-
-AI76 796 DIFFERENTIAL EFFECTS OF GLUCAGON INSULIN
AND1/EPINEPHRINE ON IN VIVO GLUCO..(U) LETTERMAN ARMY INSTOF
RESEARCH PRESIDIO OF SAN FRANCISCO CA
UNCLASSIFIED G J KLAIN ET AL OCT 86 LAIR-226 F/G 6/1 NL
EEEEEEEEEEEEEEEEEEEEEEEEEEI
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11111 .0 ~ 8
11112 1 1.
MICROCOPY RESOLUTION TEST CHARTNATIONAL BUREAU OF
STANDARDS-1963-A
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* INSTITUTE REPORT NO. 226
,* DIFFERENTIAL EFFECTS OF GLUCAGON, INSULIN AND( EPINEPHRINE ON
IN VIVO GLUCOSE OXIDATION AND HEPATICN ENZYME ACTIVITY IN THE
RAT
GEORGE J. KLAIN, PhDand
ROBERT H. HEIMAN, 00L, MC,
DIVISION OF BIOCHEMISTRY. DEPARTMENT OF NUTRITION
ANDDEPARTMENT OF MEDICINE
DTICi" 'q S ELECTE m.S JAN 2 0 1987
October 1986
LETTERMAN ARMY INSTITUTE OF RESEARCH
PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129
Aproved Lor Pu'bli c ,Ditubutiom unlimited 0
81 ' 03
-
Differential effects of glucagon, insulin and epinephrine on in
vivoglucose oxidation and hepatic enzyme activity in the rat--Klain
and Herman
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INSTITUTE REPORT NO. 22 1 T14. TITLE (and Subtitle) S. TYPE OF
REPORT A PERIOD COVERED
DIFFERENTIAL EFFECTS OF GLLICAGON, INSULIN AND
I1NAT,EPINEPHIRINE ON IN VIVO GLUCOSE OXIDATION AND Jan 1)79 - Nov
1979HEPATIC ENZYME ACTIVITY IN THE RAT. 6. PERFORMING ORG. REPORT
NUMBER
7. AUTHORll 8. CONTRACT OR GRANT NUMBER(s)
George J. Klaln, Ph.D.
Robert H. Herman, Col, MC
9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT.
PROJECT. TASKAREA & WORK UNIT NUMBERS
Letterman Army Institute of Research 62760A, 3A762760A822 100,
075Presidio of San Francisco, CA., 94129-6800
II. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATEUS Army
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Maryland 21701 13. NUMB3ER OF PAGES
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IS. DISTRI19UTION STATEMENT (of this Report)
\ppj v, > 10o- [JV I iC reCl C;O d li StI riblt ion) i S Lil
ii jI1 .
17 'SRI.TICH SI ATEMENT (of the abstract entered in, Black 20.
If different from Ropor)
IS. SUPPLEMENTARY NOTES
IS. KEY WORDS (Continue. on, revrse side if nevegewry ad
Identify by bfock numnber)
.nsu! ini, Cl uicagon , Fninieplie, Cl UCose Oxidat ion, I';at
Iic Enzvmes, Rat.
2.ASTRACT (oatelo eveve efda if na~m andentity by bile
nuiller
after the administration of glucagon, insulin y~ epinephrine.
Compared to thecoyhrol s, glucagon J~ubled the oxidat ion of U-
(-glucose an4 enhanced
6- CgI uos~or 2 C-g u)st o)xidat ion by about 83;,, but did not
af fect oxida ~n of I- C-glucose. 1 Administration of invl in
enhanced oxidation oft- C -g ILICOS 4 by 68", 1- (;-gliiCose by
71'/, 2- i-gl ntosc by 73?/, with nioeffect on 6- C-glucose
oxidation. Eprinephrine enh-inced oxidation (If
DD IM? 17 EDITION OF I NOVGS IS OBSOLETETW i~.jjeSECURITY CL.
ASSIFIC ATION Of' THIS PAr(Ill cm,.., rel. n-d)
all\~-gc~
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UnclassifiedSECURITY CLASSIFICATION OF THIS PAG;(1Ia Data
Enemfo
6 14C-glucose by 38% and had no effect on I Or 2- 14C-glucose
oxidation.
Insulin and glucagon produced rapid reciprocal changes in the
activities ofcertain glycolytic and gluconeogenic enzymes and in
the activity of acetylCoA carboxylase, a key lipogenic enzyme.
Insulin produced a rapid increasein hepatic glucokinase,
phosphofructokinase, pyruvate kinase and gliicosc-6-phosphate
dehydrogenase, and a rapid decrease in gluclose-6-phosphaLsC andand
fructose-i, 6-diphosphatase. Glucagon produced a rapid but
reciprocalresponse in the activity of these enzymes. In addition,
giu-agon enhancedthe activity of phosphoenolpyruvate carboxykinase.
Epinephrine effects weresimilar to those produced by glucagon. Both
glucagon and epinephrine increasedthe level of cyclic AMP. The
results of this study suggest that glucagon andepinephrine
stimulate the activity of the tricarboxylic acid cycle,
whereasinsulin enhances the pentose cycle. It appears that in vivo
glucose utilizationdepends on the relative concentrations of
glucagon, insulin and epinephrine inthe target issues.
Unc lass i flied
SECURITY CLASSIFICATION OF THIS PAGE(WPgen DOt Enter*d)
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ABSTRACT . o W-
i[n vivo oxidation of specifically labeled "14-glucose and the
activity ofselected hepatic enzymes involved in glucose utilization
were studied in ratsafter the administration of glucagon, insulin
or epinephrine. Compared to thecontrols, glucayon doubled the
oxidation of U-l C-glucose and enhanced theoxidation of
6-44-glucose and 2-1T-glucose oxidation by about 83%, but didnot
affect oxidation of l-i-4-glucose. Administration of insulin
enhancedoxidation of U-144C-glucose by 68%, l-ljC-glucose by 71%,
and 2-1 C-glucose by73%, with no effect on 6-1 C-glucose oxidation.
Epinephrine enhancedoxidation of 6- 4AC-glucose by 38% and had no
effect on I- or 2-1C-glucoseoxidation.
Insulin and glucagon produced rapid reciprocal changes in the
activitiesof certain glycolytic and gluconeogenic enzymes and in
the activity of acetylCoA carboxylase, a key lipogenic enzyme.
Insulin produced a rapid increase inthe activity of hepatic
glucokinase, phosphofructokinase, pyruvate kinase
andglucose-u-phosphate dehydrogenase, and a rapid dux-rease in the
activity ofglucose-6-phosphatase and fructose-l,6-diphosphatase.
Glucagon produced arapid but reciprocal response in the activity of
these enzymes. In addition,glucagon enhanced the activity of
phosphoenolpyruvate carboxykinase. Theeffects of epinephrine were
similar to those p- uced by glucagon. Bothglucagon and epinephrine
increase:d the level of cyclic AMP. The res-its o[this study
suggest that rjucagon and epinephrine stic'iate the 3ctivity of
the(tricarboxylic acid cycle, whereas insulin enhances tile pentose
cycle Itap[petr s that in vivo glucose utilization depends on the
retiveconcentrations of glucagon, insulin and epinephrine in the
target tissues\
(.Ccezion For
fNTIS CRAM
UTIC TABU:annoutrced [JJ~tfica ton
ByDj_-t ibition /
Availability CodesAvail andlor
Dist Special
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PREFACE
A canprehensive study to delineate metabolic alterations
underlyingpathologic and nutritional hypoglycenia in humans was
initiated in 1979 in the
Departments of Nitrition and of Medicine, LAIR. However, due to
changes inthe research program and the mission of the Institute,
only the preliminaryphase of the study was canpleted. The initial
phase was concerned withhormonal effects on glucose metabolisn in
the laboratory rat. The results arepresented in the following
report.
S b
ii I
-S %' - ~ S ~
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TABLE OF coturENT.S
Preface
.......................................................... ii
Table of contents
................................................ iii
Introduction
..................................................... 1
Material and methods
............................................. 3
Animals and Anesthesia ......................................
3
Hormones and 1 4C-glucose Administration .....................
3
Hormonal Effects on Plasma Glucose Levels ...................
3
Hormonal Effects on Glucose oxidation .......................
3
Hformonal Effects on Enzyme Activity .........................
4
Results
.......................................................... 5
The Hormones ar.d Plasma Glucose Levels ......................
5
TIhe fHorones -ind Glucose2 Oxidation ..........................
5
!.sc2 hOLmones andZoym Activity - Glucayon .................
6
TVie liior-nones and Fiizyme Activity - Insulin
.................. 6
The Horrpnnes and Enzyme Activity - Epinephrint ...............
7
Disi~s'~.......................................................8
ConcLlUSions)6......................................................
11
Recoinnda2--ions
.................................................. 12
References
....................................................... 13
Apjx ,nJix
......................................................... 17
Tihiet I Effect of Glucaqon, Insulin and rEPinephrine on
Plasma
ITji~h 2 I-A fIset Of (;1iiC,-1jori 01)d Insulin on Glucos.eOx
Wdit ion.....................................................
19
-
Table 3 Effect of Epinephrine on Glucose Oxidation .........
20
Table 4 Effect of Glucagon on l- 1 4 C-Glucose and 6- 1 4
C--GlucoseOxidation in Fed and Fasted Rats
............................ 21
Table 5 Effect of Glucagon on the Activity Of Hepdtic Enzymesand
Cyclic AMP .............................................. 22
Table 6 Effect of insulin on the Activity of Hepatic Enzymesand
Cyclic AMP .............................................. 23
Table 7 Effect of Epinephrine on the Activity of HepaticEnzymes
and Cyclic AMP ......................................24
Figure 1 Metabolism of Glucose via the Glycolytic and
PentosePhosphate Pathways
..........................................25
Figure 2 Insulin and Enzyme Activity ........................
26
Figure 3 Glucagon and Enzyme Activity .......................
27
Figure 4 Epinephrine and Enzyme Activity ....................
28
iv
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DIFFERENTIAL EFFECTS OF GLUCAGON, INSULIN AND EPINEPHRINE ON
INVIVO GLUCOSE OXIDATION AND HEPATIC ENZYME ACTIVITY IN THE
RAT--Klain
I NTRODUCTI ON
Glucose is an optional substrate for tissues such as muscle,
adipose orliver as these tissues can also use fatty acids to
satisfy their energy necds.Glucose, however, is an obligate
substrate for the central nervous system,since under physiological
conditions, alternative substrates either areexcluded by the
blood-brain barrier or circulate at concentrations too low tobe
taken up in substantial quantities. Because the brain can
neithersynthesize nor store adequate supply of glucose, normal
cerebral functionrequires a continuous supply of glucose from the
circulation. Consequently,maintenance of the plasma glucose
concentration above some critical level isessential to the survival
of the brain and thus, the organisri.
Plasma glucose levels in the mamnalian or-±-.i:-n are m nt-in",
byseries of coordinated enzy.atic reactions invoi'in7 h*'-tic
synt> sis 3ndbreakdown of glyccjen. Many of the reactions are
reversible and cam-on to thesynthetic _rd degradative pathways.
These reactions are always close toequiltbriun so that the rate and
direction of flow of glucose carbons can bechan od by variatio-s in
the concentrations of substrates and/or products oft!k r, ct ii. Ih
_-ver , sore re icltions of tie i lycoly*Lc [mthway art,displact I
foE Lroin Qc;ullibrium. They relt~ise a counsidr fi! , imount of
energyas tieat, , 1nd tsr,?fore, annot be easily revel s .. To
un:;ut: the ]owsubstrates in the reverse reaction, etergy barrmrs
,rc bpu:: d uv uti rreactions which are also irreversible and are
catalyzed by different enzymes.The antgjs,-intic reactions in the
qlycolytic and thie gluconeojeni- pathwaysoper-te simultaneously
but at different rates. n2 irreversible reactionsbetween glucose
and pyruvate are: (a) the interconversion of glucose
andglucose-6-phosphate, catalyzed by glucokinase and
glucose-6-phosphatase; (b)the interconversion between
fructose-6-phosphate and fructose-diphosphatewhere
phosphofructokinase is opposed by fructose-Jdphosphatase and (c)
twoseparate reactions at the pyruvate kinase bypass. Pyruvate is
carboxylated tooxaloacetate in a reaction involving ATP hydrolysis
and catalyzed by pyruvatecarboxylase. Conversion of oxaloacetate to
phosphoenolpyravate involves adecarboxylation and phosphate
transfer from either guanosine triphosphate orinosine
5'-triphosphate, which is catalyzed by
phosphoenolpyruvatecarboxykinase.
'V'V
-
Klai---2
The activity of these key enzymes may be modulated by various
metibolicmechanisms, including the action of hormones. Hormonal
effects may be short-term or long-term. The former may be achieved
by affecting enzyme activitywithout changing the amount of enzyme
present. The latter may meet thefunctional needs of the organism by
producing additional enzyme bybiosynthesis and by changing the
degradation rates. Tnese metabolic controlmechanisms enable the
organism to remove and conserve glucose efficiently fromthe blood
stream when the intake exceeds the demand and to rapidly
formglucose from non-carbohydrate sources when the need arises. The
main hormones
affecting the key enzymes of glucose metabolism are insulin,
glucagon andepinephrine, and on a slower time scale, the growth
hormone and adrenal
corticoids (1).
In the course of glucose metabolism via the major metabolic
pathways,specific glucose carbons can be oxidized and expired as CO
Theseobservations have been utilized to estimate the contribution
of the pentosecycle to glucose metabolism (2). Several studies have
demonstrated that thehormones have marked effects on glucose
oxidation and that these effects areassociated with specific tisues
or organs. Thus, glucagon reduces U-C 1 4 -glucose oxidation both
in perfused rat liver (3,4) and in rat liver slices(5,6) but
stimulates glucose oxidation in perfused rat heart (7). The
hormonehas no effect on glucose oxidation in adipose tissue (8). In
contrast toglucagon, insulin consistently stimulates glucose
oxidation in several
systems. The hormone enhances U-Ci -glucose oxidation in rat
epididymaladipose tissue (9), but oxidation of C-1 of glucose is
stimulated to a greater
extent than oxidation of C-6 (10,11). Similar observations were
reported instudies with mammary gland preparations (12).
Administration of insulin todiabetic rats restores hepatic
oxidation of l-Cl 4 -glucose and 6-C 1 4 -glucose tothe level
observed in control rats (13). The hormone also stimulatesoxidation
of U-Cl 4 -glucose in rat diaphragm preparations (14,15).
Exogenousinsulin temporarily enhances in vivo oxidation of
U-Cl4-glucose in fed,fasted, or refed rats (16,17) and of l-Cl
4-glucose in fed rats (18).Epinephrine decreases U-C 1 4 -glucose
oxidation in liver slices from fed,fasted, or refed rats (5). No
epinephrine effect was observed in ratthymocytes (19).
Beyon' these observations, little is known about the impact of
the threehormones on glucose oxidation in the intact animal. Since
these hormonesregulate the major metabolic pathways of glucose, we
hypothesized that the
hormones would have pronounced effects on in vivo oxidation of
specific
carbons of glucose molecules. Accordingly, in the present study,
we examine-dacute effects of insulin, glucagon and epinephrine on
oxidation of -C, 4 -
glucose, _ - 1 4 C, 2-1 4C ind 6-14C. In addition, icuto
hotnon.l ,selectxi strlt(N] ic hup) Itic glycolyt ic an-.] g
lucn(eYr(Jt1 ic (./I,determined.
'p
-
Klain--3
MATERIAL AND METHODS
Animals and Anesthesia - Male Holtzman rats weighing 270-300 gm
were ul-c3in all experiments. They were individually housed at 250C
in stainless steelwire cages and fed a conmercial laboratory rat
diet. Food and water wvk,available at all times, unless otherwise
indicated. The rats woreanesthetized with pentobarbital (50 mg/kg)
administered intraperitoneally andused 10 minutes later for further
experimentation. The degree of anesthesiaappeared uniform, as none
of the rats woke up and became active duringexperimentation.
Hormone and 14C-qlucose Administration - All hormnone solutions
and 14C-glucose dissolved in saline were administered via the tail
vein. Crystaillineglucagon (Eli Lilly and Co., Indianapolis, Ind)
was dissolved (l mg/ml) in themanufacturer's diluent which
contained 14 mg/ml lactose, 1.6i glycerol, and0.2% phenol. Insulin
solution (IletinR, 40 U/ml) was further diluted, andepinephrine
bitartrate was dissolved in sterile saline. Where
applicable,glucagon was administered first, followed i:mreiatse; -
P. .- rt,
% I
" ""." , " "" "
-
Klain--4
Ho rmonil F f t urF.Ymt- A' ivi ty - Aft,,r t-o *ilxhznn
w,;q)ndIpruximltely 50 m- u t :i'z , i w t-: 1~-z iiviw i ,i I I ,
Vifc'9 (yr o t im)t
-coo ied in ice-col l nod A S'm jI I piecce of iu.'" Wi- ;
1atIo.rth*incision and kept mo. -;t 41!, sA ile to prey. .;it
(ityingj ot the sur face o1 Lhe
l iver . Additional liver smples wr- collected 5, 10, 20 and 40
minutes afterthe hormonal injection. All 1 i%.'er s;-.ples were
r-,n Icxny taiken fromi di ffterontper ipheral sites of tn- c r,
I~( . o sci,,plts cot)t i j uouo t o ipL ev iOLI3 !;.imp I r,,site
were taken. Ten percent liver haog~enat.-s reprepired in 0.25 M
2~ sucrose solution, isin3 ir; .1l-qiass tissue hogun-izer. Art
a' iuot of thehoinoyenate was centrifui~i ftr 3D minutes at
1011,000 x g in a refrigeratedcentrifuge. Enz,,.7e- ass s .o.,rt--
performed on the diiluted homogenate or on aclear supernatint fli.
Tt- following me thod s were applied for thedeter-mi nat ion of
enzyme i--iv.- ies: yglucose-6-phosph-ita3se, the method of
Coriarnd Cori (21); glicokins, the method of Vinuela et al (22);
fructose-1,6-diphosohatdise, the mtwo! RiC~er (23);
phosphofrictokina-se, the method ofLea ar~d WalIke r (2 4);
pytruvite k ina-jse, the methodI of Bucher and Pfleiderer(25);
phosphoenolpri'ut.- cairt:9o'
-
Klain--5
RESULTS
The Hormones and Plasma Glucose Levels - From the zero time to 5
minutes
after administration of glucagon, the glucose level increased by
approximately48% (Table 1). The maximal effect occurred 10 minutes
after glucagonadministration. A marked hyperglycemia was maintained
for an additional 30minutes. The glucose level 5 minutes after
insulin administration decreasedby about 52%, when compared to that
at the zero time. Thereafter, severehypoglycemia was observed
throughout the entire 40-minute experimental period.Plasma glucose
levels increased by approximately 25% in 5 minutes afterepinephrine
administration. Again, the levels renained elevated during
theentire experimental period.
The Hormones and Glucose Oxidation - In general, the effects of
glucagonand insulin on the oxidation of various glucose carbons
were rapid. Theeffects were apparent 5 minutes after an
administration of either hormone, andpersisted throughout the
entire experimental period (Table 2). In comparisonto the controls,
glucagon doubled the 14CO2 rodu * ior from U-14C-glucose, andilsul
in increasd oxidation by about 680 8 uriui the 40-minute
experimentalperioxI. When giucagon and insulin were adminit.ered in
combination, r.,additi,, effect on glucose oxidation was observwd o
Tl;b, 2). The ef[ect oeach norrnone on plasna glucose was as
expected: glucagon increased theglucose level and insulin decreased
it. Insulin, when given together withg li'l.aj(n, reduced the
glucose level.
GlicIgon had no significant effect on 1l-l'C-glucose oxiiation.
Incobt~tra3t, irsulin enhanced oxidation by about 71% over the
control values.effect by insulin was observed when glucagon and
insulin were aministered v.co-[1ination, indicating that glucagon
reversed insulin's effect on 1-14C-glucose oxidation. Each hormone
enhanced oxidation of 2- 14C-glucose, glucagornby 8out 30% and
insulin by about 68%, when con.i --I1 to th3 contr.)! values.No
further effect was observed when the hortrones were administered
incombination. Glucdgon increased oxidation of 6-14C-glucose by 83%
over thecontrols; insulin had no effect. Glucose oxidation remained
elevated evenafter administration of both hormones. Thus, in:;ul in
did not overconeglt',ijon effect on 6- 1 4 C-glucoso oxidation. In
qenural, the effects of thetwo hormones on the oxidation of various
glucose± carbons wx-re rapid. Theeffects were apparent 5 minutes
after an administration of either hormone, andpersisted throughout
the entire experimental period.
4
* J
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Klain--6
Table 3 suTruarizes the effect of epinephrine on the oxidation
of specificcarbons of glucose. Compared to the control values,
epinephrine did notaffect 1-1 4C- or 2-1 4C-glucose oxidation. The
hormone did enhance oxidationof 6-14C-glucose by about 27% over the
controls. For each instance, however,the hormone increased plasna
glucose levels by approximately 22% over thecontrol values.
14 The data in Table 4 show that fasting alone decreased the
oxidation of 1-C-glucose by about 57% when campared to data for the
fed animals (Group 3 vs
Group 1). Glucagon had no effect on the oxidation of
1-14C-glucose either inthe control or in the fasting rats. Fasting
animals depressed the oxidationof 6- 1 4 C-glucose by approximately
97% when compared to fed animals (Group 7vs. Group 5). As expected,
glucagon enhanced 6- 1 4C-glucose oxidation in fedrats (Group 6 vs.
Group 5). The hormone also stimulated 6- 1 4C-glucoseoxidation in
fasted rats (Group 8 vs. Group 7). However, this effect was
lesspronounced than the effect observed in fed rats. Glucagon
increased plasmaglucose levels in fed rats. In contrast, the
hormones had no eftct onglucose levels in fasting rats.
The Hormones and Enzyme Activity - Glucagon. Within 5 minutes
ifterinjection of the hormone, there was a significant increase in
the activitiesof the key gluconeogenic enzymes,
glucose-6-phosphatase, fructose-l,6-diphosphatase and
phosphoenolpyruvate carboxykinase (Table 5). In contrast,the
hormone reduced the activities of two key glycolytic enzymes,
pyruvatekinase and phosphofructokinase and the activities of
glucokinase and acetylCoA carboxylase. The effect of glucagon
persisted for at leist fortyminutes. The activity of
glucose-6-phosphate dehydrogenase did not change.Five minutes after
glucagon administration, a fourfold increase in theconcentration of
cyclic AMP was observed. This effect persisted throughoutthe entire
40-minute exp rimental period.
The Hormones and Enzyme Activit - Insulin The honnorie produce~d
cingein enzyme activity reciprocal to those produced by glcagon
(Table 6).GLucokinase, phosphofructok ianse, pyruvate kinase,
glucosk-6-pho.sphattedehydrogenase and acetyl CoA carboxylase
activity increased significantlywithin five minutes of insulin
administration. In contrast., glucos,2-6-phosphatase and
fructose-l,6-diphosphatase activity dec-re.ised. The insulineffect
persisted for at least 20 minutes. The hormone had no effect on
thelevels of cyclic AMP or the activity of phosphoenolpyruvate
carboxykinase.
-
Klain--7
The Hormones and Enzyme Activity - Epinephrine -
Epinephrineadministration produced a rapid, significant increase in
the activity offructose-l,6-diphosphatase and phosphoenolpyruvate
carboxykinase (Table 7).The hormone reduced the activity of
glucokinase, phosphofructokinase, pyruvatekinase and acetyl CoA
carboxylase. The effect of epinephrine persisted forat least 10
minutes. The hormone had no effect on the activity of
glucose-6-phosphatase or glucose-6-phosphate dehydrogenase. Five
and ten minutes afterepinephrine administration, a twofold increase
in the level of cyclic AMP wasobservd.
-
Klain--8
DISCUSSION
There are many difficulies inherent in interpreting pathways of
glucosemetabolism on the basis of CO production from specifically
labeled glucosein a metabolically active single tissue (2). These
difficulties are magnifiedenormously when one attempts to interpret
a similar set of data from in vivostudies. It is well established
that administration of any of the threehormones evokes compensatory
changes in systemic levels of other hormone:s andmetabolites, which
in turn, markedly alter a number of metabolic parameters(33). Thus,
during the 40-minute experimental period, a variety of
compensatory changes may have taken place which could have
complicated theinterpretation of the observed changes. Furthermore,
thr contribution ofindividual organs or tissues to the overall
production of 4CO2 is unknown.Presumably, the most significant
hormonal effects occurred in the liver,muscle, and kidney.
In the course of 14C-glucose catabolism through the glycolytic
pathway,pyruvate can enter the tricarboxylic acid (TCA) cycle via
two differentpathways: (a) by condensation with 0 2 , catalyzed by
pyruvate carboxylase, toform oxaloacetate, or (b) by oxidative
decarboxylation, catalyzed by pyruvatedehydrogenase, to form acetyl
CoA. In the latter step C-3 and C-4 of glucoseare released as h 4Ce
. In the ICA cycle, isocitric dehydrogenase and alpha-ketoglutaric
dehydro~nase release C-2 and C-5 of glucose during the secondturn
of the cycle and C-1 and C-6 during the third and subsequent turns.
Inthe conversion of oxaloacetate to phosphoenolpyruvate, catalyzed
byphosphoenolpyruvate carboxykinase, C-2 and C-5 are released as
14CO2 after thefirst turn of the cycle, but as C-I and C-6 in the
subsequent turns. In thepentose phosphate pathwayf catalyzed by
phosphogluconate dehydrogenase, C-1 of2ucose is released as 4CO. .
In the subsequent reactions, the remaining
CO2 is metabolized as Pyf uvate through the ICA cycle (Figure
1). The rateat any moment at which CO is produced is determinN by
the specificradioactivity of the precursgr carbon destined to form
*CO2 and the fluxthrough the decarboxylation reactions.
-
Klain--9
increased in vitro formation of 14C02 from C-6 relative to C-1
glucosehas been interpreted as increased activity of the TCA cycle
(2). Thus, theresults of our study suggest that glucagon enhances
the TCA cycle turnover.As the hormone reduces the activity of
acetyl CoA carboxylase, the key enzy-nein the synthetic pathway of
fatty acids, an increased flux of pyruvate throughthe TCA cycle
enhances C-6 oxidation. In the fasting animal, the
TCA-cycleturnover i ; markedly decreased and pyruvate enters the
cycle primarily asoxaloacetate (34), thus increasing the amount of
cycle intermediates. Withrespect to the economy of the starving
organism, increasing the amount of theTCA-cycle intermediates from
pyruvate and therefore, ul t ima tel y froncarbohydrate, makes
sense, since these intermdxiatos can be uscd for synthesis
minino acid and proteins. This process further dilutes the
radioactivity inCO2 . The fed rat has no need to manufacture anino
acids; it can therefore
afford to burn the pyruvate directly through decy .oxylation and
reactions ofthe TCA cycle. Therefore, a large fraction of CO,
observed in fasting islikely produced by conversion of oxaloacetate
'to phosphoenolpyruvate,catalyzed by phosphoenolpyruvate
carboxykinase which resporyds to glucagon(Table 5). Another avenue
of glucose catabolism is the glucuronic acidpathway, a pathway in
which C-6, but not C-l, ,is oxidized to CO2 (?).How.,!;r, in this
study no efftct of glucagon on 'CO2 fonation froin U- C-glucurena-
,,ns observed. It would ap§.2Jr ti no sig3nificance can beatt ehei
t- the qotribution of the glcuron~c tci athway tc th, f
I n-r. ,,xid ation of 1- 14C-qluc-ose inducked bv insulin
confirms similarfi_1,i .s ot , et -k i (l ,1Wh and si!l 1,2t ; in
incrci,, t ,J ctivity of th, pentos,-icy( ','. Thi:; cot m "; A is
cons1 i t,nt wi th the f f L2ct of insulin on thacI tv I t y n 7 [
u ;,-' -[: oAh t,? dehyd rxien .
ts i', li, ft.and ji.icagon stimulate oxidation of 2-
14C-glucose. It hasbe'n darsr .t_ t , it rear:,,i, i:,.nt of
Uucos.? c abons occurs via the nentosecyclk 36 . C-2 of gl',:ose ;.
rando(xiztxl into a- r d C-3 ot tructose-6-phosphate arn therefore
into C-I and C-3 of glucose-6-phosphate upon thecompletion of tne
cycle. C-I of glucose-6-phosphate is oxidized to CO2 in thepentose
cycle and -3 iU the 'CA cycle (Figure 1). Indeed, in vitro
studiesdemonstratudJ that the gre.atest randcmization of glucose
carbons in the pentosecycle occurs in the prisence of insulin
(37).
-
Klain--lO
Both epinephrine and glucagon stimulate oxidation of 6-1
4C-glucose.However, the stimulation by epinephrine was less
pronounced than that byglucagon. In this respect, the action of the
two hormones is similar to theireffect on the activity of several
gluconeogenic enzymes (Table 7, ref 38).Factors such as the dosage
level of epinephrine, the half-life of the hormone,or possibly
different physiologic mechanisms may account, at least in part,for
the less pronounced effect of epinephrine, in comparison to
glucagon, on6-1 4C-glucose oxidation.
Alterations in the glucose oxidative pathways were accompanied
bypronounced changes in the activities of glycolytic and
gluconeogenic enzymes.In general, glucagon and epinephrine
stimulated gluconeogenesis and decreased
4 glycolysis, whereas insulin enhanced glycolysis and synthesis
of fLtty aicidsand reduced gluconeogenesis. The rapid and
antagonistic effects of the th'thormones on carbohydrate metabolism
are consistent with the previous. StudIlsof insulin, glucagon and
epinephrine on these pathways (5,6,38,39).
Since the rate-limiting step in the glycolytic pathway in the
liver is aphosphofructokinase step, the rapid change in the
activity of this enzymewould explain the effects of glucagon and
insulin on glycolysis. Theactivities of other glycolytic enzymes,
glucokinase and pyruvate kinase arealso rapidly changed in a
reciprocal manner by insulin and glucagon (Figure2,3). Glucagon and
epinephrine act as inducers of the key gluconeojenicenzymes
glucose-6-phosphatase, fructose- 1,6-diphosphatase
andphosphoenolpyruvate carboxykinase (Figure 3,4). In contrast,
insulin acts asa suppressor. It is well established that
carbohydrate and fat metabolism inliver are intimately coupled. The
synthesis of fatty acids is maximil ill thelivers of animals fed a
high carbohydrate diet. A reverse sitaittl(11 i.:,rapidly obtained
upon administration of glucagon (8). There i,; qiner dagreement
that the limiting step in the synthesis of fatty jcids ir; it
thq-level of acetyl CoA carboxylase. In this study, we have shown
that .jlacainor epinephrine and insulin exert a reciprocal control
over the activity of th.,key lipogenic enzyme, in addition to the
key enzymes of carbohydratemetabolism.
The effects of glucagon and epinephrine on enzyme activity
areaccompanied by a significant increase in the concentration of
cyclic AMP,suggesting that the effects are mediated by this
nucleotide, presumably byactivation-deactivation of cyclic
AMP-dependent protein kinases (40). Indeed,it has been reported
that glucagon increases the extent of phosphorylation ofthe
glycolytic enzymes, rendering them less active (41,42). Thc effect
ofinsulin is less clear. As others have reported (39), insulin does
not Ird]ui-,,a change in cyclic NMP, suggesting that insulin does
not rneliit, it:;intracellular action via cyclic AMP. The hormone
may exet.t iL; e ff,-t vi iseveral mediators different from c-AMP
(43).
-
Kla-n--l -
It is important to extrapolate the activity of an enzyme assayi
inbroken-cell preparations to its presumed activity in the intact
cell ortissue, particularly when the enzyme is membrane-b.)und or
latent, and hkencesubject in vivo to a degree of constraint
difficult to predict. This pruI]._3nis further magnified in the
liver. Several studies using histochLniciltechniques or
microdissection have found differences in the distribution
ofenzymes among hepatocytes of the liver acinus (44-47). According
to thesestudies, acinar zone-i hepatocytes are predominantly eng
aged ingluconeogenesis, while cells in acinar zone 3 participate
predominantly inglycolysis. There is no sharp division between
gluconeogenic and glycolytichepatocytes. Enzymes corresponding to
each pathway can be detected in bothzones. Only the predominance of
one or another rate-limiting enzyme systemwould make the zone
either gluconeogenic or glycolytic. In addition, otherparameters
that have not been determined at the zone level, such as
theconcentration of substrates, coenzymes, activators, inhibitors
and oxyrjen ineach zone may play important roles in determining
these metabolic fluxes invivo. In our exvperiments, tissue samples
were randomly selected from the-jrilphery of the liver lobes, thus
minimizing the variability and the effectof metabolic zonation on
the outcone of this study.
CON, LUS I oNS,
.......... ... r dcnonstr.ites that insulin acts
antaigenistically to glucagon indin controlling blood glucose
concentrat ns in ,namjials. The rapid
0.,-,of -nse hannones on blood glucose is, at Kaist in part,
explained byI'- I ilos in the activiti -s of several strateg i c
glycolytic ar
'I! ii 7nzymc systu-ns in -he liver. Acconp-',: ig the rapid
changjes iCti ;itiJS ar'e pronounced changes in the flow of glucose
carbons via
tie *r-,,.'. reoxylic acid and the pentose phosphate cycles. The
pru :,t t- "., j i. : tha.t in vivo oxidation of specific carbon; f
jlacsc . A. i,-it ct lt. vc coccuntration.; of insulin, gluca(jon,
nd lc)inephLi le in tr
-
Klain--12
RECOMMENDATIONS
The critical biochemical and physiolcxjical factors affectinq
theconcentration of plasma glucose in humans subjected to a variety
of stressosshould be delineated.
-
Klain--13
REFERENES
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metabolism in liver.In: Dickens F, Randle PJ and Whelan WJ, eds.
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2. Katz J, Wood HG. The use of glucose-C 1 4 Ior the evaluation
of thepathways of glucose metabolism. J Biol Chun 1960;
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3. Williamson JR, Garcia A, Renold AE, Cahill G'. Studies on the
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5. Meikle AW, Klain Gd, Hannon JP. Inhibition of glucose
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and fasted-refed rats byglucagon, epinephrine and cyclic
adenobne-3',5'-,.lqhosphate. Proc Soc ExpBiol :-Id
1973;143:379-391.
6. F lain G.-, Weisir P,-. C(-, ngk2'- ''I _J
-
Klain--14
13. Milstein SW. Oxidation of specifically labeled glucose by
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18. Young JM, Weser E. Effects of insulin on the metabolisn of
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19. Boyett JD, Hofert JF. Studies concerning the inhibition of
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20. Washko ME, Rice E3. Determination of glucose by an improved
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23. Racker E. Fructose-l,6-diphosphatase from spinach leaves.
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24. Lea MA, Walker DG. Factors affecting hepatic glycolysis and
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25. Bucher T, Pfleiderer G. Pyruvate kinase from muscle. In:
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26. Nordlie RC, Lardy HA. Mammalian liver phosphoenolpyruvate
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27. Shrago E, Lardy HA. Paths of ca~bon in gluconc"jenesi.i and
li[xxienosis.VI . Conversion of precuriors to pho.phoenolpyruvte in
liver Cyt ;'0)1. ,]Biol Ch(in 1906;241:6,1-668.
-
Klain--15
28. Horecker BL, Kornberg A, Smyrniotis, PZ. Glucose-6-phospha
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30. Gilman AG. A protein binding assay for
adenosine-3',5'-cyclicmonophosphate. Proc Natl Acad Sci USA
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31. Lowry HO, Rosebrough NJ, Farr AL, Randall AL. Protein
measurement withthe Folin phenol reagent. J Biol Chem
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32. Winer BJ. Statistical principles in experimental design. 2nd
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33. Lefebvre PJ, Unger RH. Glucagon. New York: Pergamon Press,
1972:151-173.
34. Freedman AD, Graff, S. The metabolism of pyruvate in the
tricarboxylicacid cycle. J Biol Chem 1958;233:292-295.
35. Winegrad Al, Shaw WN, Lukens FDW, Stadie iC, i ivlId AE.
Effects o thormone in vitro on the met3bolisn of glucose ii t .T
ihrxse tissue. ,] Bio.Chin 1959;234:1922-1928.
36. WoW ] HG, Kaitz J. Tnc distribution of 1 4C i[i 1ncxosc
phosph.t; an thcA -, LOL. recylinj in the [xntost cycle. J Biol C.
l' '5;21,3:1279-i282.-7. L:n 1. : K J :' ,, B !rt-r;ch GEI, WThte
LW, Wi 1 , RI. Horrnon-1 rtvyt1at 02
gof ucL5Q .2t0Lic. 1oSin inL a :ipose tissue in vitro. A:in NY
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38. St if21 FB, Taunton DD, Green HL, Hermniu RH. ipid
reciprocal c ginqes inlit ti . ,nzy:: r,:tivities following
epinephri,. -injoction. ' '1 h1 },.;24 : ,Z,,0-7244.
39. Taunton LD, Stifel FB, Greene HL, Herman RH. Rapid
reciprocal changes inrat I, ,p.tic iglycclytc enzyme ani fructose
di-.hosrFitise activities followinginsulI in tiv gluc,jon
injection. J Biol Chun 1974;24):7228-7239.
40. %' in AC, hf- injs HC Jr, Grtengard P. Pr,.2tein km.,,,; . i
tl bra-,.An:ni. !3nachm I'h5;54:931-976.
i ','.. -¢ ' .- ., '""' ',
-
Klain--16
41. Kagimoto T, Uyeda K. Regulaton of rat liver
phosphofructokinase byglucagon-induced phosphorylcation. Arch
Biochem Biophys 1980;203:792-799.
42. Marie J, Buc H, Simon MP, Kahn A. Phosphorylation of htunan
erythrocytepyruvate kinase by soluble cyclic AMP-dependent protein
kinases. Eu r JBiochem 1980;108:251-260.
43. Cheng K, Larner J. Intracellular mediators of insulin
action. Annu RvPhysiol 1985;47:405-424.
44. Shank RE, Morrison G, Cheng CH, Karl I, Schwartz R. Cell
heterogeneitywithin the hepatic lobule (quantitative
histochemistry). J Histochem Cytochem1959; 7:237-239.
45. Schumacher HH. Histochemical distribution pattern of
respiratory enzymesin the liver lobule. Science
1957;125:501-503.
46. Guder WG, Schmidt U. Liver cell heterogeneity. The
distribution ofpyruvate kinase and phosphoenolpyruvate
carboxykinase (GrP) in tht, liverlobule of fed and starved rats.
Hoppe Seylers Z Physiol Chem 1976;357:1793-1800.47. Schrnidt U,
Schnidt H, Guder WG. Liver cell heterogeneity. Thedistribution of
fructose biphosphatase in fed and fasted rats and in man.
Hoppe Seylers Z Physiol Chem 1978;359:193-198.
41
l1. i,.__
.-% ,4.
-
.v..Kl.a in--i 7
APPENDI X
Tabl-c I Effect of Gluc.ijon, Insulin and Epinephirmilu oni
Plasma Glucose Levels.
*Table 2 Effect of Glucagon and Insulin on Glucose
Oxidation.
Table 3 Effect of Epinephrine on Glucose Oxidation.
Table 4 Effect of Glucagon on l-1 C-Glucoske and 6-'4C--Glucose
Oxidationin Fed and Fasted Rats.
Table 5 Effect of Glucagon on the Activity of Hepiiti,: Enzymes
and Cyclic AMP.
Table 6 Effect of Insulin on the Activity of Hepatic Enzymes and
Cyclic AMP.
Table 7 Effect of Epinephrine on the Activity of Hepa,,tic
Enzymes and CyclicAMP.
Figure 1 Metabolismn of Glucose via the Glycolytic -3n Pentose
PhosphateP.-thva ys.
F i k* Fijr ( 2 I nsulin ,n] Enzjfvc, Activity.
Fjue " P-cqo K nImi2 Activity.
E'4 rf- Epinephrine ir.] Enzymne Ativity.
-
Klain-i1
w4 CO r4 CDm (
W a' 1-4 U') N- ID mC *+1
C V
* Ca +~ +1 + +1 +-4: CC In N E~O
=0
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ro -4 :4 CD 00 N n cc(' ~-4 0l LIC1 N .
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nf' 000 "0 10
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U) Klain--I 90
4: V * * . .0 * .- 4(% .. .
U~ +1+1+1+1 +1+1+1+1 +1+1+1+1 +1+1+1+1
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Co co. 1 -4 -4 0 Ar-C4% -44t-r4C
ro 011++ +1+1+1+1 +I+I+I++1+1+1+1'0 r~.N-4 a% Lfl '.Oa%,-i in'OD
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C) C 0- qU, %V D U) ,-4 r- M C -4 r-1-4r-4 U)C)% nrC1 0 . . . .
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Kial'n--20
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Klain--2 1
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Klain--24
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FIGURE 1 Klain--25
METABOLISM OF GLUCOSE VIA THE GLYCOLYTICAND THE PENTOSE
PHOSPHATE PATHWAYS
I CIC
2 C 2Cf PENTOSE-P PATHWAYI 1 C02
3 L2 3 456 45 64 C -~4 C C-C-C-C-C- -. C-c-C-S C 5 C PENTOSE-P
GLYCERALDEHYDE-P
1 16 C 6 C
GLUCOSE GLUCOSE-6-P 2 3 2 4 5 6
1 21 3 4 5 6C-C-C-C-C-C -'2 3 3 4 5 6
GLYCOLYTIC FRUCTOSE-6-P C-C-C-C-C-CPATHWAY 3 342
FRUCTOSE-6-P
1,6 2,5 341,6 2,5
c-c-C--L-- c-c- S-CoAPYRUVATE ACETYL-CoA
2,s C 2, 5 C 2,5 CI C)I C
1,6 C 2 1.6 C C 2 1,6 C
C -- C C1,6 C TURN 1
C CC
C C
C 2,5 C 2,5 1,6 C
C ~ 2 C ~2 1,6 CTRICARBOXYLIC 1 2,5 1I
ACDCCE1,6 C-c 1,6 C 1,6 C TURN 21,6 C 1,6 C 1.6 C
2,5 C. 2,5 C
C 1,6 C 1,6 1,6 C
C 2 C 2 1,6 C1 1,67 1
1,6 C-C ----- 4'1,6 C - --- 1,6 C TURN 3, I 11,6 C 1,6 CL 1,6
C
1,6 C 1,6 CCITRATE a-KETOGLUTARATE OXALOACETATE
-
Klain--26
LU
0~~
Lu L
L 4 z
4A~ 0
UU~ U
I//U 0
I/ Z 0 a0 II
CL Lu z
U 0 au LZ~~ ~ ~ L0L l- z:L 0
Ix CL
)0- L00 Z
z ND _uLu 00
z K- 0
s4 U >a-U 0
'Luu
Luz
>- 4-
CLu
-
Klain--27
Lu
0
%n X 4--------------------on-
4.4
' o4 U
LUCLCL '0 %
~Li
LU L
OzCLJ LU
ceL LU 'o LJL-U 0L. 0 0 ui
ma C LJ 0 o- 0 Z:
o D '0 L LU.. j1. 0~ D
z LUSLA.
0L "IfU
0U ~
LU 40 LU
0. U.
-
Klain--28
- 0
'uj LUvi Ln
z- - - - - - - - - - - -
00. 0
'oU
0 0 4
LU OU
IdLU 0
LUL OnC U
*AILUI Z 0vZLU T0 $A0
_LL 0 Z LU
m_ _ _ Z~ CL
0 a 6 -- W$ 0 I . D - - - -- - - - -- - x UW6~L Zo 0ULj0'ncLV0.
LU>0uCL z NU
x _
r44~~ CL I xa E1LU I I,-- -+- )/
z~~~L /UCe
LA. 4 /LU /
LU 0 >
IL 0U 0LU0 CieL
CLI- _ _._o _ _
LU&A- U9
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-
Klain- -29
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