaction, P = 0.13). The relative mRNA abundance levels of the

mitochondrial isoform of PEPCK (PEPCK-M) decreased already at low infusion level (10 %

NEL requirement) in a quadratic manner (P = 0.01), and the expression of the cytosolic

isoform of PEPCK (PEPCK-C) tended to decrease linearly (P = 0.07) with increasing dose.

Corresponding to the decreases in the mRNA abundance for PEPCK, the enzyme assay

showed a linear decrease in PEPCK activity with increasing glucose dose (P = 0.03).

However, the decreases in PEPCK mRNA abundance and enzyme activity occurred in both

the SI and GI groups with no significant treatment × dose interactions, indicating time-

dependent or infusion volume-dependent changes with no relationship to glucose treatment. A

linear decrease in both groups with no treatment × dose interaction was also observed for

FBPase mRNA abundance. However, FBPase activity remained stable in the SI group despite

the decreasing mRNA levels but was decreased in the GI group at the highest dose level of

30% NEL requirement (treatment × quadratic dose, P = 0.02). Treatment did not affect the

mRNA abundance of G6-Pase; although a quadratic decrease with increasing dose (P = 0.02)

was observed for both treatments. Similarly to PEPCK-M, the steepest decrease in the mRNA

abundance of G6-Pase occurred already at low infusion dosages of 10% NEL requirement.

However, the activity of G6-Pase was not affected by treatment, dose or their interactions. As

such, the changes of mRNA abundance and enzyme activity were uncoordinated for FBPase

and G6-Pase, indicating that mRNA abundance had no measurable influence on the activity of

these enzymes.

The significantly or numerically decreased activities of FBPase and PC and the decreased

transcription of PC in the GI group were restored to pre-infusion values by d 32, i.e. at 4 d

after suspending the infusion (Table 2). Similarly, PEPCK mRNA abundance and activity was

restored to pre-infusion values except for a slight remaining reduction in PEPCK activity in

the SI group (P = 0.02). However, the activity of G6-Pase was decreased in the GI but not the

SI group after stopping infusion (P = 0.02), although G6-Pase mRNA abundance increased to

pre-infusion values at the same time.


[45]


[46]

DISCUSSION

Hepatic gluconeogenesis is a key component of total glucose entry in cattle (Young, 1977;

Huntington et al., 2006). Approximately 60 - 85% of the glucose entry is utilized by the

mammary gland in lactating dairy cows (Annison et al., 1974; Bickerstaffe et al., 1974).

Consequently the kinetics and regulation of gluconeogenesis are an area of great interest with

regard to milk production. The fact that a number of previous studies failed to detect a

positive influence of surplus glucose supply on milk production (Amaral et al., 1990; Hurtaud

et al., 1998; Al-Trad et al., 2009) supports the concept that gluconeogenesis is mostly

functioning at a very appropriate level with regard to lactation requirements (Al-Trad et al.,

2009).

As with any biochemical pathway, the regulation of gluconeogenesis may occur at one or

more of the following levels; (1) regulation of substrate supply, (2) regulation of activity of

catalytic enzymes, and (3) regulation of end-product utilization. Of these, glucogenic

substrate supply is considered the control point best amenable to feeding management

(Veenhuizen et al., 1988; Overton et al., 1999). Increasing the entry rate of propionate and

glucogenic amino acids from the portal-drained viscera has become a successful strategy to

support cattle in periods of glucose shortage; especially in the critical period postpartum. Such

strategies include measures to increase DMI, to modify fermentation patterns, or to directly

add glucogenic precursors to the diet (Overton and Waldron, 2004). On the other hand,

upregulation of glucose oxidation or anabolism are primary adaptations of ruminants

themselves in times of excessive supply of glucogenic precursors (Judson and Leng, 1973a;

Veenhuizen et al., 1988) or glucose (Amaral et al., 1990; Rigout et al., 2002). However, data

is limiting on how the regulation of catalytic activity is utilized to meet imbalances between

glucogenic precursor/glucose availability and glucose demand.

The present study was designed to evaluate the effect of increasing glucose supply on four

key hepatic gluconeogenesis enzymes in mid-lactating dairy cows. The intention was to

explore the whole range of nutritionally relevant surplus glucose. When assuming postruminal

starch digestion as the major way to provide glucose directly to dairy cows and proceeding

from maximum rates of postruminal starch digestion in the order of ~5 kg/d (McCarthy et al.,

1989; Taylor and Allen, 2005), a functional maximum uptake capacity (fMUC) of the small

intestine can be calculated in the range of ~2.8 kg glucose/d according to the formula of Cant

et al. (1999). In the cows of the present study, this fMUC was modeled by the infusion dose


[47]

of 30% NEL requirement (i.e., 2.65 kg glucose/d). It became clear that this infusion dose may

not only be representative for the intestinal limit of glucose absorption. It is also a dose at

which cows show dysregulation of glucose homeostasis as evidenced by elevated

concentrations of blood glucose and insulin. Obviously, the limit of intestinal absorption of

glucose and the ability to use the absorbed glucose appear highly coordinated in dairy cows.

The present study additionally modeled the whole range of gluconeogenic requirement in

order to meet the glucose demand. Diets were formulated to be low in starch and sugar to

provide basal conditions with minimized direct entry of glucose from the digestive tract.

Consequently, hepatic gluconeogenesis had to meet the complete demand for glucose entry at

the beginning of the experiment. By contrast, the infusion dose of 2.65 kg glucose/d at the end

of the GI period completely accounted for the glucose demand, which would be 2.27 kg/d at

27.8 kg ECM/d according to the formula by Danfær (1994). The latter means that

gluconeogenesis was, in theory, completely dispensable at an infusion dose of 30% NEL

requirement.

The main finding was that the effect of GI on gluconeogenic enzyme activity was very

moderate. Enzymatic activity decreased (or tended to decrease) for only FBPase and PC, and

only at extremely high levels of surplus glucose supply (i.e. infusion of 30% NEL

requirement). Moreover, both decreases were rapidly reversible within 4 days after the end of

infusion. We further elucidated that the glucose-induced decreases in enzyme activity were

not linearly related to decreased mRNA abundance because mRNA abundance and enzyme

activity changed in a coordinated manner only for PEPCK with a tendency for PC, indicating

that the activities of FBPase and G6-Pase are predominantly regulated by translational and

posttranslational events.

Three of the investigated enzymes, PEPCK, FBPase and G6-Pase, belong to the common

gluconeogenic pathway while PC serves to shuttle lactate and glucogenic amino acids into

this pathway (Pilkis and Granner, 1992; Jitrapakdee and Wallace, 1999). Unlike in

monogastric species, lactate and glucogenic amino acids are not the dominating glucogenic

precursors in ruminants where propionate is quantitatively most important (Amaral et al.,

1990; Huntington et al., 2006). Propionate enters the common gluconeogenic pathway at the

level of PEPCK after initial conversion by propionyl-CoA carboxylase to oxaloacetate

(Halarnkar and Blomquist, 1989). Accordingly, PEPCK has been identified as a main rate-


[48]

limiting enzyme involved in glucose production from propionate in dairy cows (Greenfield et

al., 2000). Although the rate of propionate utilization for hepatic gluconeogenesis was not

measured in the current study, the non-significant treatment × dose interaction for the

expression and activity of PEPCK provides indirect support that infused glucose did not

specifically compromise gluconeogenesis from propionate at the PEPCK level. Previous

glucose metabolism studies in ruminants confirm this suggestion by showing that hepatic

propionate extraction efficiency and capacity for glucose synthesis from propionate are very

resistant to changes in glucose supply (Judson and Leng, 1973b; Baird et al., 1980) and

insulin levels (Brockman, 1990; Eisemann and Huntington, 1994; Huntington et al., 2006).

Therefore, our enzyme measurements in context with previous investigations on glucose

metabolism support the view that the liver of ruminants has a high metabolic priority to utilize

propionate for gluconeogenesis (Brockman, 1990; Huntington et al., 2006). This conclusion is

not surprising because over 90% of portal propionate is cleared by the liver and most of this

amount is used for hepatic glucose synthesis (Armentano, 1992). Efficient propionate

clearance and metabolism might not only be important for the glucose balance in lactating

dairy cows but may also prevent adverse consequences of propionate accumulation in the

blood (e.g. decreased feed intake; Allen et al., 2005).

In contrast to PEPCK, mRNA abundance of PC tended to be specifically suppressed by GI.

This confirmed earlier findings that PC mRNA abundance is most consistently changed with

varying levels of glucose or energy supply in ruminants (Bradford and Allen, 2005; Velez and

Donkin, 2005; Loor et al., 2006). Coincidence of a decrease in the mRNA abundance of PC

(by 51% at 30% NEL requirement) with a numerical decrease in PC activity at the highest

dose of glucose (by 49% at 30% NEL requirement) might suggest some contribution of

decreased PC transcription to the decreased enzyme activity. It may be speculated that the

observed decrease in PC enzyme activity, although not statistically significant, might have

contributed to a decreased deamination of amino acids because PC is required for hepatic

glucose synthesis from glucogenic amino acids (Greenfield et al., 2000; Velez and Donkin

2005). A decreased deamination of amino acids, in turn, was suggested by the decreased

serum BUN concentration in the GI group and has the unique metabolic advantage of sparing

amino acids for protein synthesis pathways (e.g. muscle or milk protein synthesis).

An enzyme activity significantly affected by GI was FBPase but only at the highest dose of

infused glucose (i.e. 30 % of the daily NEL requirement, equating to 2.65 kg surplus


[49]

glucose/d). The linear treatment × dose interaction was only evident at the enzyme activity

level and not at the mRNA level, indicating posttranscriptional regulation of enzyme activity.

FBPase is the enzyme that releases fructose 6-phosphate from the gluconeogenic pathway

(Pilkis and Granner, 1992) which, after conversion to glucose 6-phosphate, can release

glucose by the action of G6-Pase (see below). FBPase thus controls the overall output of

gluconeogenesis regardless of the precursors utilized. Consequently, the decrease in FBPase

activity might be seen as an effective measure to counteract hyperglycemia occurring at very

high infusion levels. The decrease in FBPase activity could be directly related to the

hyperglycemia because excess glucose, together with insulin, stimulates the intracellular

accumulation of fructose 2,6-bisphosphate, which is a potent competitive inhibitor of FBPase

(Pilkis et al., 1988; Pilkis and Granner, 1992). Additionally, a rapid proteasomal degradation

of FBPase has been demonstrated in yeast upon exposure to glucose (Gancedo, 1971; Brown

and Chiang, 2009). The rapid translocation of FBPase to the nucleus observed in cultured rat

hepatocytes after exposure to glucose or insulin (Yáñez et al., 2004) could indicate that

similar proteasomal degradation of FBPase is possible in the mammalian liver during

hyperglycemia.

Apart from investigating the dose effects of glucose, the second intention of the present

study was to test the response in gluconeogenic capacity after withdrawing an extremely high

glucose load. It became evident that PC mRNA abundance and FBPase activity were fully

restored within only four days after withdrawing GI. This supports the view that

gluconeogenic enzymes in cattle liver adapt rapidly to changes in glucose supply. On the

other hand, it indicates that the dysregulation or maladaptation of gluconeogenic enzymes

described in postparturient dairy cows are not a primary insufficiency of carbohydrate

metabolism but are likely secondary to disturbances of lipid metabolism (Rukkwamsuk et al.,

1999; Murondoti et al., 2004). The latter conclusion does not change when considering that

withdrawing high-dose GI led to posttranscriptional downregulation of G6-Pase in the present

study because the physiological activity of G6-Pase is regulated by the intracellular

concentration of its substrate (glucose 6-phosphate; Van Schaftingen and Gerin, 2002). The

decrease in the activity of G6-Pase after stopping glucose infusion could thus be related to an

increased availability of glucose 6-phosphate if one acknowledges that glucose 6-phosphate

does not only emerge from gluconeogenesis but also from glycogenolysis (Nordlie et al.,

1999; Van Schaftingen and Gerin, 2002). Since the liver accumulates excess amounts of

glycogen during increasing GI (Al-Trad et al., 2009), the increased utilization of this glycogen


[50]

after stopping the infusion could lead to increased availability of glucose 6-phosphate and,

subsequently, to a reduction in G6-Pase activity.

In conclusion, our study demonstrated that increases of glucose supply have, in general, no

negative effect on the activity of key gluconeogenesis enzymes in mid-lactating dairy cows.

Only very high dosages selectively suppress PC mRNA abundance and FBPase activity. Both

effects were fully reversed within only 4 d after the end of high-dose GI. The latter indicates

that glucose metabolism is rather robust with regard to changes in glucose supply in either

direction, at least, in the absence of other metabolic disturbances.

ACKNOWLEDGMENTS

The authors thank C. Benson, A. Schmidt-Mähne and I. Urbansky for technical help, and

G. F. Schusser for infrastructural support. The work was supported by Pfizer Animal Health,

Sandwich, UK.

REFERENCES

Agca, C., R. B. Greenfield, J. R. Hartwell, and S. S. Donkin. 2002. Cloning and

characterization of bovine cytosolic and mitochondrial PEPCK during transition to

lactation. Physiol. Genomics. 11:53-63.

Al-Trad, B., K. Reisberg, T. Wittek, G. B. Penner, A. Alkaassem, G. Gäbel, M. Fürll, and J.

R. Aschenbach. 2009. Increasing intravenous infusions of glucose improve body

condition but not lactation performance in mid-lactation dairy cows. J. Dairy Sci.

92:5645-5658.

Allen, M. S., B. J. Bradford, and K. J. Harvatine. 2005. The cow as a model to study food

intake regulation. Annu. Rev. Nutr. 25:523-547.

Amaral, D. M., J. J. Veenhuizen, J. K. Drackley, M. H. Cooley, A. D. McGilliard, and J. W.

Young. 1990. Metabolism of propionate, glucose, and carbon dioxide as affected by

exogenous glucose in dairy cows at energy equilibrium. J. Dairy Sci. 73:1244-1254.

Annison, E. F., R. Bickerstaffe, and J. L. Linzell. 1974. Glucose and fatty acid metabolism in

cows producing milk of low fat content. J. Agric. Sci. 82:87-95.


[51]

Armentano, L. E. 1992. Ruminant hepatic metabolism of volatile fatty acids, lactate and

pyruvate. J. Nutr. 122:838-842

Baird, G. D., M. A. Lomax, H. W. Symonds, and S. R. Shaw. 1980. Net hepatic and

splanchnic metabolism of lactate, pyruvate and propionate in dairy cows in vivo in

relation to lactation and nutrient supply. Biochem. J. 186:47-57.

Ballard, F. J., and R. W. Hanson. 1967. Phosphoenolpyruvate carboxykinase and pyruvate

carboxylase in developing rat liver. Biochem. J. 104:866-871.

Bartley, J. C., and A. L. Black. 1966. Effect of exogenous glucose on glucose metabolism in

dairy cows. J. Nutr. 89:317-328.

Bickerstaffe, R., E. F. Annison, and J. L. Linzell. 1974. The metabolism of glucose, acetate,

lipids and amino acids in lactating dairy cows. J. Agric. Sci. 82:71-85.

Bradford, B. J., and M. S. Allen. 2005. Phlorizin administration increases hepatic

gluconeogenic enzyme mRNA abundance but not feed intake in late-lactation dairy

cows. J. Nutr. 135:2206-2211.

Brockman, R. P. 1990. Effect of insulin on the utilization of propionate in gluconeogenesis in

sheep. Br. J. Nutr. 64:95-101.

Brown, C. R., and H. L. Chiang. 2009. A selective autophagy pathway that degrades

gluconeogenic enzymes during catabolite inactivation. Commun. Integr. Biol. 2:177-

183.

Cant, J. P., P. H. Luimes, T. C. Wright, and B. W. McBride. 1999. Modeling intermittent digesta flow

to calculate glucose uptake capacity of the bovine small intestine. Am. J. Physiol. 276:G1442-

G1451.

Crabtree, B., S. J. Higgins, and E. A. Newsholme. 1972. The activities of pyruvate

carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in muscles

from vertebrates and invertebrates. Biochem. J. 130:391-396.

Danfær, A. 1994. Nutrient metabolism and utilization in the liver. Livest. Prod. Sci. 39:115-

127.

Eisemann, J. H., and G. B. Huntington. 1994. Metabolite flux across portal-drained viscera,

liver, and hindquarters of hyperinsulinemic, euglycemic beef steers. J. Anim. Sci.

72:2919-2929.

Gancedo, C. 1971. Inactivation of fructose-1,6-diphosphatase by glucose in yeast. J.

Bacteriol. 107:401-405.


[52]

Greenfield, R. B., M. J. Cecava, and S. S. Donkin. 2000. Changes in mRNA expression for

gluconeogenic enzymes in liver of dairy cattle during the transition to lactation. J.

Dairy Sci. 83:1228-1236.

Gröhn, Y., and L. A. Lindberg. 1982. Methodological aspects of the microscopy of bovine

liver biopsies. J. Comp. Pathol. 92:567-578.

Halarnkar, P. P., and G. J. Blomquist. 1989. Comparative aspects of propionate metabolism.

Comp. Biochem. Physiol. B. 92:227-231.

Huntington, G. B., D. L. Harmon, and C. J. Richards. 2006. Sites, rates, and limits of starch

digestion and glucose metabolism in growing cattle. J. Anim. Sci. 84(E Suppl.):E14-

E24.

Hurtaud, C., H. Rulquin, and R. Verite. 1998. Effects of graded duodenal infusions of glucose

on yield and composition of milk from dairy cows. 1. Diets based on corn silage. J.

Dairy Sci. 81:3239-3247.

Jitrapakdee, S., and J. C. Wallace. 1999. Structure, function and regulation of pyruvate

carboxylase. Biochem. J. 340:1-16.

Judson, G. J., and R. A. Leng. 1973a. Studies on the control of gluconeogenesis in sheep:

effect of propionate, casein and butyrate infusions. Br. J. Nutr. 29:175-195.

Judson, G. J., and R. A. Leng. 1973b. Studies on the control of gluconeogenesis in sheep:

effect of glucose infusion. Br. J. Nutr. 29:159-174.

Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-

time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402-408.

Lomax, M. A., G. D. Baird, H. W. Symonds, and C. B. Mallinson. 1977. The effect of

glucose infusion on liver metabolism in the dairy cow in vivo. Proc. Nutr. Soc.

36:74A.

Loor, J. J., H. M. Dann, N. A. Guretzky, R. E. Everts, R. Oliveira, C. A. Green, N. B.

Litherland, S. L. Rodriguez-Zas, H. A. Lewin, and J. K. Drackley. 2006. Plane of

nutrition prepartum alters hepatic gene expression and function in dairy cows as

assessed by longitudinal transcript and metabolic profiling. Physiol. Genomics 27:29-

41.

Marcus, C. J., A. M. Geller, and W. L. Byrne. 1973. Studies on bovine hepatic fructose 1,6-

diphosphatase. Substrate inhibition and the kinetic mechanism. J. Biol. Chem.

248:8567-8573.


[53]

Matsubara, C., Y. Nishikawa, Y. Yoshida, and K. A. Takamura. 1983. A spectrophotometric method

for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-

coenzyme A oxidase. Anal. Biochem. 130:128-133.

McCarthy, R. D. Jr., T. H. Klusmeyer, J. L. Vicini, J. H. Clark, and D. R. Nelson. 1989. Effects of

source of protein and carbohydrate on ruminal fermentation and passage of nutrients to the

small intestine of lactating cows. J. Dairy Sci. 72:2002-2016.

McMurray, C. H., W. J. Blanchflower, D. A. Rice. 1984. Automated kinetic method for D-3-

hydroxybutyrate in plasma or serum. Clin. Chem. 30:421-425.

Murondoti, A., R. Jorritsma, A. C. Beynen, T. Wensing, and M. J. Geelen. 2004. Activities of the

enzymes of hepatic gluconeogenesis in periparturient dairy cows with induced fatty liver. J.

Dairy Res. 71:129-134.

Mills, S. E., D. C. Beitz, and J. W. Young. 1986. Evidence for impaired metabolism in liver

during induced lactation ketosis of dairy cows. J. Dairy Sci. 69:362-370.

Nordlie, R. C., J. D. Foster, and A. J. Lange. 1999. Regulation of glucose production by the

liver. Ann. Rev. Nutr. 19:379-406.

NRC, 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci. Washington,

DC.

Overton, T. R., J. K. Drackley, C. J. Ottemann-Abbamonte, A. D. Beaulieu, L. S. Emmert,

and J. H. Clark. 1999. Substrate utilization for hepatic gluconeogenesis is altered by

increased glucose demand in ruminants. J. Anim. Sci. 77:1940-1951.

Overton, T. R., and M. R. Waldron. 2004. Nutritional management of transition dairy cows:

strategies to optimize metabolic health. J. Dairy Sci. 87:(E Suppl.):E105-E119.

Peterson, J. I., and D. S. Young. 1968. Evaluation of the hexokinase/ glucose-6-phosphate

dehydrogenase method of determination of glucose in urine. Anal. Biochem. 23:301-

316.

Pilkis, S. J., M. R. el-Maghrabi, and T. H. Claus. 1988. Hormonal regulation of hepatic

gluconeogenesis and glycolysis. Ann. Rev. Biochem. 57:755-783.

Pilkis, S. J., and D. K. Granner. 1992. Molecular physiology of the regulation of hepatic

gluconeogenesis and glycolysis. Annu. Rev. Physiol. 54:885-909

Reynolds, C. K., G. B. Huntington, H. F. Tyrrell, and P. J. Reynolds. 1988. Net portal-drained

visceral and hepatic metabolism of glucose, L-lactate, and nitrogenous compounds in

lactating Holstein cows. J. Dairy Sci. 71:1803-1812.

Rigout, S., S. Lemosquet, J. E. van Eys, J. W. Blum, and H. Rulquin. 2002. Duodenal glucose

increases glucose fluxes and lactose synthesis in grass silage-fed dairy cows. J. Dairy

Sci. 85:595-606.


[54]

Rukkwamsuk, T., T. Wensing, and M. J. Geelen. 1999. Effect of fatty liver on hepatic

gluconeogenesis in periparturient dairy cows. J. Dairy Sci. 82:500-505.

SAS, 2002. User’s Guide: Statistics. Version 9.1.3. SAS Inst., Inc., Cary, NC, USA.

Seal, C. J., and C. K. Reynolds. 1993. Nutritional implications of gastrointestinal and liver

metabolism in ruminants. Nutr. Res. Rev. 6:185-208.

Swanson, M. A. 1950. Phosphatases of liver. I. Glucose-6-phosphatase. J. Biol. Chem.

184:647-659.

Talke, H., and G. E. Schubert. 1965. Enzymatic urea determination in the blood and serum in

the Warburg optical test. Klin. Wochenschr. 43:174-175.

Taylor, C. C., and M. S. Allen. 2005. Corn grain endosperm type and brown midrib 3 corn

silage: site of digestion and ruminal digestion kinetics in lactating cows. J. Dairy Sci.

88:1413-1424.

Temple, R. C., P. M. Clark, D. K. Nagi, A. E. Schneider, J. S. Yudkin, and C.N. Hales. 1990.

Radioimmunoassay may overestimate insulin in non-insulin-dependent diabetics. Clin.

Endocrinol. (Oxf.) 32:689-693.

Thompson, J. R., G. Weiser, K. Seto, and A. L. Black. 1975. Effect of glucose load on

synthesis of plasma glucose in lactating cows. J. Dairy Sci. 58:362-370.

Van Schaftingen, E., and I. Gerin. 2002. The glucose-6-phosphatase system. Biochem J.

362:513-532.

Veenhuizen, J. J., R. W. Russell, and J. W. Young. 1988. Kinetics of metabolism of glucose,

propionate and CO2 in steers as affected by injecting phlorizin and feeding propionate.

J. Nutr. 118:1366-1375.

Velez, J. C., and S. S. Donkin. 2005. Feed restriction induces pyruvate carboxylase but not

phosphoenolpyruvate carboxykinase in dairy cows. J. Dairy Sci. 88:2938-2948.

Yáñez, A. J., M. Garcia-Rocha, R. Bertinat, C. Droppelmann, I. I. Concha, J. J. Guinovart,

and J. C. Slebe. 2004. Subcellular localization of liver FBPase is modulated by

metabolic conditions. FEBS Lett. 577:154-158.

Young, J. W. 1977. Gluconeogenesis in cattle: significance and methodology. J. Dairy Sci.

60:1-15.

CHAPTER‐5: MANUSCRIPT‐III

[55]

5. Manuscript-III: Activity of hepatic but not skeletal muscle carnitine

palmitoyltransferase enzyme is depressed by intravenous glucose infusions in lactating

dairy cows*

B. Al-Trad1, T. Wittek2, G. Gäbel3, M. Fürll2, K. Reisberg3, and J.R. Aschenbach1

1Institute of Physiology and Pathophysiology, University of Veterinary Medicine Vienna,

1210 Vienna, Austria 2Clinic for Large Animal Internal Medicine, University of Leipzig, D-04103 Leipzig,

Germany 3Institute of Veterinary Physiology, University of Leipzig, D-04103 Leipzig, Germany

Running head: Carnitine palmitoyltransferase during glucose infusion

Address for correspondence:

Prof. Dr. Jörg R. Aschenbach, Institute of Physiology and Pathophysiology, University

of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria

Phone: +43 1-25077 4500

Fax: +43 1-25077 4590

e-mail: [email protected]

Journal of Animal Physiology and Animal Nutrition (2010); doi: 10.1111/j.1439-

0396.2010.00993.x

*Studies were performed at the University of Leipzig


[56]

Summary A positive energy balance in dairy cows prepartum may decrease hepatic carnitine

palmitoyltransferase (CPT) enzyme activity, which might contribute to disturbances of lipid

metabolism postpartum. The purpose of the present study was to investigate whether skeletal

muscle CPT activity can also be downregulated during positive energy balance. Midlactating

dairy cows were maintained on intravenous infusion of either saline (control) or glucose

solutions that increased linearly over 24 d, remained at the 24-d level until d 28, and were

suspended thereafter. Liver and skeletal muscle biopsies, as well as four diurnal blood

samples, were taken on days 0, 8, 16, 24, and 32, representing infusion levels equivalent to

0%, 10%, 20%, 30%, and 0% of the net energy for lactation (NEL) requirement, respectively.

Glucose infusion increased serum insulin concentrations on d 16 and 24 while plasma glucose

levels were increased at only a single time point on d 24. Serum beta-hydroxybutyric acid

(BHBA) concentrations decreased between d 8 and 24; whereas changes in non-esterified

fatty acids (NEFA) were mostly insignificant. Total lipid contents of liver and skeletal muscle

were not affected by treatment. Hepatic CPT activity decreased with glucose infusion (by

35% on d 24) and remained decreased on d 32. Hepatic expression levels of CPT-1A and

CPT-2 mRNA were not significantly altered but tended to reflect the changes in enzyme

activity. In contrast to the liver, no effect of glucose infusion was observed on skeletal muscle

CPT activity. We conclude that suppression of CPT activity by positive energy balance

appears to be specific for the liver in midlactating dairy cows.

Keywords: dairy cow, carnitine palmitoyltransferase, liver, skeletal muscle, glucose infusion


[57]

Introduction During periods of negative energy balance, such as early lactation, massive mobilization of

non-esterified fatty acids (NEFA) from adipose tissue is common in high-yielding dairy cows.

This situation may trigger the development of metabolic disorders like fatty liver and ketosis.

Fatty liver develops when hepatic import of NEFA exceeds the capacity to oxidize NEFA or

to export them after re-esterification as very low density lipoproteins (Grummer, 1993; Bobe

et al., 2004). Consequently, inefficient hepatic β-oxidation of long chain fatty acids (LCFA)

could be one of the predisposing factors for fatty liver in lactating dairy cows (Emery et al.,

1992; Drackley, 1999; Murondoti et al., 2004).

The β-oxidation of LCFA is localized to mitochondria, and the flux of LCFA into the

mitochondrial matrix is considered the rate-limiting step for their degradation (McGarry and

Brown, 1997; Eaton, 2002). This influx step is mediated by the carnitine-acylcarnitine

translocase system which is composed of the carrier protein, carnitine-acylcarnitine

translocase, and two mitochondrial carnitine palmitoyltransferases (CPT) (for review see

McGarry and Brown, 1997; Kerner and Hoppel, 2000). CPT-1 on the outer membrane of

mitochondria catalyzes the initial reaction of activated LCFA (LCFA-CoA) with carnitine to

form the transportable substrate, acylcarnitine; while CPT-2 is required to release LCFA-CoA

from acylcarnitine inside the mitochondrial matrix. The two steps catalyzed by CPT largely

determine transport rates (McGarry and Brown, 1997; Eaton, 2002). Therefore, the regulation

of hepatic CPT function is considered an important intervention point for the prevention and

treatment of lipid-related metabolic disorders in dairy cows (Drackley, 1999). So far, nutrition

has been acknowledged as a potential intervention strategy because hepatic CPT activity

increased during negative energy balance (Dann and Drackley, 2005; Douglas et al., 2006)

and decreased with a high-energy diet (Aiello et al., 1984).

What remained unclear, however, is the responsiveness of CPT in other tissues to changes

in energy supply. Apart from liver and mammary tissue, only muscle tissue can contribute

significantly to the oxidation of LCFA, which can be relevant for disorders of lipid

metabolism. In human and rat, prolonged increases in systemic glucose supply lead to an

increase in muscle fat content and decreases in muscle CPT activity and fatty acid oxidation

(Krebs and Roden, 2004; Cahová et al., 2007). These metabolic changes are closely

associated with the development of insulin resistance, the major factor in the pathogenesis of

type-2 diabetes (Dobbins et al., 2001; Cahová et al., 2007). The development of peripheral


[58]

insulin resistence can also be observed in non-lactating cows when excess energy is provided

during the dry period (Holtenius et al., 2003). If insulin resistance persists into lactation, it

might contribute to metabolic disorders during the periparturient period (e.g. fatty liver and

ketosis; Hayirli, 2006). Therefore, possible changes in skeletal muscle CPT activity relative to

the energy balance could be of relevance for the postpartum health status in dairy cows. To

elucidate whether CPT of liver and skeletal muscle respond in a co-ordinate manner to

positive energy balance, their catalytic activities were compared during steadily increasing

glucose infusions in the present study.

Materials and methods Animals, management and experimental design

The experimental design has been described in detail in an earlier report (Al-Trad et al.,

2009). It utilized procedures pre-approved by the local authorities, the Regierungspräsidium

Leipzig (reference 24-9168.11, TVV 49/06). In short, twelve midlactating dairy cows (2nd -

4th month of gestation) were aseptically fitted with a 14-ga, 20-cm jugular catheter (Cavafix

Certo Splittocan 338, Melsungen, Germany) which was replaced every 8 d. Cows were

assigned into two groups balanced for actual lactation performance and actual days in milk.

The saline (n = 6) and glucose infusion groups (n = 6) received continuous jugular infusions

of 0.9% NaCl and 40% glucose solutions, respectively (both from Serumwerk Bernburg,

Bernburg, Germany), over a period of 28 d. The glucose infusion dose was calculated for each

animal separately as a percentage of their daily energy requirement for maintenance and

lactation (based on net energy for lactation, NEL) according to the recommendations of the

National Research Council (2001) (see Calculations and Statistical Analysis). To allow for

day-by-day adaptation to the surplus glucose supply, glucose infusion started at 1.25% of the

NEL requirement at d 1 and gradually increased by 1.25% each day until a maximum dose of

30% of the NEL requirement was reached at d 24. This maximum dose equalled to 2.65 ± 0.19

kg glucose per cow per day. Then the infusion dose was maintained at 30% of the NEL

requirement until d 28. No infusions were made between d 29 and d 32. The volumes of

saline infusions were calculated the same way like the volumes of glucose infusions based on

the NEL requirement of the individual cow. Twice daily (at 0600 h and 1500 h), cows were

offered a components diet based on grass haylage (60.2% of total dry matter, DM) and a

concentrate supplement containing a commercial concentrate mix for lactating dairy cows

(Multilac; 27.2% of total DM; Leikra GmbH, Leipzig, Germany) and soybean meal (12.6% of

total DM; Al-Trad et al., 2009). The diet was composed to include little sugar and starch in


[59]

order to minimize the direct entry of glucose from the gastrointestinal tract and, thereby, to

assess the whole relevant range of surplus glucose supply. This also resulted in a slightly

negative energy balance when no glucose was infused (see below). The diet contained 6.72

MJ NEL/kg DM and had the following approximate composition (g/kg DM); crude protein

202.1, utilizable protein 162.5, ether extract 33.2, crude ash 95.4, acid detergent fibre 231.9,

neutral detergent fibre 379.0, sugar 43.7, starch 112.6, K 22.1, Na 1.0, Ca 7.6, P 5.0, Mg 2.4.

Because the cows did not decrease feed intake during glucose infusion, energy balance

increased in the glucose infusion group from -14.6 to 35.4 MJ/d between d 0 and d 24; while

it remained unchanged in the saline group during the same period (from -10.4 to -1.8 MJ/d;

Al-Trad et al., 2009). Cows were milked daily at 0630 and 1600 h. The energy-corrected milk

yield amounted to 31.5 and 29.4 kg/d in the saline and glucose infusion group, respectively, at

the start of the experiment (Al-Trad et al., 2009).

Sample collection

Liver and skeletal muscle biopsies were obtained between 1000 h and 1200 h on d 0, 8, 16,

24, and 32. Liver biopsies were performed following the procedure of Gröhn and Lindberg

(1982). Approximately 500 mg of liver tissue was aseptically collected under local anesthesia

(5ml Isocain 2%, Selectavet, Weyarn-Holzolling, Germany) using a 2.5-mm wide, 25-cm

long biopsy needle (Model Berlin, Walter Veterinär-Instrumente, Rietzneuendorf, Germany).

Liver samples were washed immediately in ice-cold saline and one portion (~100 mg) for

mitochondrial isolation was suspended in ice-cold homogenization buffer containing 20 mM

4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES), 100 mM sucrose and 0.25

mM ethylenediaminetetraacetic acid (EDTA), adjusted to pH 7.4 using NaOH. Another

portion of liver samples (~300 mg) was transferred into tubes with 3 mL RNAlater (Qiagen,

Germantown, MD) for RNA isolation. The remaining tissue was washed immediately in ice-

cold saline, snap-frozen in liquid nitrogen, and stored at -80 °C for total lipid content analysis.

Skeletal muscle biopsies were taken after subcutaneous and intramuscular local anesthesia

under aseptical conditions via a ~ 5 cm scalpel incision over the gluteus medius muscle.

Incisions for muscle biopsies were made alternating on the left and right side, and were closed

using a simple continuous suture for muscle and subcutaneous tissue and a Ford interlocking

suture (Reverdin) for skin closure. For mitochondrial isolation, approximately 500 mg of

muscle tissue was washed in ice-cold saline and suspended in ice-cold homogenization buffer

(containing 120 mM KCl, 5 mM Tris-HCl, 1 mM EDTA, pH 7.4). For total lipid content


[60]

analysis, ~ 200 mg skeletal muscle was washed immediately in ice-cold saline, snap frozen in

liquid nitrogen, and stored at -80 °C.

To monitor changes of blood metabolites concentrations over the infusion period, blood

samples were collected via puncture of the coccygeal vein at 1000, 1600, 2200 and 0400 h on

days 0, 8, 16, 24, and 32 before each biopsy. Tubes with anticoagulant (lithium heparin,

Sarstedt, Nümbrecht, Germany) were used to obtain plasma for glucose analysis. Tubes with a

clotting activator (Sarstedt) were used for serum preparation for insulin, β-hydroxybutyric

acid (BHBA) and NEFA analysis. Heparinized tubes were immediately placed on ice, and

transported to the laboratory within 30 min after sampling. Plasma was separated from whole

blood by centrifugation at 4 °C and 3 000 × g for 15 min. Blood tubes without heparin were

allowed to sit for 30 min at room temperature and centrifuged at room temperature (3 000 × g,

15 min) to separate serum. All plasma and serum samples were stored at –20°C until further

analyses.

Analysis of blood metabolites

Plasma glucose and serum NEFA and BHBA concentrations were assayed on a Hitachi 912

automatic analyzer (Boehringer Mannheim, Mannheim, Germany) using commercial kits

obtained from Roche Diagnostics GmbH (Mannheim, Germany) for glucose and from

Randox Laboratories Ltd. (Crumlin, UK) for NEFA and BHBA measurements. A radiometric

immunoassay was used for serum insulin analysis, using self-prepared calibration standards.

The latter were produced by diluting bovine insulin in the human insulin-free serum provided

with the test (INS-IRMA; BioSource Europe SA, Nivelles, Belgium).

Analysis of CPT activity

Mitochondria from liver (Mizutani et al., 1999) and skeletal muscle (Power and Newsholme,

1997) were prepared immediately after the biopsy by differential centrifugation as described

previously. Mitochondria were stored at -80 °C until analysis. After thawing, CPT activity

was measured by following the release of CoA-SH from palmitoyl-CoA based on the

colorimetric procedure described by Bieber et al. (1972). The assay medium comprised in a

final volume of 0.9 ml: Tris-HCl-DTNB buffer (containing 116 mM Tris HCl, 2 mM EDTA,

0.25 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), pH 8.0), 0.04 mM palmitoyl-CoA and

1.25 mM L-carnitine. The reaction was initiated by the addition of 0.1 ml of tissue

homogenate which was mixed immediately by inversion. The rates of CoA release were


[61]

followed at room temperature by monitoring the changes in absorbance at 412 nm on a

Thermo Scientific EvolutionTM 100 spectrophotometer (Thermo Fisher Scientific, Dreieich,

Germany), using the molar extinction coefficient for 5-thio-2-nitrobenzoate (TNB; 13,600 M-1

cm-1) as conversion factor. Control samples without L-carnitine solution were run for each

sample. The CPT activity was calculated as the difference between the rates in the presence

and absence of L-carnitine, and expressed as nanomoles of CoA release/min/mg of

mitochondrial protein under the above assay conditions. Activity of CPT was normalized to

mitochondrial protein contents which were determined by the protein assay method of Smith

et al. (1985).

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

RNA was extracted from liver tissues using Trizol reagent (Invitrogen, Carlsbad, CA)

according to the manufacturer's instructions. The quantity of RNA was determined by

measuring the absorbance at 260 nm and purity was determined by measuring the A260/A280

ratio which was > 1.85 for all preparations. Integrity of RNA was assessed by agarose gel

electrophoresis. Total RNA (1 µg) was reverse transcribed using oligo-d(T)15 primer in a 20-

µl reaction according to the manufacturer's instructions (AMV-RT kit, Roche Diagnostics,

Mannheim, Germany).

A comparative analysis of hepatic CPT-1A and CPT-2 mRNA levels between treatment

and control groups was carried out by qRT-PCR using β2-microglobulin (B2M) as

unregulated reference gene. PCR primers and dual-labelled fluorescent probes were designed

using the web-based quantitative PCR probe design software provided by MWG Biotech AG

(Ebersberg, Germany; http://www.eurofinsdna.com) and synthesized by the same company.

Since no sequence information was available for bovine hepatic CPT-1A at the time of

analysis, a primer set derived from the ovine sequence (GenBank accession Y18387.1;

forward primer: 5′-ATG ACG GCT CTG GCA CAA GA-3′; reverse primer: 5′-TGG ATG

GTG TCT GTC TCC TC-3′ was applied first to amplify and sequence a respective bovine

cDNA fragment. This unique bovine CPT-1A sequence was submitted to GenBank

(FJ415874) and used for the selection of primers and probes in the qRT-PCR experiments.

GenBank accession numbers and the derived primers and probes for qRT-PCR were as

follows: B2M (GenBank BC118352.1; forward primer: 5′-AGC GTC CTC CAA AGA TTC

AAG T-3′; reverse primer: 5′-GGA TGG AAC CCA TAC ACA TAG CA-3′; probe: 5′-FAM–

CAC CAG AAG ATG GAA AGC CAA ATT ACC TGA A-BHQ1-3′), CPT-1A (GenBank


[62]

FJ415874.1; forward primer: 5′-CTG GAC CGG GAG GAA ATC-3′; reverse primer: 5′-CCG

AGA AGT ATT AAA CAT GCG C-3′; probe: 5′-ROX-TTC TGG GGT CTA CGA TTC

CGC TCT GCT-BHQ2-3′), and CPT-2 (GenBank BC105423.1; forward primer: 5′-TGT

GAG TGC CTC TGA AAT CC-3′; reverse primer: 5′-CAC TAG TCA GAT ACG AAA

GCG G-3′; probe: 5′-JOE-TAC ATT CTG TCA GAC AAT AGC CCG GCC C-TAM-3′). To

ensure the specificity of the primers and probes, an ethidium-bromide stained agarose gel

(1%) was run in which a single band of expected molecular size was obtained.

RNA quantification by qRT-PCR was carried out using Rotor-Gene 6000 (Corbett

Research, Sydney, Australia). For each sample, the target gene and the control gene were run

under duplex reaction conditions in duplicate. The following reagents were used for

amplification in 20 μl final volume: 1 μl of sample cDNA, 2μl Mg-free 10X buffer, 0.75 U

Dynazyme II DNA polymerase (Finnzymes, Finland), 5.5 mM MgCl2, 0.3 mM dNTP,

900 nM of each primer and 150 nM of each probe for CPT-1A and CPT-2, 200 nM of each

primer and 50 nM of each probe for B2M. Amplification conditions for quantification were:

95 °C for 2 min and 45 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. After the

amplification efficiency of each target and reference gene was validated, the relative gene

expression levels were determined by the 2-ΔΔCT method as described by Livak and

Schmittgen (2001). With this method, levels of genes expression were normalized to B2M

and expressed as relative values using a single sample obtained from one cow in the saline

treatment group at d 0 as an interplate calibrator.

Liver and skeletal muscle total lipid content

Samples of frozen liver and skeletal muscle were analyzed for concentrations of total lipid by

the gravimetric method after chloroform-methanol extraction (Floch et al., 1957).

Calculations and statistical analyses

Net energy required for maintenance and lactation (MJ/day) was calculated as 4.184 ×

([BW0.75 × 0.08] + milk yield (kg) × [(0.0929 × fat %) + (0.0563 × true protein %) + (0.0395 ×

lactose %)]) according to the NRC (2001). Based on the energy requirement, infusion dose

(kg glucose/day) was calculated as follows; designated dose level × NEL/(15.6 MJ/kg).

Statistical analyses were conducted using the Sigma Stat software package (version 3.5;

SPSS, Chicago, IL). Normally distributed data were tested using one-way repeated-measures


[63]

ANOVA followed by Dunnett's post hoc analysis. In cases of non-normal distribution of the

data, the Friedman repeated-measures ANOVA on ranks, followed by Dunnett's test, was

used. Results were considered statistically significant when P < 0.05.

Results Plasma glucose concentrations remained in the physiological range without significant diurnal

variations when glucose infusion doses varied between 0 - 20% NEL requirements (i.e. from d

0 to d 16; P > 0.05; Fig. 1). A period of marked hyperglycaemia was only observed at the

highest infusion dose (30% NEL requirement) on day 24. This single period occurred at 1600

h, i.e. 1 h postprandially. Increases in serum insulin concentration were observed initially at

night time on d 16 (2200 h and 0400 h; P < 0.01; Fig. 2) but extended to the afternoon (1600

h; P < 0.01) when glucose infusion increased to 30% NEL requirement on day 24.

Serum BHBA levels started to decrease by glucose infusion already on d 8 between 1600 h

and 0400 h (P < 0.05), and were lower than initial values at all day times on d 16 and d 24 in

the glucose infusion group (P < 0.01; Fig. 3). A similar trend for glucose-induced decreases in

serum concentration was also observed for NEFA; but this numerical decrease reached

statistical significance (P < 0.05) only for a single time point (2200 h) on d 16 (Fig. 4).

The activity of hepatic CPT decreased by 35% when glucose dose increased from 0% to

30% NEL requirement between d 0 and d 24 (P < 0.01; Table 1). This depression in hepatic

CPT activity persisted until four days after the end of the infusion period (d 32; P < 0.01,

Table 1). In contrast to the liver, no effect of glucose infusion was observed on skeletal

muscle CPT activity (P > 0.1; Table 1).

To elucidate whether the decrease in the hepatic CPT activity during glucose infusion was

due to downregulation of CPT gene transcription, the mRNA expression of the two hepatic

CPT isoforms, CPT-1A and CPT-2, was measured by qRT-PCR. These analyses revealed

numerical decreases in the mRNA abundance of CPT-1A (by 28%) and CPT-2 (by 42%) that

were comparable to the glucose-induced decrease in hepatic CPT activity (35%); but none of

the decreases in mRNA abundance reached statistical significance (P > 0.1; Table 1). No

attempt was made to measure the gene expression level of muscular CPT (i.e. CPT-1B and

CPT-2) given the unchanged enzyme activity of CPT in skeletal muscle. No significant

changes were observed in liver and skeletal muscle lipid contents (P > 0.1; Table 1).


[64]

0 8 16 24 32 40

Glu

cose

, mm

ol/L

0

2

4

6

81000 h

0 8 16 24 32 40

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cose

, mm

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0 8 16 24 32 40

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0

2

4

6

82200 h

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0 8 16 24 32 40

Glu

cose

, mm

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2

4

6

80400 h

Figure 1. Mean plasma glucose

concentrations for blood samples taken the

day before each biopsy at 1000 h, 1600 h,

2200 h, and 0400 h for cows treated with

glucose (■) or saline (●).

Open symbols depict post-infusion values.

Data are expressed as means ± SEM. *P <

0.05 vs. day 0.

0 8 16 24 32 40Insu

lin, p

mol

/L

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4001000 h

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Insu

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1200

1600

2000

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400

800

12002200 h

**

**

Day

0 8 16 24 32 40Insu

lin, p

mol

/L

0

4000400 h** **

**

Figure 2. Mean serum insulin concentrations

for blood samples taken before each biopsy at

1000 h, 1600 h, 2200 h, and 0400 h for cows

treated with glucose (■) or saline (●).


Data are expressed as means ± SEM. **P <

0.01 vs. day 0.


[65]

0 8 16 24 32 40

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0.3

0.6

0.9

1.2

1.52200 h

****

*

Day

0 8 16 24 32 40

BH

BA

, mm

ol/L

0.0

0.3

0.6

0.9

1.2

1.50400 h

**

*

***

***

Figure 3. Mean serum concentrations of

beta-hydroxybutyric acid (BHBA) for blood

samples taken before each biopsy at 1000 h,

1600 h, 2200 h, and 0400 h for cows treated

with glucose (■) or saline (●).


Data are expressed as means ± SEM. */**P <

0.05/0.01 vs. day 0.

0 8 16 24 32 40

NE

FA, µ

mol

/L

0

100

200

3001000 h

0 8 16 24 32 40

NE

FA, µ

mol

/L

0

100

200

3001600 h

0 8 16 24 32 40

NE

FA, µ

mol

/L

0

100

200

300

4002200 h

*

Day

0 8 16 24 32 40

NE

FA, µ

mol

/L

0

100

200

300

400

5000400 h

Figure 4. Mean serum concentrations of

non-esterified fatty acid (NEFA) for blood

samples taken before each biopsy at 1000 h,

1600 h, 2200 h, and 0400 h for cows treated

with glucose (■) or saline (●).


Data are expressed as means ± SEM. *P <

0.05 vs. day 0.


[66]

Table 1. Liver and skeletal muscle CPT activity, expression levels of hepatic CPT-1A and CPT-2 mRNA, and total lipid contents in liver and skeletal muscle for glucose and saline treatment groups.

Treatment Day 0

(0% NEL)

Day 8

(10% NEL)

Day 16

(20% NEL)

Day 24

(30% NEL)

Day 32

(0% NEL) SEM

CPT Activity (nmol/min/mg mitochondrial protein)

Glucose 29.9 28.0 24.0 19.6** 18.3** Liver

Saline 26.3 24.7 21.9 22.0 25.0 2.4

Glucose 6.9 7.2 6.7 6.3 7.0 Skeletal muscle

Saline 7.6 7.5 8.1 6.6 7.4 1.6

relative mRNA expression1 2−ΔΔCT

Glucose 0.71 0.65 0.44 0.51 0.57 Hepatic CPT-1A

Saline 0.52 0.70 0.38 0.60 0.61 0.13

Glucose 0.63 0.63 0.55 0.37 0.70 Hepatic CPT-2

Saline 0.67 0.47 0.49 0.58 0.75 0.12

Total Lipid (% wet weight)

Glucose 3.55 3.77 3.73 4.64 4.35 Liver

Saline 3.50 4.05 3.61 3.43 3.60 0.24

Glucose 3.62 3.62 3.18 4.09 3.27 Skeletal muscle

Saline 3.04 3.90 3.63 3.17 3.22 0.22

1Data are expressed as means with pooled SEM. **P < 0.01 vs. day 0. 1Data are expressed as the normalized ratio of gene expression relative to β2-microglobulin mRNA level using one sample from saline treatment group as an interplate calibrator.


[67]

Discussion The present study was intended to provide insight into the responses of liver and skeletal

muscle CPT activity to gradual increases in glucose availability, brought by intravenous

glucose infusion in lactating dairy cows. To assess the concurrent changes in metabolism,

blood concentrations of glucose, insulin, BHBA and NEFA were simultaneously monitored.

The latter analyses revealed a continuous increase in serum insulin concentration with

increasing glucose infusions, while plasma glucose concentrations were only elevated in

postprandial blood samples during the highest infusion dose. When considering that the

maximum amount of postruminal digestible starch has been estimated with ~2 kg/d in dairy

cows (Matthé et al., 2001; Gäbel and Aschenbach, 2004), it might not be surprising that a

glucose dose of ~1.8 kg/d (i.e. 20% NEL requirement) was tolerated by the cows of the

present study while a dose of 2.6 kg/d (i.e. 30% NEL requirement) led to hyperglycaemia. The

occurrence of temporal hyperglycaemia on d 24 thus indicates that we explored the whole

relevant dose range of surplus glucose supply. On the other hand, an observed trend for

decreases in serum NEFA concentration indicates that glucose infusion interacted with lipid

metabolism, likely via an insulin-mediated inhibition of fatty acid mobilization from adipose

tissue (Vernon, 1980). That decreases in serum NEFA concentration reached statistical

significance only at a single time point on d 16 becomes plausible if one considers that the

decrease in adipose tissue lipid release during glucose infusion is generally paralleled by a

decrease in lipid oxidation (Treacher et al., 1976). With regard to the liver, such decreased

fatty acid oxidation was evident from the decreased serum BHBA concentrations as will be

discussed in more detail below.

In many species, it has been well established that the main regulatory step of fatty acid

oxidation is CPT-1 (Aiello et al., 1984; McGarry, 1998; Bonnefont et al., 2004; Morash et al.,

2008). In fatty acid synthesizing tissues like liver, short-term regulation of CPT-1 is inversely

coupled to the endogenous production of fatty acids; which is based on an allosteric inhibition

of CPT-1 by the first metabolite of de novo fatty acid synthesis, malonyl-CoA (Bonnefont et

al., 2004; Morash et al., 2008). Tissues without fatty acid synthesizing capacity, like skeletal

muscle, are yet able to perform this first step of fatty acid synthesis and specifically produce

malonyl-CoA as a signaling molecule to inhibit CPT-1 short-term (McGarry, 1998).

Intermediate to long-term regulation of CPT-1 activity is primarily transcriptional (McGarry

and Brown, 1997) but can also involve changes in mitochondrial membrane fluidity. Such

changes in membrane fluidity have been shown to affect CPT-1 activity and its malonyl-CoA


[68]

sensitivity (Kolodziej and Zammit, 1990) and are likely the pathway by which insulin

increases the malonyl-CoA sensitivity of CPT-1 and down-regulates fatty acid oxidation

(Zammit, 1999). In contrast to the well defined role of CPT-1, the role of CPT-2 in the

regulation of fatty acid oxidation is less clear. CPT-2 is insensitive to short-term regulation by

malonyl-CoA (Declercq et al., 1987), less dependent on membrane-enzyme interaction than

CPT-1 (Declercq et al., 1987; Woeltje et al., 1987), and had been rather stably expressed at

different ontogenetic and metabolic states during previous studies in rats (Bonnefont et al.,

2004).

The enzyme activity assay applied in the present study measures the intermediate to long-

term regulation of total CPT activity. This intermediate to long-term regulation sets the

margins within the animal can respond to changing metabolic demands. The freeze-thawing

step of our protocol partly damages mitochondrial membranes and thus allowed for combined

analyses of CPT-1 activity localised to the outer and CPT-2 activity localized to the inner

mitochondrial membrane (Woeltje et al., 1987). The results obtained with this method showed

that glucose load had a regulatory effect on only hepatic but not skeletal muscle CPT activity.

The intermediate to long-term character of the depressive effect on hepatic CPT activity was

evident from its persistence for four days after stopping the surplus glucose supply (i.e. until d

32).

As regards the cause of glucose-induced depression of hepatic CPT activity, the elevation

in serum insulin level observed between d 16 and d 24 may provide one possible explanation.

As discussed above, insulin has been suggested to increase the malonyl-CoA sensitivity of

CPT-1 via a modification of the mitochondrial membrane fluidity (Zammit, 1999), which

could implement a decrease in baseline enzyme activity, too (Morash et al., 2008). On the

other hand, transcriptional regulation likely also contributed to the decreased hepatic CPT

activity. Unfortunately, the performed qRT-PCR experiments faild to provide statistical proof

for this. Nevertheless, qRT-PCR revealed numerical decreases in the hepatic expression of

both CPT-1A (by 28 %) and CPT-2 (by 42%), which were in the same range like the observed

decrease in enzyme activity (by 35%) between d 0 and d 24.

Our finding of decreased hepatic CPT activity with possibly transcriptional causes after

surplus energy supply is in agreement with previous studies showing increased CPT activity

or mRNA levels by prepartum feed restriction of dairy cows (Douglas et al., 2006; Loor et al.,


[69]

2006). Although one might expect that skeletal muscle CPT would be affected in a similar

manner by energy balance, the missing effect of glucose infusion on skeletal muscle CPT

seems to be compatible with metabolic priorities in the high-yielding dairy cow.

The transition of dairy cows from a positive energy balance prepartum to a negative energy

balance postpartum requires mobilization of body fat reserves, evidenced by a rise of NEFA

concentration in blood plasma (Bell, 1995; Vernon, 2005). Besides the mammary gland where

NEFA are predominantly used for milk fat synthesis (Bell, 1995), liver and skeletal muscle

are major NEFA-utilizing tissues (Heitmann et al., 1987; Hocquette and Bauchart, 1999;

Drackley et al., 2001). Skeletal muscle differs from liver because it utilizes NEFA exclusively

for its own energy metabolism. By contrast, liver clears NEFA from the systemic circulation

in excess of its own demand. It converts part of them to water-soluble ketone bodies (e.g.

BHBA and acetoacetate) that can be used as energy substrates by other tissues (Heitmann et

al., 1987; Emery et al., 1992; Drackley et al., 2001), and re-esterifies the remaining part to

triglycerides (Hocquette and Bauchart, 1999; Drackley et al., 2001). The hepatic conversion

of NEFA to ketone bodies includes mitochondrial β-oxidation of the imported LCFA as an

essential step (Kerner and Hoppel, 2000; Drackley et al., 2001; Vernon, 2005). Thereby,

hepatic fatty acid oxidation plays a key role not only for the control of blood NEFA levels but

also for the provision of adequate amounts of ketone bodies. From a teleological point of

view, it appears thus plausible that hepatic fatty acid oxidation has a higher metabolic priority

for regulation and requires larger plasticity compared to fatty acid oxidation in skeletal

muscle.

The intracellular level of fatty acids itself appears to be the prevailing signal for the

metabolic regulation of CPT activity. Their effect is essentially mediated via peroxisome

proliferator-activated receptor (PPAR)-α as has been shown for CPT-1B in murine

cardiomyocytes (Brandt et al., 1998). A higher functional activity of PPAR-α in liver

compared to skeletal muscle would thus provide a plausible hypothesis for the negative effect

of glucose infusion on hepatic but not skeletal muscle CPT activity. Indeed, a recent study by

Waters et al. (2009) failed to detect an involvement of PPAR-α in the regulation of Δ9-

desaturase in beef muscle, which could point to a low activity of PPAR-α in beef muscle

tissue. However, more studies are warranted to test the applicability of the above hypothesis

for the lactating dairy cow.


[70]

It should finally be noted that a low level of CPT regulation in skeletal muscle could

contribute to the development of peripheral insulin resistance in the postpartum dairy cow.

Failure to upregulate CPT activity in muscle with increasing availability of NEFA during

times of negative energy balance would promote the accumulation of intracellular lipids. It

has been shown in other species that such accumulation of intracellular lipids leads to insulin

resistance (Krebs and Roden, 2004) due to suppression of insulin signalling (Cahová et al.,

2007). Albeit muscle tissue may appear to have a minor share in energy partitioning during

peak lactation, it may become important when lipid utilization by other tissues (e.g. liver)

becomes impaired.

Acknowledgement We wish to thank Gerald F. Schusser, Carsten Benson and the animal care staff of the Clinic

for Large Animal Internal Medicine for their support. We are very grateful to Anke Schmidt-

Mähne and Ines Urbansky for help during samplings and laboratory analyses. The work was

supported by Pfizer Animal Health.

References

Aiello, R. J.; Kenna, T. M.; Herbein, J. H., 1984: Hepatic gluconeogenic and ketogenic

interrelationships in the lactating cow. Journal of Dairy Science 67, 1707-1715.

Al-Trad, B.; Reisberg K.; Wittek T.; Penner G. B.; Alkaassem A.; Gäbel G.; Fürll M.;

Aschenbach J. R., 2009: Increasing intravenous infusions of glucose improve body

condition but not lactation performance in mid-lactation dairy cows. Journal of Dairy

Science 92, 5645-5658.

Bell, A. W., 1995: Regulation of organic nutrient metabolism during transition from late

pregnancy to early lactation. Journal of Animal Science 73, 2804-2819.

Bieber, L. L.; Abraham, T.; Helmrath, T., 1972: A rapid spectrophotometric assay for

carnitine palmitoyltransferase. Analytical Biochemistry 50, 509-518.

Bobe, G.; Young, J. W.; Beitz, C., 2004: Invited review: pathology, etiology, prevention, and

treatment of fatty liver in dairy cows. Journal of Dairy Science 87, 3105-3124.

Bonnefont, J. P.; Djouadi, F.; Prip-Buus, C.; Gobin, S.; Munnich, A.; Bastin, J., 2004:

Carnitine palmitoyltransferases 1 and 2: biochemical, molecular and medical aspects.

Molecular Aspects of Medicine 25, 495-520.


[71]

Brandt, J. M.; Djouadi, F.; Kelly, D. P., 1998: Fatty acids activate transcription of the muscle

carnitine palmitoyltransferase I gene in cardiac myocytes via the peroxisome proliferator-

activated receptor alpha. Journal of Biological Chemistry 273, 23786-23792.

Cahová, M.; Vavrínková, H.; Kazdová, L., 2007: Glucose-fatty acid interaction in skeletal

muscle and adipose tissue in insulin resistance. Physiological Research 56, 1-15.

Dann, H. M.; Drackley, J. K., 2005: Carnitine palmitoyltransferase I in liver of periparturient

dairy cows: effects of prepartum intake, postpartum induction of ketosis, and periparturient

disorders. Journal of Dairy Science 88, 3851-3859.

Declercq, P. E.; Falck, J. R.; Kuwajima, M.; Tyminski, H.; Foster, D. W.; McGarry, J. D.,

1987: Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system.

I. Use of inhibitors. Journal of Biological Chemistry 262, 9812-9821.

Dobbins, R. L.; Szczepaniak, L. S.; Bentley, B.; Esser, V.; Myhill, J.; McGarry, J. D., 2001:

Prolonged inhibition of muscle carnitine palmitoyltransferase-1 promotes intramyocellular

lipid accumulation and insulin resistance in rats. Diabetes 50, 123-130.

Douglas, G. N.; Overton, T. R.; Bateman, H. G. 2nd; Dann, H. M.; Drackley, J. K., 2006:

Prepartal plane of nutrition, regardless of dietary energy source, affects periparturient

metabolism and dry matter intake in Holstein cows. Journal of Dairy Science 89, 2141-

2157.

Drackley, J. K., 1999: Biology of dairy cows during the transition period: the final frontier?

Journal of Dairy Science 82, 2259-2273.

Drackley, J. K.; Overton, T. R.; Douglas, G. N., 2001: Adaptations of glucose and long-chain

fatty acid metabolism in liver of dairy cows during the periparturient period. Journal of

Dairy Science 84(E Suppl), E100-E112.

Eaton, S., 2002: Control of mitochondrial β-oxidation flux. Progress in Lipid Research 41,

197-239.

Emery, R. S.; Liesman, J. S.; Herdt, T. H., 1992: Metabolism of long chain fatty acids by

ruminant liver. The Journal of Nutrition 122(3 Suppl), 832-837.

Floch, J.; Lees, M.; Sloane-Stanley, G. H., 1957: A simple method for the isolation and

purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497-

499.

Gäbel, G.; Aschenbach, J. R., 2004: Adaptation und Regulation resorptiver Prozesse im

Gastrointestinaltrakt von Wiederkäuern. Übersichten in Tierernährung 32, 149-181.

Gröhn, Y.; Lindberg, L. A., 1982: Methodological aspects of the microscopy of bovine liver

biopsies. Journal of Comparative Pathology 92, 567-578.


[72]

Grummer, R. R., 1993: Etiology of lipid-related metabolic disorders in periparturient dairy

cows. Journal of Dairy Science 76, 3882-3896.

Hayirli, A., 2006: The role of exogenous insulin in the complex of hepatic lipidosis and

ketosis associated with insulin resistance phenomenon in postpartum dairy cattle.

Veterinary Research Communications 30, 749-774.

Heitmann, R. N.; Dawes, D. J.; Sensenig, S. C., 1987: Hepatic ketogenesis and peripheral

ketone body utilization in the ruminant. The Journal of Nutrition 117, 1174-1180.

Hocquette, J. F.; Bauchart, D., 1999: Intestinal absorption, blood transport and hepatic and

muscle metabolism of fatty acids in preruminant and ruminant animals. Reproduction

Nutrition Development 39, 27-48.

Holtenius, K.; Agenäs, S.; Delavaud, C.; Chilliard, Y., 2003: Effects of feeding intensity

during the dry period. 2. Metabolic and hormonal responses. Journal of Dairy Science 86,

883-891.

Kerner, J.; Hoppel, C., 2000: Fatty acid import into mitochondria. Biochimica et Biophysica

Acta 1486, 1-17.

Kolodziej, M. P.; Zammit, V. A., 1990: Sensitivity of inhibition of rat liver mitochondrial

outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and

temperature-induced changes in membrane fluidity. Biochemical Journal 272, 421-425.

Krebs, M.; Roden, M., 2004: Nutrient-induced insulin resistance in human skeletal muscle.

Current Medicinal Chemistry 11, 901–908.

Livak, K. J.; Schmittgen, T. D., 2001: Analysis of relative gene expression data using real-

time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408.

Loor, J. J.; Dann, H. M.; Guretzky, N. A.; Everts, R. E.; Oliveira, R.; Green, C. A.;

Litherland, N. B.; Rodriguez-Zas, S. L.; Lewin, H. A.; Drackley, J. K., 2006: Plane of

nutrition prepartum alters hepatic gene expression and function in dairy cows as assessed

by longitudinal transcript and metabolic profiling. Physiological Genomics 27, 29-41.

Matthé, A.; Lebzien, P.; Hric, I.; Flachowsky, G.; Sommer, A, 2001: Effect of starch

application into the proximal duodenum of ruminants on starch digestibility in the small

and total intestine. Archiv für Tierernährung 55, 351-369.

McGarry, J. D., 1998: Glucose-fatty acid interactions in health and disease. American Journal

of Clinical Nutrition 67(3 Suppl), 500S-504S.

McGarry, J. D.; Brown, N. F., 1997: The mitochondrial carnitine palmitoyltransferase system.

From concept to molecular analysis. European Journal of Biochemistry 244, 1-14.


[73]

Mizutani, H.; Sako, T.; Toyoda, Y.; Kawabata, T.; Urumuhang, N.; Koyama, H.; Motoyoshi,

S., 1999: Preliminary studies on hepatic carnitine palmitoyltransferase in dairy cattle with

or without fatty liver. Veterinary Research Communications 23, 475-480.

Morash, A. J.; Kajimura, M.; McClelland, G. B., 2008: Intertissue regulation of carnitine

palmitoyltransferase I (CPTI): mitochondrial membrane properties and gene expression in

rainbow trout (Oncorhynchus mykiss). Biochimica et Biophysica Acta 1778, 1382-1389.

Murondoti, A.; Jorritsma, R.; Beynen, A. C.; Wensing, T.; Geelen, M. J., 2004: Unrestricted

feed intake during the dry period impairs the postpartum oxidation and synthesis of fatty

acids in the liver of dairy cows. Journal of Dairy Science 87, 672-679.

National Research Council., 2001: Nutrient Requirements of Dairy Cattle. 7th revised edition

National Academy of Science Washington, DC.

Power, G. W.; Newsholme, E. A., 1997: Dietary fatty acids influence the activity and

metabolic control of mitochondrial carnitine palmitoyltransferase I in rat heart and skeletal

muscle. The Journal of Nutrition 127, 2142-2150.

Smith, P.; Krohn, R.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.;

Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C., 1985: Measurement of protein

using bicinchoninic acid. Analytical Biochemistry 150, 76–85.

Treacher, R. J.; Baird, G. D.; Young, J. L., 1976: Anti-ketogenic effect of glucose in the

lactating cow deprived of food. The Biochemical Journal 158, 127-134.

Vernon, R. G., 1980: Lipid metabolism in the adipose tissue of ruminant animals. Progress in

Lipid Research 19, 23-106.

Vernon, R. G., 2005: Lipid metabolism during lactation: a review of adipose tissue-liver

interactions and the development of fatty liver. Journal of Dairy Research 72, 460-469.

Waters, S. M.; Kelly, J. P.; O'Boyle, P.; Moloney, A. P.; Kenny, D. A., 2009: Effect of level

and duration of dietary n-3 polyunsaturated fatty acid supplementation on the

transcriptional regulation of Delta9-desaturase in muscle of beef cattle. Journal of Animal

Science 87, 244-252.

Woeltje, K. F.; Kuwajima, M.; Foster, D. W.; McGarry, J. D., 1987: Characterization of the

mitochondrial carnitine palmitoyltransferase enzyme system. II. Use of detergents and

antibodies. Journal of Biological Chemistry 262, 9822-9827.

Zammit, V. A., 1999: Carnitine acyltransferases: functional significance of subcellular

distribution and membrane topology. Progress in Lipid Research 38, 199-224.

CHAPTER‐6: GENERAL DISCUSSION

[74]

6 General Discussion

Although infusions into the stomach or intestine would be a more physiological route of

glucose entry, such studies are biased by the high utilization of glucose within the portal-

drained viscera (REYNOLDS et al. 1994) and may further be biased by the release of

gastrointestinal hormones that affect glucose metabolism and food intake (e.g. glucagon-like

peptide-1; HOLST 2007). Therefore, the effect of glucose dose at the level of intermediary

metabolism can only be assessed by using an intravenous infusion route like in the present

study. The dose of infused glucose was increased slowly and gradually from 0% of NEL

requirement at d 0 to 30% at d 24 (i.e. to 2.65 ± 0.19 kg glucose per cow at d 24). In addition

to testing the dose effect of surplus glucose, this gradual infusion protocol proofed to be

advantageous in several aspects. The most important advantage is probably that the

potentially confounding effects occasionally observed in constant level infusion protocols,

such as decreased DMI and fall in the blood phosphorus concentration (hypophosphatemia),

could be avoided (DHIMAN et al. 1993; GRÜNBERG et al. 2006). In the present study,

hypercaloric intravenous GI did not affect feed intake even when the infusion dose amounted

to 2.65 kg glucose per day (Chapter 3, Table 2). Consequently, when interpreting data, the

infused glucose can be considered true surplus supply of glucose and energy as indicated by

the increase in the energy balance status of GI cows (Chapter 3, Table 2). A minimal direct

entry of glucose from feed can be expected because the diet was low in starch and sugar

(Chapter 3, Table 1). Based on this minimal direct entry at the beginning of the infusion

period, glucose supply was gradually increased until hyperglycemia, hyperinsulinemia and

glucosuria were evident at the end of the infusion period. Consequently, the infusion protocol

explored the entire range of nutritionally relevant surplus glucose.

6.1 Glucose Supply Is Not a Limiting Factor for Lactation Performance in Midlactating

Dairy Cows

Effects of excessive glucose supply on milk yield and components have attracted considerable

attention in dairy cows. Regarding results obtained from a number of previous studies

conducted in lactating dairy cows, responses of milk yield to an exogenous glucose supply

were inconclusive (KNOWLTON et al. 1998; HURTAUD et al. 2000). The present finding

that excessive glucose supply had no effect on milk yield (Chapter 3, Table 2) is in agreement

with a number of previous studies in which intravenous (AMARAL et al. 1990; KIM et al.

2000) or postruminal GI (CLARK et al. 1977; LEMOSQUET et al. 1997) did not

significantly increase milk output. This result in the context with previous investigations


[75]

could support the hypothesis that the failure of GI to enhance milk production could be

independent of the route of glucose administration. In contrast to the present study, however,

a previous study by RIGOUT et al. (2002b) had observed dose effects of duodenal GI on milk

yield. Since the present study using increasing doses of intravenous glucose did not show the

same effect, it seems appropriate to conclude that the metabolic fate of glucose, including its

conversion to milk lactose, may be modified by metabolic and/or hormonal signals from the

portal-drained viscera when absorbed from the intestine.

Milk lactose is considered the primary osmoregulator of milk (LINZELL and PEAKER

1971; RIGOUT et al. 2002b); hence, an increase in glucose availability for mammary gland

lactose synthesis could be a mechanism for increased milk yield during additional glucose

supply (KNOWLTON et al. 1998; RIGOUT et al. 2002b). In the present study, the missing

effects of GI on milk lactose yield (Chapter 3, Table 2) might be a plausible explanation for

the absent effect of glucose supply on milk yield. It is noteworthy that the present study was

conducted over a long time period and the infusion dose was gradually increased over time.

Therefore, it can be assumed that the gradual increases in the glucose doses with a long-term

infusion period play a potential role in enhancing metabolic and/or hormonal adaptation

mechanisms to the excessive glucose supply (REYNOLDS et al. 1994; KNOWLTON et al.

1998) and, as a consequence, tissues glucose storage and/or utilization rate increased without

affecting mammary gland glucose uptake and metabolism. This assumption of day-by-day

adaptation is supported, in part, by the sudden decrease in milk lactose output after glucose

withdrawal (Chapter 3, Table 2).

Previous studies showed a negative effect of GI on milk fat yield (FISHER and ELLIOT

1966; HURTAUD et al. 2000) which was related to the decrease in blood precursors for milk

fat synthesis (acetate, BHBA, and NEFA; MCCLYMONT and VALLANCE 1962).

Unfortunately, a similar depression in milk fat yield could neither be proofed nor disproofed

in the present study, mainly because a decrease in milk fat percentage and yield occurred also

in the SI group (Chapter 3, Table 2). It is likely, however, that the differences in serum

concentrations of NEFA, and BHBA (serum acetate was not measured) might have been too

small to induce significant changes in milk fat percentage between the two treatments. On the

other hand, it appears that an increase in energy input by high-dose GI enhanced milk protein

synthesis (Chapter 3, Table 2). This result is in agreement with the almost consistent increase

in the milk protein yield observed with postruminal or intravenous glucose administration


[76]

(AMARAL et al. 1990; RULQUIN et al. 2004). The decreases in serum BUN and in the gene

expression of the key enzyme involved in hepatic glucose synthesis from glucogenic AA (i.e.

PC, as outlined below) support the concept that the increase in protein synthesis in the

mammary gland might have been due to the sparing effect of excessive glucose on AA

breakdown, particularly on glucogenic AA breakdown (REYLONDS et al. 1994;

VANHATALO et al. 2003). In addition, hormonal effects of increased insulin concentration

on mammary gland AA uptake could also have contributed to the increase in milk protein

yield (MACKLE et al. 2000). However, the increase in milk protein yield observed in this

experiment is still not of practical significance in view of the large amount of infused glucose

(> 2kg/day) needed to obtain this effect.

Presupposing that the diet offered in this study provided minimal direct entry of glucose

from the portal-drained viscera (Chapter 3, Table 1), it follows that endogenous glucose

output from the liver was sufficient to fully meet the demands of milk synthesis already in the

absence of GI. This further allows the conclusion that glucose availability can hardly be a

limiting factor for milk yield and milk energy output in mid lactating dairy cows if energy and

protein requirements are met. Consequently, dairy cows on an energy-balanced diet and

sufficient protein supply do not direct excess glucose to increased lactation performance.

6.2 Dysregulation of Blood Glucose Occurs Postprandially at High Glucose Loads

The effect of GI on blood glucose and insulin concentrations was more obvious in the daily

mean values of blood samples collected every 8 d compared with blood samples collected at

1000 h every 2 d. Indeed, frequent blood samples (i.e. day profiles) might be necessary to

detect transient increases in blood glucose and insulin concentrations in response to GI in

dairy cows. Blood samples collected at 1000 h every 2 d showed a treatment effect on insulin

concentration and a quadratic day × treatment interaction on both glucose and insulin

concentrations (Chapter 4, Fig. 1A and B). Nevertheless, the blood glucose and insulin

concentrations largely remained within the physiological range in the GI-treated group with

only occasional increases. Further, the daily minimum values of glucose and insulin

concentrations showed no treatment effects and no significant treatment × dose interactions

(Chapter 3, Table 3), indicating that daytimes with almost balanced glucose homeostasis

occurred still when glucose was infused at the highest dose level of 30% NEL requirement.

Since glucosuria was not present up to an infusion dose of 20% NEL requirements and the

infused glucose did not translate into higher milk production, one can presume that the


[77]

increase in glucose uptake and storage by peripheral tissues, such as liver, skeletal muscle and

adipose tissue might enable the cows to deal with the excess supply of glucose in order to

maintain glucose homeostasis (see Section 6.6.).

At day 24 and with a GI accounting for 30% NEL requirements, the daily maximum values

of plasma glucose increased by 130% and the maximum serum insulin concentrations

increased 17-fold (Chapter 3, Table 3) which led to a significant linear treatment effect and

treatment × day interaction in the daily mean values recorded every 8 d for both glucose and

insulin concentrations (Chapter 3, Fig. 1A and 1B). In fact, these increases were not constant

during the day but mostly due to the postprandial hyperglycemia and hyperinsulinemia

observed in four out of six GI cows (i.e. at 1600 h on d 24; Chapter 5, Fig. 1 and Fig. 2).

Alterations in peripheral insulin sensitivity may have been responsible for the observed

postprandial hyperglycemia and hyperinsulinemia as will be discussed in the next section. In

conclusion, surplus glucose between 0-20% NEL requirements was well tolerated by the cows

of the present study. The latter would be equivalent to the maximum capacity for postruminal

starch digestion and absorption (i.e. ~2 kg/d; GÄBEL and ASCHENBACH 2004). Increasing

the infusion dose from 20 to 30% NEL requirement was no longer tolerated by the animal and

led to periods of obviously dysregulated glucose homeostasis.

6.3 Gradual Increases in Glucose Supply Cause Insulin Resistance in a Dose-Dependent

Fashion

As mentioned above, glucose homeostasis was impaired in GI cows only at high infusion

doses of 30% NEL requirement as indicated by the postprandial hyperglycemia/

hyperinsulinemia. Glucosuria was also detected at the same period in GI cows. To test

whether these metabolic changes are a consequence to peripheral insulin resistance

(HOSTETTLER-ALLEN et al. 1994), RQUICKI was calculated in the present study from the

plasma concentration of glucose, and the serum concentrations of insulin and NEFA

(Chapter 3, Calculations and Statistical Analysis). RQUICKI has been used mainly in humans

as a measure for peripheral insulin sensitivity, with lower values being indicative of decreased

insulin sensitivity (PERSEGHIN et al. 2001; RABASA-LHORET et al. 2003). Recently

RQUICKI has been introduced as a suitable monitor for insulin resistance in dairy cows since

glucose tolerance tests are difficult to perform and to interpret in adult ruminants due to

continuing ruminal fermentation and due to high rates of insulin-insensitive glucose uptake

into peripheral tissues (HOLTENIUS and HOLTENIUS 2007). Significant treatment × dose


[78]

interaction was observed for RQUICKI based on a dose-dependent decrease in the index

value when the infusion dose increased from 0 to 30% NEL requirements (Chapter 3, Fig. 1C).

Decreased insulin sensitivity in the present study was associated with a plateau in liver

glycogen content between d 8 and d 16 (Chapter 3, Fig. 3A). The latter indicate that the

hepatic uptake of glucose and its conversion to glycogen started to become compromised at

the time when insulin resistance developed. It may appear contradictory that muscle glycogen

stores also increased in the present study despite insulin resistance (Chapter 3, Fig. 3B).

However, ruminant animals differ from most non-ruminant species in that they express high

levels of GLUT1 in their skeletal muscle which is constitutively present and does not require

insulin stimulation (DUHLMEIER et al. 2005). Thereby, hyperglycemia alone is sufficient to

increase glucose entry (and glycogen storage) in skeletal muscle. Nonetheless, the ability of

hyperglycemia to compensate for insulin resistance has also been observed in muscle of

humans and rats under hyperglycemic, hyperinsulinemic conditions (COMMERFORD et al.

2001). The decrease in insulin sensitivity might be related to the increase in body weight

found in GI cow (Chapter 3, Table 2), since insulin sensitivity is disturbed in obese cows

(HOLTENIUS and HOLTENIUS 2007). However, insulin sensitivity was restored in the

current study within only 4 days of glucose withdrawal as demonstrated by complete reversal

of both RQUICKI and liver glycogen content.

6.4 Nutritionally Relevant Increases of Glucose Supply Have No Negative Effect on the

Enzymatic Capacity for Gluconeogenesis

To test whether there was a decrease in the enzymatic capacity for hepatic gluconeogenesis in

GI cows corresponding to the increase in glucose utilization and storage, the effect of

increasing intravenous GI on the activities and relative mRNA expressions of four rate-

limiting gluconeogenesis enzymes was investigated, namely PEPCK, PC, FBPase and G6-

Pase (Chapter 4, Table 2). PEPCK has been proposed as the rate-determining enzyme

involved in hepatic glucose production from propionate in dairy cows (GREENFIELD et al.

2000). Despite the increase in glucose availability in the present study, the total PEPCK

activity and the relative mRNA expression levels of its two isoforms (PEPCK-C and PEPCK-

M) showed no dose × treatment interaction, suggesting that infused glucose did not affect

hepatic capacity for glucose synthesis from propionate. This result supports the view that the

liver of dairy cows has a high metabolic priority to utilize propionate for gluconeogenesis

regardless of the physiological and nutritional status (HUNTINGTON et al. 2006).


[79]

Between infusion doses of 0-20% NEL requirement, the effect of glucose supply on the

other gluconeogenic enzymes activities and mRNA expressions was very moderate (Chapter

4, Table 2). However, at non-physiologically high levels of surplus glucose supply (i.e.

infusion of 30% NEL requirement), a treatment × dose interaction was noted for PC relative

mRNA expression with a numerical decrease in PC activity (linear treatment × dose

interaction, P = 0.13). This finding is consistent with previous reports where PC mRNA

expression is most consistently changed with changing levels of glucose or energy supply in

ruminants (BRADFORD and ALLEN 2005; LOOR et al. 2006). Lactate and some of the

glucogenic AA are metabolized first to pyruvate and then directly to OAA through an ATP-

dependent reaction catalyzed by PC. Therefore, the decrease in PC mRNA level with non-

physiologically high glucose loads might mirror a decrease in hepatic gluconeogenic capacity

from lactate and glucogenic AA. A decreased gluconeogenesis from AA would be expected to

decrease hepatic urea production which was demonstrated in the present study by the

decreased serum BUN concentration in the GI group (Chapter 4, Fig. 2A).

A part of the decrease in PC mRNA level in the present study can be explained by the

increases in insulin concentrations during the period of high glucose load, mainly, the

observed postprandial hyperinsulinemia (JITRAPAKDEE and WALLACE 1999). PC activity

requires acetyl-CoA as an activator. Therefore, the activity of this enzyme is enhanced under

condition of high fatty acid oxidation, presumably, as a result of increased intramitochondrial

levels of acetyl-CoA (TUTWILER and DELLEVIGNE 1979). In contrast, reduced rates of

fatty acid β-oxidation are thought to inhibit hepatic gluconeogenesis by lowering PC activity

(TUTWILER and DELLEVIGNE 1979; CHOW and JESSE 1992). Previous studies have

reported that insulin depressed the hepatic capacity for LCFA oxidation (JESSE et al. 1986a;

ANDERSEN et al. 2002). It may be speculated, therefore, that the observed decrease in PC

enzyme activity in the present study, although not statistically significant, might have been a

result of the increased plasma insulin concentration, leading to decreased hepatic fatty acid β-

oxidation as will be discussed in Section 6.8.

The second enzyme activity significantly affected by GI was FBPase but only at the

highest dose of infused glucose and with only posttranscriptional downregulation (Chapter 4,

Table 2). FBPase is postulated to be a key regulatory enzyme that controls the overall output

of gluconeogenesis (ENGELKING 2004; PILKIS et al. 1988). Consequently, the decrease in

FBPase activity might represent one of the homeostatic mechanism that is partially able to

limit the development of hyperglycemia due to high glucose load. The decrease in FBPase


[80]

activity could be directly related to the hyperglycemia because excess glucose, together with

insulin, stimulates the intracellular accumulation of fructose 2,6-bisphosphate, which is a

powerful competitive inhibitor of FBPase (PILKIS et al. 1988). Additionally, a rapid

proteasomal degradation of FBPase has been demonstrated in yeast upon exposure to glucose

(BROWN and CHIANG 2009). The rapid translocation of FBPase to the nucleus observed in

cultured rat hepatocytes after addition of glucose or insulin (YÁÑEZ et al. 2004) could

indicate that similar proteasomal degradation of FBPase is possible in the mammalian liver

during hyperglycemia.

The aforementioned changes in PC mRNA expression and FBPase activity were fully

restored within only four days after withdrawing GI as will be discussed in Section 6.9.

However, posttranscriptional downregulation of G6-Pase activity was observed in the present

study after withdrawing high-dose GI. This decrease in the activity of G6-Pase could be

related to the increased intracellular availability of its substrate (i.e. glucose 6-phosphate)

derived from glycogen breakdown after stopping GI (VAN SCHAFTINGEN and GERIN

2002). In conclusion, moderate increases of glucose supply (i.e. nutritionally relevant

increases) have no negative effect on the activity and mRNA expression of key

gluconeogenesis enzymes in mid-lactating dairy cows. Only high, non-nutritionally relevant

increases of glucose supply which lead to hyperglycemia and hyperinsulinemia affect some of

the hepatic gluconeogenesis enzymes (i.e. PC, FBPase).

6.5 Excessive Glucose Has a Protein-Sparing Effect

Quadratic day × treatment interaction was detected for the serum BUN concentration from 2-

d blood samples (Chapter 4, Fig. 2A). In addition, the daily minimum, maximum, and AUC

values for BUN showed numerically larger decreases in GI cows compared to SI cows

(Chapter 3, Table 3), indicating a protein-sparing effect. A similar response with a decrease of

BUN concentration has been described earlier during GI studies (VIK-MO et al. 1974;

OBITSU et al. 2000). The decrease in BUN in the GI group was probably a reflection of the

improvement of body nitrogen retention through a decrease in the hepatic uptake and

deamination of glucogenic AA. This has likely promoted the cellular uptake of AA for protein

synthesis and, as a consequence, led to decreased hepatic urea synthesis (OBITSU et al.

2000). In the present experiment, the shuttle of AA from hepatic gluconeogenesis pathway to

protein synthesis pathways is supported by the increase in milk protein output (see Section

6.1) and the decrease in PC expression level (see Section 6.4).


[81]

6.6 Surplus Glucose Is Used Mainly by Glycogen and Fat Synthesis Pathways

Accelerated glucose storage as glycogen (liver and skeletal muscle) and TAG (adipose

tissues) are the generalized responses to the excessive glucose supply in the current study. It

had previously been demonstrated in other studies that glucose uptake and utilization by body

tissues was accelerated with increased glucose supply in dairy cows (AMARAL et al. 1990;

RIGOUT et al. 2002b). The same effect was also observed in the case of abomasal starch

infusion (KNOWLTON et al. 1998). As the supply of energy increased by hypercaloric GI in

the present study, GI cows moved into a positive energy status, stored excess energy as fat,

and gained BW (Chapter 3, Table 2). The increase in BFT observed in the present study

supports the view that the increase in BW was in large part due to an increase in adiposity. In

line with that, previous estimation indicates that each 1-mm changes in the BFT equates

approximately 5 kg changes in total body fat content (SCHRÖDER and STAUFENBIEL

2006). If this estimation is applied to the results of the present study, then it can be calculated

that ~78% of the increases in the BW came from the increase in body fat depositions. This

assumption is supported by the observations that lipogenesis from glucose and other

substrates (acetate, lactate) is enhanced in times of increased glucose availability in ruminants

(BALLARD et al. 1972; VERNON 1980). Additionally, a very small fraction of the observed

BW gain was also attributable to glycogen storage in liver and skeletal muscle (Chapter 3,

Fig. 3). Over all, the increase in BW, BFT and the capacity of liver and skeletal muscle to

accumulate glycogen in the present study, coupled with a decrease in intermediate lipid and

protein metabolism, all suggest that the rate of glucose uptake and utilization by peripheral

tissues was increased with increased glucose supply.

6.7 Surplus Glucose Diverts Lipid from Energy-Generating Pathways into Anabolic

Pathways

Significant dose × treatment interaction was present for the minimum values of serum NEFA

concentrations (Chapter 3, Table 3) and a trend toward a treatment × quadratic dose

interaction was observed for serum NEFA concentrations measured in 2-d blood samples

(Chapter 4, Fig. 3C), indicating a decrease in the mobilization of body fat and an improved

EB status for GI cows. Similarly, daily mean (Chapter 3, Fig. 2A) and maximum values, and

the daily AUC of serum BHBA concentration (Chapter 3, Table 3) showed linear dose ×

treatment interactions, demonstrating a decrease in BHBA concentration with increasing dose

of glucose. Glucose is known for its direct and indirect antiketogenic effects (TREACHER et

al. 1976; AMARAL et al. 1990). These effects include: (1) decreasing the supply of free fatty


[82]

acids to the liver by stimulating lipogenesis and inhibiting lipolysis in adipose tissue and (2)

eliciting metabolic alterations within the liver that alter enzyme activities and availability of

substrates that are involved in hepatic ketogenesis (TREACHER et al. 1976). With regard to

the liver, such effect on the activity of enzymes involved in hepatic fatty acid oxidation was

evident in the present experiment from the decreased CPT activity as will be discussed in next

section. Collectively, the depressive effect of GI on serum NEFA and BHBA concentrations

indicates that the anabolism analysed in Section 6.6 was, to a significant part, due to shifts in

lipid metabolism from energy generating pathways to anabolic pathways.

6.8 Hepatic but Not Skeletal Muscle CPT Activity Is Sensitive to the Changes in Energy

Balance Status

The hepatic fatty acid oxidation process is likely involved in the adaptation of the dairy cows

to changes in energy supply. This is evident from previous observations that CPT activity

increased or tended to increase during periods of NEB (DANN and DRACKLEY 2005;

DOUGLAS et al. 2006) and decreased with a high-energy diet (AIELLO et al. 1984).

Consistent with these results, gradual increase in glucose availability, brought by intravenous

GI in the present experiment, revealed a continuous decrease in hepatic CPT activity with

increasing GI (Chapter 5, Table 1). The mechanism by which hepatic CPT activity was

decreased was not investigated in this study. However, the regulation of CPT-1 activity is

thought to occur predominantly via changes in cytoplasmic malonyl-CoA concentrations

(KERNER and HOPPEL 2000). A previous report by ZAMMIT (1999) suggested that insulin

was able to increase the malonyl-CoA sensitivity of CPT-1 via a modification of the

mitochondrial membrane fluidity. In addition, CPT activity was lower in mitochondria

isolated from sheep hepatocytes preincubated with insulin (CHOW and JESSE 1992).

Therefore, the elevation in serum insulin level may provide one possible explanation to the

cause of glucose-induced depression of hepatic CPT activity. Additionally, the numerical

decreases in the hepatic mRNA expression of both CPT-1A (by 28 %) and CPT-2 (by 42%),

which were in the same range like the observed decrease in enzyme activity (by 35%)

between d 0 and d 24 likely also contributed to the decreased hepatic CPT activity via

transcriptional regulation.

Maintaining optimal hepatic CPT function during the dry period could be critical for cows

to make a smooth transition into the heavy milk production period. As hepatic CPT activity

was depressed by positive energy balance in this study, it seems possible that positive energy


[83]

balance during the dry period would negatively affect hepatic LCFA oxidation, which could

be relevant for postpartum metabolic disturbances. In line with that, a beneficial effect of

prepartum feed restriction on postpartum lipid metabolism through increased CPT activity or

mRNA levels has been described recently (DOUGLAS et al. 2006; LOOR et al. 2006).

Enhancing hepatic capacity for β-oxidation by increased CPT activity will direct LCFA

toward mitochondrial oxidation rather than esterification, thereby leading to less

accumulation of lipid and TAG in the liver (DOUGLAS et al. 2006).

The literature screening in the present work revealed no information about the changes in

skeletal muscle CPT activity relative to the energy balance in dairy cows. However, this is an

important issue since failure to upregulate CPT activity in muscle with increasing availability

of NEFA during times of negative energy balance would promote the accumulation of

intracellular lipids. It has been shown in other species that such accumulation of intracellular

lipids leads to insulin resistance (KREBS and RODEN 2004) due to suppression of insulin

signaling pathways (CAHOVÁ et al. 2007). An observation coming from the present study is

that the regulatory effect of surplus glucose on hepatic CPT activity was not observed on

skeletal muscle CPT, suggesting that muscle CPT activity maybe less sensitive to the changes

in energy supply in lactating dairy cows. In contrast to the skeletal muscle which utilizes

NEFA exclusively for its own energy metabolism, the liver clears NEFA from the systemic

circulation in excess of its own demand and converts part of them to water-soluble ketone

bodies (e.g. BHBA and acetoacetate) that can be used as energy substrates by other tissues.

Thereby, hepatic fatty acid oxidation plays a key role not only for the control of blood NEFA

levels but also for the provision of adequate amounts of ketone bodies to provide fuel to

extrahepatic tissues. From a teleological point of view, it appears thus reasonable that hepatic

fatty acid oxidation has a higher metabolic priority for regulation and requires larger plasticity

compared to fatty acid oxidation in skeletal muscle.

6.9 Adaptations Are Rapidly Reversible after Ceasing of Glucose Infusions Except for

Those of Lipid Metabolism

Apart from investigating the dose effects of glucose, another intention of the present study

was to test how quick possible changes in intermediary metabolism can be reversed after

withdrawing a non-physiologically high glucose load. The withdrawal part of the

experimental model relates to the important question if energy overfeeding prepartum may

have persistent negative effects on glucose production capacity postpartum (e.g. periods of


[84]

hypoglycaemia due to insufficient gluconeogensis). Thus, the infusion dose was maintained at

30% of the NEL requirement between d 24 and 28 (i.e. 4 d) before the post infusion samples

were collected on d 32 (i.e. 4 d after the end of the infusion period).

For parameters related to glucose intermediary metabolism, post-infusion samples showed

that daily mean values of plasma glucose concentration in the GI group had returned to

baseline values while daily mean values of serum insulin concentration remained slightly

elevated compared with baseline values at d 0 (Chapter 3, Fig. 1). Additionally, blood glucose

and insulin values from the blood samples collected every 2 d (Chapter 4, Fig 1), daily

minimum and maximum values and AUC of plasma glucose and serum insulin (Chapter 3,

Table 3) were not different between d 0 and d 32 in both groups. The decreases in RQUIKI

values (Chapter 3, Fig. 1C), hepatic PC mRNA and FBPase activity (Chapter 4, Table 2) and

the increases in the glycogen levels in liver and skeletal muscles of GI cows (Chapter 3, Fig

3) were fully reversed and returned to pre-infusion values, indicating that glucose metabolism

of dairy cattle is very robust with regard to changes in glucose supply. In this study, excess

glucose supply and sudden glucose withdrawal were both surprisingly well tolerated.

For parameters related to lipid intermediary metabolism, post-infusion values were lower

in the GI group for serum NEFA concentration (2-d samples; Chapter 4, Fig 2C), minimum

daily concentrations of serum BHBA and NEFA, and daily AUC for serum BHBA (Chapter

3, Table 3). Serum BHBA levels were also lower than initial values at 1600 h and 0400 h on d

32 (Chapter 5, Fig. 3). Moreover, the depression in hepatic CPT activity persisted until four

days after the end of the infusion period (Chapter 5, Table 1). Lower post-infusion values of

serum BHBA and hepatic CPT activity in GI cows commonly suggest that hepatic capacity

for LCFA oxidation was lower compared to d 0. It seems, therefore, that GI cows were less

dependent on their lipid body stores as a source of energy after ceasing GI. This could, in

part, be related to the availability of glycogen as an additional glucose source during the post-

infusion period in the GI group.

Taken together, the beneficial effect of surplus glucose on lactation performance is

negligible in midlactating dairy cows. Most of the infused glucose was used to increase the

body reserves of glycogen and fat. Cows adapted well to nutritionally relevant increases of

glucose supply by making adjustments in the intermediary metabolism to coordinate the

metabolic homeostasis. These adjustments mainly involved: 1) the acceleration of glucose


[85]

utilization and storage by peripheral tissues and as a consequence, 2) the diversion of protein

and lipid from energy-generating pathways into anabolic pathways. However, non-

nutritionally relevant GI doses were associated with alterations in the peripheral insulin

sensitivity and in the catalytic capacity of some hepatic gluconeogenesis enzymes (i.e. PC,

FBPase). In general, glucose homeastasis of midlactation dairy cows proofed to be very

robust to both gradual increases in glucose supply and sudden withdrawal thereof. If the

results of the present study were transferable to the postpartum cow, this may suggest that

disturbances of glucose homestasis are more likely a secondary event in metabolic

disturbances like postpartum ketosis.

CHAPTER‐7: SUMMARY

[86]

7 Summary Bahaa Al-Trad

Effects of Increasing Intravenous Glucose Infusions on Lactation Performance,

Metabolic Profiles, and Metabolic Gene Expression in Dairy Cows

Institute of Physiology, Faculty of Veterinary Medicine, University of Leipzig

Submitted in December 2009

89 Pages, 3 Manuscripts, 152 references

Keywords: Glucose infusion, Dairy cows, Lactation performance, Hepatic gluconeogenesis,

Carnitine palmitoyltransferase

Knowledge on the precise effects of surplus glucose supply in dairy cows is limited by the

lack of information on how intermediary metabolism adapts at different levels of glucose

availability. Therefore, a gradual increase of glucose supply via intravenous glucose infusion

was used in the present study to test the dose effect of surplus provision of glucose on the

metabolic status and milk production of dairy cows. Furthermore, the effects of increasing

levels of surplus glucose on mRNA expressions and activities of rate-limiting enzymes

involved in hepatic gluconeogenesis were investigated. Based on a previous finding that a

positive energy balance may decrease hepatic carnitine palmitoyltransferase (CPT) enzyme

activity, it was also of interest whether skeletal muscle CPT activity is downregulated in a

similar manner during positive energy balance.

Twelve midlactating Holstein-Friesian dairy cows were continuously infused over a 28-d

experimental period with either saline (SI group, six cows) or 40% glucose solutions (GI

group, six cows). The infusion dose was calculated as a percentage of the daily energy (NEL)

requirements by the animal, starting at 0% on d 0 and increasing gradually by 1.25%/d until a

maximum dose of 30% was reached by d 24. Dose was then maintained at 30% NEL

requirement for 5 d. No infusions were made between d 29-32. Liver and skeletal muscle

biopsies were taken on d 0, 8, 16, 24, and 32. Body weight (BW) and back fat thickness

(BFT) were recorded on biopsies days. Blood samples were taken every 2 d. In addition,

blood samples over 24 h (6-h intervals) were taken the days before each biopsy. Milk and

urine samples were taken on biopsies days.

CHAPTER‐7: SUMMARY

[87]

BW and BFT increased linearly with increasing glucose dose for GI cows. No differences

were observed in the dry matter intake, milk energy output, and energy corrected milk yield

between groups. However, milk protein percentage and yield increased linearly in the GI

group. Only occasional increases in blood glucose and insulin concentrations were observed

in blood samples taken at 1000 h every 2 d. However, during infusion dose of 30% NEL

requirements on d 24, GI cows developed postprandial hyperglycemia associated with

hyperinsulinemia, coinciding with glucosuria. The revised quantitative insulin sensitivity

check index (RQUIKI) indicated linear development of insulin resistance for the GI

treatment. GI decreased serum concentrations of beta-hydroxybutyrate (BHBA) and blood

urea nitrogen and tended to decrease the serum concentration of non-esterified fatty acids

(NEFA). Liver glycogen content increased, while glycogen content in skeletal muscle only

tended to increase by GI. No significant changes were observed in the activities and relative

mRNA expression levels of hepatic phosphoenolpyruvate carboxykinase and glucose 6-

phospatase. The activity of fructose 1,6-bisphosphatase (FBPase) and relative mRNA

expression levels of pyruvate carboxylase (PC) were decreased in the GI group but only

during the high dose of glucose infusion. Hepatic CPT activity decreased with GI and

remained decreased on d 32. The hepatic expression levels of CPT-1A and CPT-2 mRNA

were not significantly altered but tended to reflect the changes in enzyme activity. No effect

of glucose infusion was observed on skeletal muscle CPT activity. The aforementioned

adaptations were reversed four days after the end of glucose infusions except for those of BW,

BFT, and lipid metabolism (i.e. serum BHBA and NEFA concentrations, hepatic CPT

activity).

It is concluded that mid-lactation dairy cows on an energy-balanced diet direct

intravenously infused glucose predominantly to body fat reserves but not to increased

lactation performance. Cows rapidly adapted to increasing glucose supply but experienced

dose-dependent development of insulin resistance corresponding with postprandial

hyperglycemia/hyperinsulinemia and glucosuria at dosages equivalent to 30% NEL

requirements. The catalytic capacity of key hepatic gluconeogenesis enzymes in mid-lactating

dairy cows is not significantly affected by nutritionally relevant increases of glucose supply.

Only very high dosages selectively suppress PC transcription and FBPase activity. Finally, it

can be concluded that suppression of CPT activity by positive energy balance appears to be

specific for the liver in midlactating dairy cows.

CHAPTER‐8: Zusammenfassung

[88]

8 Zusammenfassung Bahaa Al-Trad

Effekte einer ansteigenden intravenösen Glukoseinfusion auf Laktationsleistung,

metabolische Profile und die Expression metabolisch relevanter Gene bei Milchkühen

Veterinär-Physiologisches Institut, Veterinärmedizinische Fakultät, Universität Leipzig

Eingereicht im Dezember 2009

89 Seiten, 3 Manuskripte, 152 Literaturangaben

Schlüsselwörter: Glukoseinfusion, Milchkühe, Laktationsleistung, Hepatische

Gluconeogenese, Carnitin-Palmitoyltransferase

Die genauen Effekte einer additiven Glukoseversorgung von Milchkühen können aufgrund

fehlender Informationen bezüglich der Anpassung des Intermediärstoffwechsels an

verschiedene Stufen der Glukoseversorgung derzeit nur schwer abgeschätzt werden. Deshalb

wurden im Rahmen der vorliegenden Studie die Dosiseffekte einer langsam ansteigenden,

intravenösen Glukoseinfusion auf den metabolischen Status und die Milchproduktion

laktierender Kühe untersucht. Zusätzlich wurden die Effekte der verschiedenen Glukosedosen

auf die mRNA-Expression und Aktivität von ratenlimitierenden Enzymen der

Glukoneogenese gemessen. Aufgrund älterer Befunde, dass eine positive Energiebilanz die

Aktivität der hepatischen Carnitin-Palmitoyltransferase (CPT) senken kann, war es weiterhin

im Rahmen dieser Studie von Interesse, ob die CPT-Aktivität der Skelettmuskulatur in

ähnlicher Weise durch eine positive Energiebilanz herunterreguliert wird.

Zwölf Holstein-Friesian-Kühe in der Mitte der Laktation wurden über 28 Tage entweder

mit 0,9%-iger NaCl-Lösung (SI-Gruppe) oder einer 40%-igen Glukoselösung (GI-Gruppe)

intravenös infundiert. Die Infusionsdosis wurde als Prozentsatz des täglichen Energiebedarfes

(NEL) errechnet. Die Infusion wurde beginnend mit 0% am d 0 langsam um 1.25%/d

gesteigert bis am d 24 eine Maximaldosis von 30% erreicht war. Die Dosis von 30% NEL-

Bedarf wurde über 5 d gehalten. Keine Infusionen erfolgten zwischen d 29 - 32. Biopsien von

Leber und Skelettmuskulatur wurden an d 0, 8, 16, 24, and 32 gewonnen. Die Entnahme von

Blutproben erfolgte aller 2 d. Zusätzlich wurden Blutproben über 24 h (6-stündige Intervalle)

jeweils am Tag vor den Biopsien genommen. An den Biopsie-Tagen wurden Körpermasse

(BW) und Rückenfettdicke (BFT) gemessen sowie Milch- und Urinproben gewonnen.

CHAPTER‐8: Zusammenfassung

[89]

BW und BFT erhöhten sich linear mit ansteigenden Glukosedosen in der GI-Gruppe.

Keine Unterschiede waren bezüglich der Trockensubstanzaufnahme, Milchenergieabgabe und

energiekorrigierten Milchleistung zwischen den beiden Gruppen zu verzeichnen. Jedoch

erhöhten sich Milchproteingehalt und -menge linear in der GI-Gruppe. Nur gelegentlich

wurden bei GI-Kühen erhöhte Konzentrationen von Glukose und Insulin in jenen Blutproben

gemessen, die in zweitägigen Abständen um jeweils 10:00 Uhr gewonnen wurden.

Demgegenüber entwickelten die Kühe der GI-Gruppe eine postprandiale Hyperglykämie und

Hyperinsulinämie während der Infusion von 30% NEL-Bedarf am d 24, was mit einer

Glukosurie gekoppelt war. Der Revised Quantitative Insulin Sensitivity Check Index

(RQUIKI) deutete auf die lineare Entwicklung einer Insulinresistenz bei GI-Behandlung hin.

Die Glukoseinfusion erniedrigte die Serumkonzentrationen von β-Hydroxybutyrat (BHBA)

sowie Harnstoff-N. Die Serumkonzentration der freien Fettsäuren tendierte ebenfalls zu einem

Abfall. Der Leberglykogengehalt erhöhte sich, wohingegen der Glykogengehalt im

Skelettmuskel nur tendenziell durch Glukoseinfusion erhöht war. Die Aktivität und relative

mRNA-Expression der hepatischen Phosphoenolpyruvat-Carboxykinase und Glukose-6-

phospatase wurden durch die Glukoseinfusion nicht verändert. Die Aktivität der Fruktose-1,6-

bisphosphatase (FBPase) und die relative mRNA-Expression der Pyruvat-Carboxylase (PC)

waren in der GI-Gruppe nur während der Infusion der höchsten Glukosedosis erniedrigt. Die

Aktivität der hepatischen CPT fiel während der Glukoseinfusion ab, was von einem

tendenziellen Abfall der mRNA-Expression der CPT-1A and CPT-2 begleitet war. Die CPT-

Aktivität im Skelettmuskel war unverändert. Alle erwähnten Glukoseeffekte waren nach

Absetzen der Glukoseinfusion (d 32) reversibel mit Ausnahme von BW, BFT und Parametern

des Lipidstoffwechsels (d.h. Serum-BHBA und -NEFA-Konzentrationen sowie hepatische

CPT-Aktivität).

Es kann der Schluss gezogen werden, dass intravenös infundierte Glukose bei Milchkühen

in der Mitte der Laktation hauptsächlich zu erhöhtem Fettansatz, nicht jedoch zu erhöhter

Milchleistung führt. Kühe passen sich schnell an die erhöhte Glukoseversorgung an, zeigen

jedoch dosisabhängig die Entwicklung einer Insulinresistenz mit postprandialer

Hyperglykämie/Hyperinsulinämie und Glukosurie. Die katalytische Kapazität von

Schlüsselenzymen der hepatischen Glukoneogenese wird durch ernährungsphysiologisch

relevante Glukosemengen nicht signifikant verändert. Erst sehr hohe Glukosedosen führen zu

einer selektiven Suppression der PC-Transkription sowie der FBPase-Aktivität. Eine

glukoseabhängige Suppression der CPT-Aktivität tritt nur in der Leber, nicht jedoch in der

Skelettmuskulatur auf.

CHAPTER‐9: REFERENCES

[90]

References

Abe H, Morimatsu M, Nikami H, Miyashige T, Saito M. Molecular cloning and mRNA expression of the bovine insulin-responsive glucose transporter (GLUT4). J Anim Sci. 1997; 75:182-8. Abe H, Kawakit Y, Hodate K, Saito M. Postnatal development of glucose transporter proteins in bovine skeletal muscle and adipose Tissue. J Vet Med Sci. 2001;63:1071-5.

Agca C, Greenfield RB, Hartwell JR, Donkin SS. Cloning and characterization of bovine cytosolic and mitochondrial PEPCK during transition to lactation. Physiol Genomics. 2002;11:53–63.

Ahmed BM, Bergen WG, Ames AK. Effect of nutritional state and insulin on hind-limb amino acid metabolism in steers. J Nutr. 1983;113:1529-43.

Aiello RJ, Kenna TM, Herbein JH. Hepatic gluconeogenic and ketogenic interrelationships in the lactating cow. J Dairy Sci. 1984;67:1707-15. Allen MS. Effects of diet on short-term regulation of feed intake by lactating dairy cattle. J Dairy Sci. 2000;83:1598-624.

Allen MS, Bradford BJ. Regulation of feed intake in transition cows: application of the hepatic oxidation hypothesis. 22nd Annual Southwest Nutrition and Management Conference; 2007 February 22-23; Tempe, Arizona; 2007. Allen MS, Bradford BJ, Oba M. Board Invited Review: The hepatic oxidation theory of the control of feed intake and its application to ruminants. J Anim Sci. 2009;87:3317-34. Amaral DM, Veenhuizen JJ, Drackley JK, Cooley MH, McGilliard AD, Young JW. Metabolism of propionate, glucose, and carbon dioxide as affected by exogenous glucose in dairy cows at energy equilibrium. J Dairy Sci. 1990;73:1244-54.

Andersen JB, Mashek DG, Larsen T, Nielsen MO, Ingvartsen KL. Effects of hyperinsulinaemia under euglycaemic condition on liver fat metabolism in dairy cows in early and mid-lactation. J Vet Med A Physiol Pathol Clin Med. 2002;49:65-71. Annison EF, Bickerstaffe R, Linzell JL. Glucose and fatty acid metabolism in cows producing milk of low fat content. J Agric Sci (Camb). 1974;82:87-95. Balcells J, Seal CJ, Parker DS. Effect of intravenous glucose infusion on metabolism of portal-drained viscera in sheep fed a cereal/straw-based diet. J Anim Sci. 1995;73:2146-55.

Ballard FJ, Hanson RW, Kronfeld DS. Gluconeogenesis and lipogenesis in tissue from ruminant and nonruminant animals. Fed Proc. 1969;28:218-31.

Ballard FJ, Filsell OH, Jarrett IG. Effects of carbohydrate availability on lipogenesis in sheep. Biochem J. 1972;126:193-200. Bauchart D, Gruffat D, Durand D. Lipid absorption and hepatic metabolism in ruminants. Proc Nutr Soc. 1996;55:39-47.


[91]

Bauman DE, Griinari JM. Nutritional regulation of milk fat synthesis. Annu Rev Nutr. 2003;23:203-27.

Bartley JC, Black AL. Effect of exogenous glucose on glucose metabolism in dairy cows. J Nutr. 1966;89:317-28.

Bartley JC, Freedland RA, Black AL. Effect of aging and glucose loading on the activities of glucose-6-phosphatase and phosphorylase of livers of cows and calves. Am J Vet Res. 1966;27:1243-8.

Bell AW. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation. J Anim Sci. 1995;73:2804-19. Bell GI, Burant CF, Takeda J, Gould GW. Structure and function of mammalian facilitative sugar transporters. J Biol Chem. 1993;268:19161-4. Bell GI, Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Fukumoto H, Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care. 1990;13:198-208.

Bergman EN. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol Rev. 1990;70:567–90. Bergman EN, Brockman RP, Kaufman CF. Glucose metabolism in ruminants: comparison of whole-body turnover with production by gut, liver, and kidneys. Fed Proc. 1974;33:1849-54.

Bickerstaff R, Annison EF, Linzell JL. The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows. J Agric Sci (Camb). 1974;82:71-85.

Bobe G, Young JW, Beitz C, Invited review: pathology, etiology, prevention, and treatment of fatty liver in dairy cows. J Dairy Sci. 2004;87:3105-24. Bradford BJ, Allen MS. Depression in feed intake by a highly fermentable diet is related to plasma insulin concentration and insulin response to glucose infusion. J Dairy Sci. 2007;90:3838-45.

Bradford, BJ, Allen MS. Phlorizin administration increases hepatic gluconeogenic enzyme mRNA abundance but not feed intake in late-lactation dairy cows. J Nutr. 2005;135:2206-11. Brockman RP. Effect of insulin on the utilization of propionate in gluconeogenesis in sheep. Br J Nutr. 1990;64:95-101.

Brockman RP. Role of insulin in regulating hepatic gluconeogenesis in sheep. Can J Physiol Pharmacol. 1985;63:1460-64.

Brockman RP, Laarveld B. Hormonal regulation of metabolism in ruminants; a review. Livest Prod Sci.1986;14:313-34. Brown CR., Chiang HL. A selective autophagy pathway that degrades gluconeogenic enzymes during catabolite inactivation. Commun Integr Biol. 2009;2:177-183.


[92]

Butler WR. Nutritional interactions with reproductive performance in dairy cattle. Anim Reprod Sci. 2000;60:449-57.

Cahová M, Vavrínková H, Kazdová L. Glucose-fatty acid interaction in skeletal muscle and adipose tissue in insulin resistance. Physiol Res. 2007;56:1-15. Chow JC, Jesse BW. Interactions between gluconeogenesis and fatty acid oxidation in isolated sheep hepatocytes. J Dairy Sci. 1992;75:2142-8. Christ B, Nath A, Bastian H, Jungermann K. Regulation of the expression of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes by glucagon and insulin. Eur J Biochem. 1988;178:373–9. Clark JH, Spires HR, Derrig RG, Bennink MR. Milk production, nitrogen utilization and glucose synthesis in lactating cows infused postruminally with sodium caseinate and glucose. J Nutr. 1977;107:631-44. Commerford SR, Bizeau ME, McRae H, Jampolis A, Thresher JS, Pagliassotti MJ. Hyperglycemia compensates for diet-induced insulin resistance in liver and skeletal muscle of rats. Am J Physiol Regul Integr Comp Physiol. 2001;281:R1380-9 Danfær A. Nutrient metabolism and utilization in the liver. Livest Prod Sci. 1994;39:115-27. Danfær A, Tetens V, Agergaard N. Review and an experimental study on the physiological and quantitative aspects of gluconeogenesis in lactating ruminants. Comp Biochem Physiol B Biochem Mol Biol. 1995;111:201-10. Dann HM, Drackley JK. Carnitine palmitoyltransferase I in liver of periparturient dairy cows: effects of prepartum intake, postpartum induction of ketosis, and periparturient disorders. J Dairy Sci. 2005;88:3851-9. Dhiman TR, Cadorniga C, Salter LD. Protein and energy supplementation of high alfalfa silage diets during early lactation. J Dairy Sci. 1993;76:1945-59.

Dobbins RL, Szczepaniak LS, Bentley B, Esser V, Myhill J, McGarry JD. Prolonged inhibition of muscle carnitine palmitoyltransferase-1 promotes intramyocellular lipid accumulation and insulin resistance in rats. Diabetes. 2001;50:123-30.

Donkin SS, Armentano LE. Insulin and glucagon regulation of gluconeogenesis in preruminating and ruminating bovine. J Anim Sci. 1995;73:546-51.

Donkin SS, Bertics SJ, Armentano LE. Chronic and transitional regulation of gluconeogenesis and glyconeogenesis by insulin and glucagon in neonatal calf hepatocytes. J Anim Sci. 1997;75:3082-7. Douglas GN, Overton TR, Bateman HG 2nd, Dann HM, Drackley JK. Prepartal plane of nutrition, regardless of dietary energy source, affects periparturient metabolism and dry matter intake in Holstein cows. J Dairy Sci. 2006;89:2141-57.


[93]

Duhlmeier R, Hacker A, Widdel A, von Engelhardt W, Sallmann HP. Mechanisms of insulin-dependent glucose transport into porcine and bovine skeletal muscle. Am J Physiol Regul Integr Comp Physiol. 2005;289:R187-97. Drackley JK. Biology of dairy cows during the transition period: the final frontier? J Dairy Sci. 1999;82:2259-73. Drackley JK, Overton TR, Douglas GN. Adaptations of glucose and long-chain fatty acid metabolism in liver of dairy cows during the periparturient period. J Dairy Sci. 2001;84(E. Suppl.): E100-E112. Eisemann JH, Huntington GB. Metabolite flux across portal-drained viscera, liver, and hindquarters of hyperinsulinemic, euglycemic beef steers. J Anim Sci. 1994;72:2919–29.

Engelking LR. Textbook of Veterinary Physiological Chemistry, 1st edition, Jackson, WY: Teton NewMedia; 2004.

El-maghrabi MR, Lange AJ, Kümmel L, Pilkis SJ. The rat fructose-1,6-bisphosphatase gene. Structure and regulation of expression. J Biol Chem. 1991;266:2115-20.

Faulkner A, Pollock HT. Effects of glucagon and alpha- and beta-agonists on glycogenolysis and gluconeogenesis in isolated ovine hepatocytes. Biochim Biophys Acta. 1990;1052:229-34.

Filsell OH, Jarrett IG, Taylor PH, Keech DB. Effects of fasting, diabetes and glucocorticoids on gluconeogenic enzymes in the sheep. Biochim Biophys Acta. 1969;184:54-63.

Fisher LJ, Elliot JM. Effect of intravenous infusion of propionate or glucose on bovine milk composition. J Dairy Sci. 1966;49:826-9.

Freetly HC, Klindt J. Changes in gut and liver glucose, lactate, insulin, and oxygen flux in mature ewes during mesenteric or abdominal vena cava glucose infusion. J Nutr. 1996;126:924-32. Frobish RA, Davis CL. Effect of Abomasal infusions of glucose and propionate on milk yield and composition. J Dairy Sci. 1977;60:204-9. Fuhrmann H, Eulitz-Meder C, Geldermann H, Sallmann HP. Zur Evaluierung von Hormon- und Metabolitprofilen nach Infusion von Glucose, Propionat und Butyrat beim Rind. Berl Münch Tierärztl Wochenschr. 1989;102:188-93. Gäbel G, Aschenbach JR. Adaptation und Regulation resorptiver Prozesse im Gastrointestinaltrakt von Wiederkäuern. Übersichten in Tierernährung. 2004;32:149-81. Goff JP. Major advances in our understanding of nutritional influences on bovine health. J Dairy Sci. 2006;89:1292-301.

Goff JP, Horst RL. Physiological changes at parturition and their relationship to metabolic disorders. J Dairy Sci. 1997;80:1260-8. Gray GM. Starch digestion and absorption in nonruminants. J Nutr. 1992;122:172-7.


[94]

Greenfield RB, Cecava MJ, Donkin SS. Changes in mRNA expression for gluconeogenic enzymes in liver of dairy cattle during the transition to lactation. J Dairy Sci. 2000;83:1228-36. Grünberg W, Morin DE, Drackley JK, Barger AM, Constable PD. Effect of continuous intravenous administration of a 50% dextrose solution on phosphorus homeostasis in dairy cows. J Am Vet Med Assoc. 2006;229:413-20. Grummer RR. Etiology of lipid-related metabolic disorders in periparturient dairy cows. J Dairy Sci. 1993;76:3882-96. Harmon DL, McLeod KR Glucose uptake and regulation by intestinal tissues: Implications and whole-body energetic. J Anim Sci. 2001;79:E59-E72.

Hocquette JF, Abe H. Facilitative glucose transporters in livestock species. Reprod Nutr Dev. 2000;40:517-33.

Hocquette JF, Ortigues-Marty I, Pethick D, Herpin P, Fernandez X. Nutritional and hormonal regulation of energy metabolism in skeletal muscles of meat-producing animals. Livest Prod Sci. 1998;56:115–43.

Hod Y, Cook JS, Weldon SL, Short JM, Wynshaw-Boris A, Hanson RW. Differential expression of the genes for the mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase. Ann N Y Acad Sci. 1986;478:31-45.

Holst JJ. The physiology of glucagon-like peptide 1. Physiol Rev. 2007;87:1409-39.

Holtenius K, Agenäs S, Delavaud C, Chilliard Y. Effects of feeding intensity during the dry period. 2. Metabolic and hormonal responses. J Dairy Sci. 2003;86:883-91. Holtenius P, Holtenius K. A model to estimate insulin sensitivity in dairy cows. Acta Vet Scand. 2007;49:29. Hostettler-Allen RL, Tappy L, Blum JW. Insulin resistance, hyperglycemia, and glucosuria in intensively milk-fed calves. J Anim Sci. 1994;72:160–73.

Huntington GB. Starch utilization by ruminants: from basics to the bunk. J Anim Sci. 1997;75:852-67.

Huntington GB, Harmon DL, Richards CJ. Sites, rates, and limits of starch digestion and glucose metabolism in growing cattle. J Anim Sci. 2006;84 Suppl:E14-24.

Hurtaud C, Lemosquet S, Rulquin H. Effect of graded duodenal infusions of glucose on yield and composition of milk from dairy cows. 2. Diets based on grass silage. J Dairy Sci. 2000;83:2952-62.

Hurtaud C, Rulquin H, Verite R. Effects of graded duodenal infusions of glucose on yield and composition of milk from dairy cows. 1. Diets based on corn silage. J Dairy Sci. 1998;81:3239-47.


[95]

Ingle DL, Bauman DE, Garrigus US. Lipogenesis in the ruminant: in vitro study of tissue sites, carbon source and reducing equivalent generation for fatty acid synthesis. J Nutr. 1972;102:609-16.

Jahoor F, Wolfe RR. Regulation of urea production by glucose infusion in vivo. Am J Physiol. 1987;253:E543-50.

Janes AN, Weekes TE, Armstrong DG. Absorption and metabolism of glucose by the mesenteric-drained viscera of sheep fed on dried-grass or ground, maize-based diets. Br J Nutr. 1985a;54:449-58.

Janes AN, Weekes TEC, Armstrong DG. Insulin action and glucose metabolism in sheep fed on dried-grass or ground, maize-based diets. Br J Nutr. 1985b;54:459-71.

Jesse BW, Emery RS, Thomas JW. Aspects of the regulation of long-chain fatty acid oxidation in bovine liver. J Dairy Sci. 1986a;69:2298-303.

Jesse BW, Emery RS, Thomas JW. Control of bovine hepatic fatty acid oxidation. J Dairy Sci. 1986b;69:2290-97.

Jitrapakdee S, Wallace JC. Structure, function and regulation of pyruvate carboxylase. Biochem J. 1999;340:1-16.

Judson GJ, Leng RA. Studies on the control of gluconeogenesis in sheep: effect of propionate, casein and butyrate infusions. Br J Nutr. 1973a;29:175-95. Judson GJ, Leng RA. Studies on the control of gluconeogenesis in sheep: effect of glucose infusion. Br J Nutr. 1973b;29:159-74.

Kaske M, Elmahdi B, Von Engelhardt W, Sallmann HP. Insulin responsiveness of sheep, ponies, miniature pigs and camels: results of hyperinsulinemic clamps using porcine insulin. J Comp Physiol [B]. 2001;171:549-56.

Kerner J, Hoppel C. Fatty acid import into mitochondria. Biochim Biophys Acta. 2000;1486:1-17.

Kim CH, Choung JJ, Chamberlain DG. The effects of intravenous administration of amino acids and glucose on the milk production of dairy cows consuming diets based on grass silage. Grass Forage Sci. 2000;55:173-80. Knowlton KF, Dawson TE, Glenn BP, Huntington GB, Erdman RA. Glucose metabolism and milk yield of cows infused abomasally or ruminally with starch. J Dairy Sci. 1998;81:3248-58.

Komatsu T, Itoh F, Kushibiki S, Hodate K. Changes in gene expression of glucose transporters in lactating and nonlactating cows. J Anim Sci. 2005;83:557-64. Krebs M, Roden M. Nutrient-induced insulin resistance in human skeletal muscle. Curr Med Chem. 2004;11:901-8.


[96]

Langhans W, Egli G, Scharrer E. Selective hepatic vagotomy eliminates the hypophagic effect of different metabolites. J Auton Nerv Syst. 1985;13:255-62.

Larsen M, Kristensen NB. Effect of abomasal glucose infusion on splanchnic and whole-body glucose metabolism in periparturient dairy cows. J Dairy Sci. 2009;92:1071-83.

Lemaigre FP, Rousseau GG. Transcriptional control of genes that regulate glycolysis and gluconeogenesis in adult liver. Biochem J. 1994;303:1-14. Lemosquet S, Rideau N, Rulquin H, Faverdin P, Simon J, Verite R. Effects of a duodenal glucose infusion on the relationship between plasma concentrations of glucose and insulin in dairy cows. J Dairy Sci. 1997;80:2854-65. Lemosquet S, Rigout S, Bach A, Rulquin H, Blum JW. Glucose metabolism in lactating cows in response to isoenergetic infusions of propionic acid or duodenal glucose. J Dairy Sci. 2004;87:1767-77.

Lindsay DB. Metabolism in the whole animal. Proc Nutr Soc. 1979;38:295-301.

Linzell JL, Peaker M. Mechanism of milk secretion. Physiol Rev. 1971;51:564-97.

Lomax MA, Baird GD, Mallinson CB, Symonds HW. Differences between lactating and non-lactating dairy cows in concentration and secretion rate of insulin. Biochem J. 1979;180:281-9. Loor JJ, Dann HM, Guretzky NA, Everts RE, Oliveira R, Green CA, Litherland NB, Rodriguez-Zas SL, Lewin HA, Drackley JK. Plane of nutrition prepartum alters hepatic gene expression and function in dairy cows as assessed by longitudinal transcript and metabolic profiling. Physiol Genomics. 2006;27:29-41.

Mackle TR, Dwyer DA, Ingvartsen KL, Chouinard PY, Ross DA, Bauman DE. Effects of insulin and postruminal supply of protein on use of amino acids by the mammary gland for milk protein synthesis. J Dairy Sci. 2000;83:93-105. Martens H. The dairy cow: Physiological facts and concerns. 13th international conference: Production diseases in farm animals; 2007 29th July-4th august; Leipzig, Germany; 2007. Matras J, Preston RL. The role of glucose infusion on the metabolism of nitrogen in ruminants. J Anim Sci. 1989;67:1642-7.

McAtee JW, Trenkle A. Metabolic regulation of plasma insulin levels in cattle. J Anim Sci. 1971;33:438-42.

McClymont GL, Vallance S. Depression of blood glycerides and milk fat synthesis by glucose infusion. Proc Nutr Soc. 1962;21:xli-xlii McDowell GH. Hormonal control of glucose homoeostasis in ruminants. Proc Nutr Soc. 1983;42:149-67.


[97]

Murondoti A, Jorritsma R, Beynen AC, Wensing T, Geelen MJ. Activities of the enzymes of hepatic gluconeogenesis in periparturient dairy cows with induced fatty liver. J Dairy Res. 2004;71:129-34. Nafikov RA, Beitz DC. Carbohydrate and lipid metabolism in farm animals. J Nutr. 2007;137:702-5.

Niijima A. Glucose-sensitive afferent nerve fibers in the liver and their role in food intake and blood glucose regulation. J Auton Nerv Sys. 1983;9:207-20.

Nocek JE, Tamminga S. Site of digestion of starch in the gastrointestinal tract of dairy cows and its effect on milk yield and composition. J Dairy Sci. 1991;74:3598-629.

Nordlie RC, Foster JD, Lange AJ. Regulation of glucose production by the liver. Annu Rev Nutr. 1999;19:379-406. Obitsu T, Bremner D, Milne E, Lobley GE. Effect of abomasal glucose infusion on alanine metabolism and urea production in sheep. Br J Nutr. 2000;84:157-63.

Ørskov ER. Starch Digestion and Utilization in Ruminants. J Anim Sci. 1986;63:1624-33.

Ortigues-Marty I, Vernet J, Majdoub L. Whole body glucose turnover in growing and non-productive adult ruminants: meta-analysis and review. Reprod Nutr Dev. 2003;43:371-83.

Owens FN. Zinn RA. Kim YK. Limits to starch digestion in the ruminant small intestine. J Anim Sci. 1986;63:1634-48. Pearce J, Unsworth EF. The effects of grass and concentrate diets on the specific activities of some enzymes of hepatic carbohydrate metabolism in sheep. Br J Nutr. 1976;35:407-11.

Pearce J, Unsworth EF. The effects of duodenal glucose infusion on some hepatic enzyme activities in sheep. Int J Biochem. 1982;14:545-7.

Pearce J, Piperova LS. The effects of duodenal glucose and dextrin infusion on adipose tissue metabolism in sheep. Comp Biochem Physiol B. 1984;78:565-7.

Pilkis SJ, El-Maghrabi MR, Claus TH. Hormonal regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Biochem. 1988;57:755-83. Perseghin G, Caumo A, Caloni M, Testolin G, Luzi L. Incorporation of the fasting plasma FFA concentration into quicki improves its association with insulin sensitivity in nonobese individuals. J Clin Endocrinol Metab. 2001;86:4776–81.

Prior RL, Scott RA. Effects of intravenous infusions of glucose, lactate, propionate or acetate on the induction of lipogenesis in bovine adipose tissue. J Nutr. 1980;110:2011-9. Rabasa-Lhoret R, Bastard JP, Jan V, Ducluzeau PH, Andreelli F, Guebre F, Bruzeau J, Louche-Pellisier C, Maitrepierre C, Peirat J, Chagné J, Vidal H, Laville M. Modified quantitative insulin sensitivity check index is better correlated to hyperinsulinemic glucose clamp than other fasting-based index of insulin sensitivity in different insulin-resistant states. J Clin Endocrinol Metab. 2003;88:4917–23.


[98]

Ramsay RR, Gandour RD, Van der Leij FR. Molecular enzymology of carnitine transfer and transport. Biochim Biophys Acta. 2001;1546:21-43.

Reynolds CK. Glucose balance in cattle. Proceedings of the 2005 Florida Ruminant Nutrition Conference; 2005 February 1-2; Gainesville, Florida; 2005.

Reynolds CK, Harmon DL, Cecava MJ. Absorption and delivery of nutrients for milk protein synthesis by portal-drained viscera. J Dairy Sci. 1994;77:2787-808. Reynolds CK, Huntington GB, Tyrrell HF, Reynolds PJ. Net portal-drained visceral and hepatic metabolism of glucose, L-lactate, and nitrogenous compounds in lactating holstein cows. J Dairy Sci. 1988;71:1803-12.

Rigout S, Lemosquet S, Bach A, Blum JW, Rulquin H. Duodenal infusion of glucose decreases milk fat production in grass silage-fed dairy cows. J Dairy Sci. 2002a;85:2541-50. Rigout S, Lemosquet S, Van Eys JE, Blum JW, Rulquin H. Duodenal glucose increases glucose fluxes and lactose synthesis in grass silage-fed dairy cows. J Dairy Sci. 2002b;85:595-606. Rulquin H, Rigout S, Lemosquet S and Bach A. Infusion of glucose directs circulating amino acids to the mammary gland in well-fed dairy cows. J Dairy Sci. 2004;87:340-9. Rose MT, Itoh F, Matsumoto M, Takahashi Y, Obara Y. Insulin-independent glucose uptake in growth hormone treated dairy cows. J Dairy Res. 1998;65:423-31. Ross JP, Kitts WD. Relationship between postprandial plasma volatile fatty acids, glucose and insulin levels in sheep fed different feeds. J Nutr. 1973;103:488-93.

Rukkwamsuk T, Wensing T, Geelen MJ. Effect of fatty liver on hepatic gluconeogenesis in periparturient dairy cows. J Dairy Sci. 1999;82:500-5. Rulquin H, Rigout S, Lemosquet S, Bach A. Infusion of glucose directs circulating amino acids to the mammary gland in well-fed dairy cows. J Dairy Sci. 2004;87:340-9.

Sasaki SI. Mechanism of insulin action on glucose metabolism in ruminants. Anim Sci J. 2002;73:423-33.

She P, Linberg GL, Hippen AR, Beitz DC, Young JW. Regulation of messenger ribonucleic acid expression for gluconeogenic enzymes during glucagon infusions into lactating cows. J Dairy Sci. 1999;82:1153–63. Schröder UJ, Staufenbiel R. Invited review: Methods to determine body fat reserves in the dairy cow with special regard to ultrasonographic measurement of backfat thickness. J Dairy Sci. 2006;89:1-14. Thompson JR, Weiser G, Seto K, Black AL. Effect of glucose load on synthesis of plasma glucose in lactating cows. J Dairy Sci. 1975;58:362-70. Treacher RJ, Baird GD, Young JL. Anti-ketogenic effect of glucose in the lactating cow deprived of food. Biochem J. 1976;158:127-34.


[99]

Tutwiler GF, Dellevigne P. Action of the oral hypoglycemic agent 2-tetradecylglycidic acid on hepatic fatty acid oxidation and gluconeogenesis. J Biol Chem. 1979. 25;254:2935-41. Yáñez AJ Garcia-Rocha M, Bertinat R, Droppelmann C, Concha II, Guinovart JJ, Slebe JC. Subcellular localization of liver FBPase is modulated by metabolic conditions. FEBS Lett. 2004;577:154-158. Young JW. Gluconeogenesis in cattle: significance and methodology. J Dairy Sci. 1977;60:1-15. Vanhatalo A, Varvikko T, Huhtanen P. Effects of casein and glucose on responses of cows fed diets based on restrictively fermented grass silage. J Dairy Sci. 86;2003:3260-70. Van Schaftingen E, Gerin I. The glucose-6-phosphatase system. Biochem J. 2002;362:513-32. Veenhuizen JJ, Russell RW, Young JW. Kinetics of metabolism of glucose, propionate and CO2 in steers as affected by injecting phlorizin and feeding propionate. J Nutr. 1988;118:1366-75. Vernon RG. Lipid metabolism in the adipose tissue of ruminant animals. Prog Lipid Res. 1980;19:23-106. Vik-mo L, Huber JT, Bergen WG, Lichtenwalner RE, Emery RS. Blood Metabolites in Cows Abomasally Infused with Casein or Glucose. J Dairy Sci.1974;57:1024-30.

Williams EL, Rodriguez SM, Beitz DC, Donkin SS. Effects of short-term glucagon administration on gluconeogenic enzymes in the liver of midlactation dairy cows. J Dairy Sci. 2006;89:693-703.

Wray-Cahen D, Metcalf JA, Backwell FR, Bequette BJ, Brown DS, Sutton JD, Lobley GE. Hepatic response to increased exogenous supply of plasma amino acids by infusion into the mesenteric vein of Holstein-Friesian cows in late gestation. Br J Nutr. 1997;78:913-30. Zammit VA. Carnitine acyltransferases: functional significance of subcellular distribution and membrane topology. Prog Lipid Res. 1999;38:199-224.

Zhao FQ, Glimm DR, Kennelly JJ. Distribution of mammalian facilitative glucose transporter messenger RNA in bovine tissues. Int J Biochem. 1993;25:1897-903.

ACKNOWLEDGEMENTS

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Acknowledgements

First of all, all praise and thanks are to Allah, God the Almighty, most beneficent and most merciful. The best and worst moments of my doctoral dissertation journey have been shared with many people. It has been a great opportunity to spend several years at the Institute of Veterinary Physiology, University of Leipzig. It really was a place where I constantly felt inspired by the intelligence and humanity surrounding me. Therefore, its members will always remain dear to me. I would like to express my sincere gratitude and appreciation to my advisor Prof. Dr. Gotthold Gäbel for his support and guidance throughout the period of this work. My very special thanks go to the person who deserving most of the acknowledgments, Prof. Dr. Jörg R. Aschenbach, University of Veterinary Medicine, Vienna. I would like to thank him for his support, friendship, encouragement and guides in every step of my thesis. I consider myself very lucky to have had the opportunity to work with him. I am indebted to him more than he knows. I would like to express my deepest thanks to Anke, Ines and Petra, the great and efficient laboratory staff of the Institute of Veterinary Physiology. This study could not have been completed without their hard work and guidance. Many thanks go also to the Institute secretary, Jana Kirchner. Thanks Jana for everything. My special thanks go to my colleagues at the Institute, Kerstin, Carola, Reiko, Marianne, Thomas, Firas and all other graduate students and staff for being not only excellent colleagues but also for their help and support on several occasions. Thanks go also to Prof. Dr. R. Cermak, PD Dr. H. Pfannkuche and Dr. K. Honscha for their assistance and support. This thesis owes very much to a number of people who are my co-authors or took the time for technical help. I want to thank Prof. Dr. Manfred Fürll, Dr. Thomas Wittek and Dr. Ahmed Alkaassem for their valuable help during samples collections and manuscript preparations. I am very grateful to Dr. Gregory Penner, University of Saskatchewan, Canada for his help when performing the statistical analysis and his helpful comments during the preparation of Manuscripts I and II. I wish to thank Prof. Dr. Gerald Schusser, Carsten Benson and the staff of Clinic for Large Animal Internal Medicine, University of Leipzig for their support during the experiments. My thanks go also to the staff of the Institute of Biochemistry for their help with real-time PCR experiments and Pfizer Animal Health, UK, for their financial support. My special thanks go to my family. My sisters and brothers were always heartening and encouraging. Most of all to my late father and to my mother, who introduced me to science, taught me the value of the hard work and gave endless support along the way.

Finally, to those who did not feel appreciated at that time – I apologize. Leipzig 2010 Bahaa Al-Trad

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