The Metabolic Effects of Intensive Insulin Therapy in Critically Ill

Patients

MB Whyte1, NC Jackson2, F Shojaee-Moradie2, DF Treacher3, RJ Beale3, RH

Jones1, AM Umpleby2

1Dept of Diabetes, Endocrinology and Internal Medicine, GKT School of Medicine, St.Thomas’

Hospital, 2Dept of Diabetes and Endocrinology, Postgraduate Medical School, University of

Surrey, 3Dept Intensive Care Medicine, St.Thomas’ Hospital, London, UK.

Running title: Metabolic Effects of Intensive Insulin Therapy

Address for correspondence

Dr. Martin Whyte

Department of Diabetes and Endocrinology

Kings College Hospital

London

SE5 9RS

Telephone: 020 32991737

Fax: 020 32991730

E mail: mbwhyte@hotmail.co.uk

Abstract word count 250

Manuscript Word count: 4363 (excluding abstract, tables and figure legends)

Abstract

Aims To investigate the effects of glycaemic control and insulin concentration on lipolysis,

glucose, and protein metabolism in critically-ill medical patients.

Methods. Patients were studied twice. In Study 1, blood glucose (BG) concentrations were

maintained between 7-9mmol/L with intravenous insulin. After Study 1 patients entered 1 of 4

protocols for 48h until Study 2: Low-insulin high-glucose, LIHG (variable insulin, BG 7–

9mmol/L), Low-insulin low-glucose, LILG (variable insulin, BG 4–6mmol/L), High-insulin high-

glucose, HIHG (insulin [2.0 mU kg-1min-1plus insulin requirement from study one], BG 7–

9mmol/L), High-insulin low-glucose, HILG (insulin [2.0 mU kg-1min-1plus insulin requirement

from study one], BG 4-6mmol/L). Age-matched healthy control subjects received two-step

euglycaemic hyperinsulinaemic clamps achieving insulin levels similar to the LI and HI groups.

Results Whole-body proteolysis was higher in patients in study 1 (P<0.006) compared to control

subjects at comparable insulin concentrations and was reduced with LI (P<0.01) and HI (P=0.001)

in control subjects, but not in patients. Endogenous glucose production rate (Ra), glucose

disposal and lipolysis were not different in all patients in study 1 compared to control subjects at

comparable insulin concentrations. Glucose Ra and lipolysis did not change in any of the study 2

patient groups. HI increased glucose disposal in the patients (HIHG, P=0.001; HILG, P=0.07

versus study 1) but this was less than in controls receiving HI (P<0.03).

Conclusions Low-dose intravenous insulin, administered to maintain BG between 7-9 mmol/L is

sufficient to limit lipolysis and endogenous glucose Ra and increase glucose Rd. Neither

hyperinsulinaemia nor normoglycaemia had any protein-sparing effect.

Keywords Insulin, glucose, glycaemic control, critical illness, lipolysis, endogenous glucose

production rate, glucose disposal, proteolysis

Abbreviations

APACHE Acute Physiology and Chronic Health Evaluation

APE atom percent excess

BG blood glucose

CRP C reactive protein

HIHG High-insulin high-glucose

HILG High-insulin low-glucose

KIC α ketoisocaproate

LIHG Low-insulin high-glucose

LILG Low-insulin low-glucose

NOLD Non-oxidative leucine disappearance

Ra Rate of appearance

Rd Rate of disappearance

TPN total parenteral nutrition

Introduction

Critically-ill patients exhibit a stress response characterised by lipolysis, protein

breakdown and hyperglycaemia. Hyperglycaemia was considered to be a beneficial,

adaptive response but recent evidence suggests that it may be detrimental.

Maintenance of normoglycaemia with either a Glucose-Insulin-Potassium infusion

(32) or variable insulin infusion (16; 55; 56) has been reported to improve patient

morbidity and mortality. Post-hoc analysis suggested that the prevailing glycaemia,

rather than insulin dose administered, effects the beneficial outcomes (57). More

recently, debate has intensified following the publication of a multi-center study that

casts doubt over the benefits of glycaemic control in a critical care environment (15).

The metabolic effects of such treatment have yet to be characterised.

Endogenous glucose production rate (Ra) has been shown to be increased by at least

twofold in critical illness, compared to healthy controls (45), however it is not known

how effective a variable insulin infusion is at lowering glucose Ra. Insulin

administration at a rate of 1mg/kg/min was required to overcome hepatic insulin

resistance in septic patients (10) but there was evidence of incomplete suppression (as

reflected by high serum IGFBP-1 levels) in surgical patients (36). Accelerated

lipolysis is also a consistent feature of critical illness and elevated fatty acids can

contribute to insulin resistance. In health, lipolysis is inhibited by low insulin levels

but in critical illness, higher doses may be required (10). The effect of maintenance

of normoglycemia with intensive insulin therapy on lipolysis is also unknown.

Critically-ill patients may experience loss of muscle protein approaching 2% per day

(19). Patient survival (20), duration of intensive care unit (ICU) admission (12), and

time to recovery of normal physiological function (59), are inversely correlated with

loss of lean body mass. Insulin’s effect on protein metabolism has been contentious

1

but it appears primarily to inhibit proteolysis (9; 18; 38; 51) although increased

protein synthesis has also been reported (6). We hypothesised that the anti-catabolic

effects of insulin might explain the benefits of insulin administration to critically-ill

patients.

We have studied the effect of maintaining normoglycaemia with intravenous insulin

on the rate of lipolysis, glucose and protein metabolism in patients requiring tertiary-

level support.

Research Design and Methods

The study protocol was approved by the regional ethics committee. Twenty-five

mechanically-ventilated patients were recruited from the ICU at St.Thomas’ Hospital,

London. Assent was obtained from the patients’ families before their entry into the

study. Patients newly admitted to the ICU (within 36 h) and likely to require

ventilatory support for at least 3 days were considered for inclusion.

Patients were excluded if they had a surgical procedure prior to ICU admission

requiring general anaesthesia or regional block. Patients with diabetes mellitus, acute

or chronic pancreatitis, liver disease, and those receiving oral steroids within one-

month prior to entering the ICU were excluded. Liver disease was defined as any one

of: history of oesophageal varices, biopsy (or ultrasonographic) evidence of cirrhosis,

International normalised ratio (INR) > 1.4, bilirubin concentration more than twice

the normal range, ALT, AST, γGT, or ALP concentration over three times the normal

range. 13 patients were admitted with primary respiratory dysfunction, 3 with sepsis

from a non-respiratory cause, 2 with neurological illness, 2 with cardiac disease and 5

2

with other illnesses. Patients received a variety of vasopressor agents, steroids and

anaesthetic agents (Table 1). Severity of illness was determined by the Acute

Physiology and Chronic Health Evaluation (APACHE) II score.

All patients were studied on two occasions, 48 h apart. Patients receiving enteral

nutrition (Nutrison multifibre™, Nutricia, Zoetermeer, Holland), had the feed stopped

8 h prior to Study 1. All patients received intravenous 20% dextrose (25kcal kg-1day-

1) for at least 8 h before Study 1 (range 10-18 h). Study 1 took place within 36 h of

ICU admission with blood glucose (BG) concentrations maintained between 7-9

mmol/L with intravenous Actrapid insulin (Novo-Nordisk, Bagsvaerd, Denmark).

The insulin infusion was adjusted at hourly intervals to maintain BG between 7 and

9mmol/L for at least 10 h before study 1 (range 10-18 h; figure 1). After study 1

patients entered 1 of 4 protocols for 48h until Study 2: Low-insulin high-glucose,

LIHG (variable insulin infusion to maintain BG 7 – 9mmol/L), Low-insulin low-

glucose, LILG (variable insulin to maintain BG 4 – 6mmol/L), High-insulin high-

glucose, HIHG (insulin infusion rate 2.0mU kg-1min-1 plus insulin requirement from

Study 1), with additional dextrose, as required, to maintain BG 7 –9mmol/L), High-

insulin low-glucose, HILG (insulin infusion rate 2.0mU kg-1min-1 plus insulin

requirement from Study 1) with additional dextrose, as required, to maintain BG 4 -

6mmol/L (figure 1).

Study Protocol (Patients).

Study 1. After baseline sampling, a prime of 170mg of [6,6 2H2]glucose, 0.15mg / kg

of [2H5]glycerol, 1mg / kg of [1-13C]leucine and 0.2 mg / kg [13C]NaHCO3 (CK Gases

Ltd, UK, subsidiary of Cambridge Isotope Laboratories, Massachusetts) was

3

administered (33) followed by continuous infusion of (1.7mg/min) [6,6 2H2]glucose,

(0.61mg kg-1hr-1) [2H5]glycerol and (1mg kg-1hr-1) [1-13C]leucine, for 180 min. During

the 3 h tracer infusion, glucose infusion rate remained constant and if necessary,

intravenous insulin was adjusted every 30min to maintain BG concentrations.

Indwelling arterial and central venous lines were used for blood sampling and for

tracer infusion, respectively. BG was determined on heparinised samples using a

point-of-care Omni 2 blood gas analyser (AVL/Roche, Welwyn Garden City, UK).

Blood samples were taken at baseline and at 150, 160, 170, 175 and 180 min (steady

state) to measure the isotopic enrichment of glucose, glycerol and α-keto-isocaproate

(αKIC) and hormone and metabolite concentrations. Samples were stored at -70˚C

until analysis.

Expired air was collected from the exhaust-port of the ventilator, at the same intervals

as blood sampling to measure isotopic enrichment, and stored in 250ml aluminium-

coated bags (QuinTron® Instrument Company, Milwaukee, Wisconsin USA),

pending transfer into evacuated, septum-capped 10ml glass tubes (Exetainer, Labco

Ltd, High Wycombe), for the measurement of 13CO2 by isotope ratio mass

spectrometry (IRMS). Pulmonary gas exchanges were measured by an open-circuit

indirect calorimeter (Deltatrac™ II metabolic monitor, Datex-Ohmeda, Finland).

Data acquisition was performed until steady-state values were reached over a 30-60

min period, defined as requiring no change in ventilatory settings, no requirement for

large volumes of fluids, and institution or cessation of vasopressors. The values of

VO2 (oxygen consumption) and VCO2 (carbon dioxide production) and the calculated

respiratory quotient (RQ) represent an average of the data obtained over 30 min, with

a coefficient of variation of <10%.

4

After Study 1, enteral feeding (1 kcal/ml; 16% protein, 49% carbohydrate, 35% fat)

was instituted immediately, unless a clinical decision was made not to do so. The

feed was supplemented by intravenous 50% dextrose, as required, to achieve

glycaemic targets. Patients were fed via nasogastric tube according to a standard

feeding protocol. It was the policy of the local ICU to use total parenteral nutrition

(TPN) only if specifically indicated, as such, no patient received TPN. Eight hours

prior to the second isotope study the enteral feed was stopped and replaced with

isocaloric intravenous 20% or 50% dextrose (depending on fluid requirements.)

Study 2. The isotope infusion and sampling protocol of the second isotope study was

identical in methodology to Study 1.

Study Protocol (Control subjects). Five healthy age-matched control subjects (M4,

F1) were studied to quantify the normal dose response effect of insulin on suppression

of proteolysis, lipolysis and glucose production and increase in glucose disposal. All

volunteers were in good health and gave informed consent to the study. Subjects

were fasted from midnight and studied throughout in a semi-recumbent position.

Following baseline blood sampling, a 9 h, primed, constant infusion of [6,6

2H2]glucose, (1.7mg/min), [2H5]glycerol (0.61mg kg-1hr-1), [1-13C]leucine (1mg kg-

1hr-1) was commenced using an IVAC syringe pump (Alaris, San Diego). Insulin was

infused at 0.5mU kg-1min-1 between 180-360 min and 2.0mU kg-1min-1 from 360-540

min. Blood samples were taken at baseline, 150, 160, 170, 175 and 180 min (first

steady-state), 330, 340, 350, 355 and 360 min (second steady-state) and at 510, 520,

530, 535 and 540 min (third steady state) (Figure 2). BG was determined every 5 min

(Yellow Springs Instruments, Yellow Springs, OH) and maintained within 5% of

5

5mmol/L by adjustment of a 20% glucose infusion (IVAC Volumetric pump, Alaris

systems, San Diego) (13), spiked with [6,6-2H2]glucose (‘hot glucose infusion

method’, using 8mg/g glucose for step 1 and 10 mg/g glucose for step 2).

Experimental Methods. Plasma α-KIC enrichment was measured as a quinoxalinol-

tertiarybutyldimethylsilyl derivative as previously described (17). Using a reciprocal

pool model (35), plasma α-KIC enrichment was considered a measure of intracellular

leucine enrichment. 13C enrichment of breath CO2 was measured on a SIRA series II

continuous-flow IRMS (VG Isotech, Cheshire, UK) modified with a Roboprep G+

inlet system (Europa Scientific). The isotopic enrichment of plasma glucose was

determined by the penta-O-trimethylsilyl-D-glucose-o-methoxamine derivative (46)

and glycerol as the tris-trimethylsilyl derivative (60). Plasma and infusate glucose

concentrations were determined using a Clandon Scientific Glucose analyser (Yellow

Springs Instruments, Yellow Springs, OH). Blood lactate concentration was

measured by point-of-care AVL/Roche Omni2 blood gas analyser. Serum NEFA

concentrations were determined spectrophotometrically by Cobas analyser (Roche,

Welwyn Garden City, UK) using a kit from Wako Chemicals Inc (Alpha

Laboratories, Hampshire, UK). Plasma and infusate glycerol concentrations were

measured by direct colorimetry (Randox Laboratories, Crumlin, Co.Antrim, UK) on a

Cobas analyser. Total cholesterol concentration was measured using a kit from ABX

Diagnostics (Montpellier, France) on a Cobas analyser. Serum cortisol concentration

was measured by an automated chemiluminescent assay (Bayer Diagnostics,

Tarrytown, USA). Serum insulin concentrations were measured by an in-house

double-antibody radioimmunoassay (47). C-peptide concentration was measured

using a double-antibody radioimmunoassay (RIA) kit (LINCO Research Inc.

6

St.Charles, Missouri, USA). C-Reactive Protein (CRP) was measured with an

immunoturbidometric assay (Beckman Synchron LX20).

Calculations. The rates of appearance and disappearance of a substrate were

calculated from the dilution of labelled substrate in plasma using a mono-

compartment model and Steele’s equations (2) modified for stable isotopes. During

physiologic and isotopic steady-state, the production rate (Ra) of unlabelled substrate

can be calculated by:

Ra (µmol kg-1min-1) = (Ei / Ep -1) x I

Where Ei = enrichment of infusate (Atom % excess, APE), Ep = enrichment of

substrate in plasma (APE), and I = infusion rate (µmol kg-1min-1). Total glucose Ra

was assumed to equal peripheral glucose disposal (Rd). The endogenous glucose

production rate was estimated by subtraction of the unlabelled glucose infusion rate

from the total glucose Ra. Propofol (Diprivan®) contains glycerol at 22.5mg/ml and

soybean oil at 100mg/ml. We have assumed that 30% of the triglyceride composition

of soybean oil is hydrolysed to glycerol (25). Endogenous glycerol Ra was calculated

by subtracting the rate of glycerol appearance from propofol from the total glycerol

Ra. Leucine oxidation rate was calculated from the CO2 Ra and expired 13CO2 as

previously described (34).

Ox (µmol kg-1min-1) = (ECO2 x VCO2) / EKIC x 0.899

ECO2 is the atoms percent excess of 13CO2 in expired air, EKIC is the [13C]KIC

enrichment, and 0.899 is a recovery factor that accounts for the fraction of 13CO2

formed on oxidation of the tracer but not released from the bicarbonate pool (53).

Non-oxidative leucine disappearance rate (NOLD, a measure of protein synthesis)

7

was calculated by subtracting leucine oxidation rate (Rox) from leucine Ra. Net

protein balance was estimated using the equation (NOLD – Ra), with the assumption

of 8g of leucine for 100g whole body protein (22).

Commercially available dextrose for intravenous infusion contains carbon with a high

natural abundance of 13C isotope: this could affect calculation of leucine oxidation

when first administered. As the patient group received at least 8 h of intravenous

dextrose prior to the 13C-leucine infusion, the rate of 13C release by oxidation of

exogenous carbohydrate would have equilibrated before 13C leucine was infused.

Basal samples were collected prior to the isotope infusion thus allowing correction for

this in the subsequent measurement of leucine oxidation. However, in the control

group dextrose was infused after infusion of the isotopes so the contribution of 13C

isotope to 13CO2 could not be corrected for and leucine oxidation could not be

measured.

Statistical analysis. The primary endpoint was leucine Ra. With 6 patients in each

group the study was powered to detect a difference of 20% in leucine Ra between

Study 1 and Study 2 in the patients with a power of 80% at a two-sided 5 % level of

significance. This was based on previous studies of protein metabolism in ICU in both

surgical patients (26) and septic medical patients (5) whereby the SD for leucine Ra

was between 6 and 16%. Studies have reported an SD for leucine Ra in healthy

subjects to be 8.5% (18). With n=5 in the control subjects the study was powered to

detect a difference of 9.5% in leucine Ra between insulin doses with a power of 80%

at a two-sided 5 % level of significance. The Statistical Package for the Social

Sciences (version 12.0 for Windows software; SPSS Inc, Chertsey UK) was used for

statistical analyses. Between-group comparisons were analysed for statistical

8

significance using repeated-measures ANOVA. Post-hoc analysis was conducted

using Bonferroni’s multiple-comparison test. For within-group comparisons of study

1 to study 2, a paired t test was used. Results are expressed as mean values SEM

unless stated otherwise.

9

Results

Group Characteristics (table1)

The 4 groups differed significantly only as intended with respect to glucose

concentration and insulin infusion rates. The age (60.0 ± 1.3 years) and BMI (25.0 ±

0.9kg/m2) of control subjects were not different to the patients. All subjects survived

the duration of the study although one patient in the HIHG group subsequently died.

Treatment protocols were well tolerated with no short-term adverse effects seen on

gas exchange, renal, hepatic or cardiovascular status. Two glucose measurements

were <3mmol/L during the study but none were <2.2mmol/L. The control subjects’

plasma glucose was 5.03 ± 0.14mmol/L at baseline; 4.34 ± 0.16mmol/L with LD

insulin; and 4.24 ± 0.19 with HD insulin (NS).

Hormone and Plasma Metabolite Concentrations

Serum insulin, plasma C-peptide and serum cortisol concentrations are shown in

Table 2. In the LI study 2 patient groups; insulin concentrations were not different

from the LI control study. In the HI study 2 patient groups, insulin concentrations

were not different from the HI control study. Plasma lactate, serum NEFA, plasma

glycerol and total cholesterol levels are shown in Table 3. Plasma NEFA, glycerol

and total cholesterol concentrations were not different in study 2 compared to study 1

in any of the patient groups. Plasma total cholesterol was significantly lower in all

patient groups (at both study 1 and study 2 compared with fasting control subjects;

P<0.01). Plasma NEFA concentrations were lower in study 1 in all patients (n=25)

when compared to fasting control subjects (NEFA: 0.22 ± 0.04 vs. 0.67 ±

0.11mmol/L, P=0.02) but were higher than in the control study LI step where insulin

levels were comparable (NEFA: 0.04 ± 0.005mmol/L; P<0.001).

10

Kinetic Data

In the control subjects leucine Ra, a measure of proteolysis (figure 3), was reduced

from basal levels by 11.6 ± 1.3% with LI (from 1.78 ± 0.05 to 1.57 ± 0.03µmol kg -

1min-1; P=0.01) and by 21.7 ± 0.9% (to 1.39 ± 0.04µmol kg-1min-1) with HI (P<0.001).

In all patients in Study 1 (n=25), leucine Ra was not different from the fasting control

subjects but was higher than control subjects with comparable insulin concentrations

(LI) (2.00 ± 0.13 vs.1.57 ± 0.03µmol kg-1min-1; P=0.006). Leucine Ra was higher in

the HILG group in Study 2 than control subjects receiving HI (2.1 ± 0.15 vs. 1.39 ±

0.40µmol kg-1min-1; P=0.005). Comparing Study 2 to Study 1, none of the four

treatment regimens altered the rates of leucine Ra, NOLD and leucine oxidation

(figure 3 and table 4) with the result that net protein balance was unaffected (figure 4).

Furthermore, leucine Ra of the combined LI groups (n=13) did not change from study

1 to study 2 (2.07 ± 0.19 vs. 2.13 ± 0.15µmol kg-1min-1); likewise, leucine Ra of the

combined HI groups (n=12) was unchanged from study 1 to study 2 (1.94 ± 0.21 vs.

2.28 ± 0.19µmol kg-1min-1).

In the control subjects, glucose Ra was reduced from basal values with LI (P=0.07)

and was completely suppressed with HI (P=0.003; NS different from zero) (table 5).

Glucose Rd was increased with LI and HI compared to basal (P < 0.001). In all

patients at Study 1 (n=25) endogenous glucose Ra was lower and glucose Rd was

higher than the control subjects during fasting (P<0.001) but were not different from

the LI step in the controls which had comparable insulin levels. In Study 2,

endogenous glucose Ra was lower than Study 1 in the HILG group but was no

different in the other patient groups. Glucose Rd increased in Study 2 in both HI

11

groups (P=0.001 for HIHG; P=0.07 for HILG) compared to Study 1 but this was

lower than glucose Rd in the control study HI step which had comparable insulin

levels, (HILG group study 2, P<0.01; HIHG group study 2, P<0.03).

Glycerol Ra was reduced from basal by LI in the control subjects (P <0.001), with no

further suppression with HI (Table 4). In all patients in Study 1, glycerol Ra was not

different from control subjects receiving LI, and did not suppress further in any group

in Study 2. The glycerol Ra data were not significantly influenced by whether the

patients did or did not receive propofol.

12

Discussion

In this study we sought to determine the effects of glycaemic control and insulin

concentration on intermediary metabolism in critically-ill medical patients, and for the

first time, to measure the effect of insulin on protein turnover in critically ill medical

patients. The results demonstrate that low dose intravenous insulin, administered to

maintain BG between 7-9 mmol/L is sufficient to limit lipolysis and endogenous

glucose Ra and increase glucose Rd as compared to fasting control subjects.

Complete suppression of endogenous glucose Ra was only achieved in the HILG

group. Neither normoglycaemia nor hyperinsulinaemia had any effect on whole body

protein synthesis or proteolysis.

Protein loss characterises the catabolic response to critical illness: this is thought to

arise both from increased proteolysis (24) and diminished protein synthesis (58). The

use of leucine Ra in this study as a measure of proteolysis allows for a more accurate

estimation of protein flux than urinary nitrogen balance (31). Our data show that rates

of proteolysis in patients at study 1 were higher than in control subjects who received

similar insulin doses, even though endogenous glucose production rate was

suppressed which would reduce the requirement for gluconeogenic precursors. This

finding is consistent with previous observations in both normal subjects and surgical

patients that low-dose glucose administration (causing a doubling of insulin

concentration) does not reduce proteolysis (23) (42). Our data also show that the

administration of insulin to achieve supra-physiological concentrations did not

attenuate protein breakdown and had no effect on net protein balance. This finding

was not anticipated. In healthy subjects, insulin inhibits proteolysis in a dose-

dependent manner (51), as we confirmed from our control studies. Although this was

13

a small study, the findings suggest that neither tight glycaemic control or high doses

of insulin are likely to have any clinically significant benefit in terms of protein

balance in critical illness.

Stress-induced hyperglycaemia of critical illness has been attributed to insulin

resistance either at the liver (27) or skeletal muscle (1). Whilst it has been reported

that intensive insulin treatment to maintain tight glycaemic control between 4.4-

6.1mmol/l is unable to overcome hepatic insulin resistance, as assessed by high levels

of serum IGFBP-1 in critically-ill patients (36), our data show that suppression of

hepatic glucose Ra to low levels can occur with an insulin infusion of approximately

1mU kg-1min-1. This is in accord with recent studies in critically-ill patients which

also used kinetic techniques (10; 52) and showed that glycaemic control with an

insulin infusion is achieved by suppression of hepatic glucose production. In study 2,

patients in the HILG group had a level of glycaemia and insulinaemia which was

similar to control subjects receiving HI enabling a direct comparison of rates of

glucose disposal. Glucose Rd was lower than in the control subjects, suggesting the

presence of peripheral insulin resistance - a feature that has previously been reported

both in sepsis and trauma (40; 43) .

As expected, serum cortisol was higher in the patient groups than controls. It is not

yet known how important this is for the induction of peripheral insulin resistance

associated with critical-illness, as over 30 other factors have been proposed (4).

These may be grouped as either hormonal responses (for example glucocorticoid

hypersecretion or low circulating IGF-1) (54) or cytokine-mediated (including TNFα,

14

IL-6) (4). It is noteworthy that attempts to manipulate the cytokine cascade have not

affected the physiologic response to illness (11).

Lipolysis is a consistent feature of critical-illness (30) making the effect on lipolysis

of exogenous insulin an area of interest. In this study, the contribution of exogenous

glycerol was subtracted from the total glycerol Ra to give the endogenous glycerol

Ra. Although the contribution of exogenous glycerol was small (providing less than

5% of the total glycerol Ra) because the rate of release of glycerol from the hydrolysis

of soybean oil within propofol could not be measured directly, the endogenous

glycerol Ra is a very close approximation and not an exact measurement. In study 1

lipolysis (as measured by glycerol Ra and NEFA concentrations) was suppressed by

insulin and no further suppression occurred with HD insulin or tight glycaemic

control. Cardiac NEFA metabolism requires more oxygen per unit energy yield than

glucose, which could therefore have a deleterious effect on myocardial performance

in the hypoxic setting (3). Suppression of NEFA concentrations in the presence of

increased insulin will result in glucose being the prime substrate for myocardial

metabolism which may be beneficial. It is of interest that suppression of NEFA

concentration occurred without the necessity of achieving normoglycaemia.

Hypocholesterolaemia confers a poor prognosis in critically-ill patients (21). We

found that none of the treatment regimens normalised cholesterol, although a study

period of up to 1 week might have been required to do so (37).

A possible limitation to this study is the difference in study duration of the control and

patient groups. Bed-rest itself may cause insulin resistance after 5 to 6 days (48)

although not after 24 hours (39): this might have biased the data of the control group

15

(recumbent for 9-hours). However, it has also been shown that the insulin

responsiveness of protein metabolism after bed-rest remains intact (44) suggesting

that the conclusions drawn from the control studies were valid. The 48 hour duration

of clamp (plus an 8 hour lead-in to study 1) of the patient groups was based on the

expected length of stay of such patients in our ICU being 3 days, which is similar to

reported data (56). Effects of insulin on glucose and glycerol turnover in critically ill

patients have been reported within 500 minutes (10; 52) but we considered that an

effect on leucine turnover would take up to 72 hours (7).

We did not perform studies in the fed state as it would have required using total

parenteral nutrition which is a means of feeding seldom used in our ICU due to

infectious complications (29). It is possible that amino acid supplementation

combined with insulin might have increased whole-body protein synthesis as reported

in healthy subjects (8; 50). This was likely to have been a direct effect of the amino

acids as in the only study examining the action of insulin on protein turnover during

an amino acid clamp (41), it was found that insulin could suppress leucine Ra by 19%

but it had no effect on NOLD. However, systemic insulin infusion, despite

hypoaminoacidaemia, is capable of suppression of leucine Ra (14): as was also seen

in our control studies.

Inherent to the study design was that standard treatments be provided to obtain a

clearer understanding of the effect of glycaemic control in a setting familiar to

physicians and intensivists, this meant that the use of glucocorticoids could not be

controlled between the patient groups. However, we were unable to detect any

difference in leucine Ra at either study 1 or study 2 when patients were categorised as

16

receiving glucocorticoids or not and neither was any correlation seen between cortisol

concentration and leucine Ra. This study was of medical ICU admissions only and

therefore it remains to be seen whether tight glycaemic control can exert a protein-

sparing effect in surgical ICU cases.

To summarise, the use of a variable dose of insulin to maintain blood glucose below

6mmol/L in a medical group of critically-ill patients led to suppression of lipolysis

and near full reduction of hepatic glucose production and increased peripheral glucose

uptake. These effects were also seen with a higher level of glycaemia (7-9mmol/l),

suggesting intravenous insulin which achieves moderate glucose levels rather than

tight glycaemic control is sufficient to reduce fatty acid levels and overproduction of

glucose by the liver. Hyperinsulinaemia increased peripheral glucose disposal but this

failed to reach values seen in health; additionally, hyperinsulinaemia could not

attenuate protein loss. There appears to be a differential effect of resistance to insulin

action in skeletal muscle compared to adipose tissue or the liver. The intracellular

insulin signalling network is highly complex and includes cross-talk between

pathways but the Ras/MAPK pathway has been implicated as being responsible for

the growth and mitogenic effects of insulin (49). It may be that this pathway is

particularly susceptible to interruption during critical illness, perhaps by cytokine

action. Alternatively, it has been shown from studies of knockout mice that IRS-1 has

the predominant role in skeletal muscle and IRS-2 in the liver (28). Furthermore, data

suggests that IRS-1 knockouts affect protein synthesis whereas IRS-2 knockouts

primarily affect glucose regulation (49). It may be hypothesized that insulin receptor

subtypes are differentially affected in critical illness. To conclude, this study suggests

17

that any beneficial effect of tight glycaemic control on morbidity and mortality is not

due to protein sparing.

Acknowledgements

This study was supported by a grant from the Guy’s and St.Thomas’ Charitable

Foundation. The authors wish to thank William Jefferson, Premila Croos and Joanne

Crunden for their technical assistance with this study. We also acknowledge the help

of John Smith, Tony Sherry and the all the clinical staff at the Intensive Care Unit.

18

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31

Figure 1

Subject recruited to study Study 1 Study 2

LILG

LIHG

HILG

HIHG

10 - 18 h 48 h

Blood glucose7 – 9 mmol/L

32

Figure 2

Primed, constant infusion of stable isotopes of leucine, glucose and glycerol

Insulin 0.5 mU kg-1min-1

Insulin 2.0 mU kg-1min-1

0 330 360 510 540 min

Blood samples

Indirect calorimetry

Variable glucose infusion

150 180

33

Figure 3. Leucine Ra

34

Figure 4. Net protein balance

35

Figure Legends

Figure 1. Study protocol for patients. Low-insulin high-glucose, LIHG (variable

insulin with BG of 7 – 9mmol/L; n=7), Low-insulin low-glucose, LILG (variable

insulin with BG of 4 – 6mmol/L; n=6), High-insulin high-glucose, HIHG (high dose

insulin infusion [2.0 mU kg-1min-1] plus the insulin requirement from study one with

BG of 7 – 9mmol/L; n=6), High-insulin low-glucose, HILG (high dose insulin

infusion [2.0 mU kg-1min-1] plus the insulin requirement from study one with BG of 4

– 6mmol/L; n=6).

Figure 2. Study protocol for control subjects.

Figure 3. Rate of leucine appearance (leucine Ra) in the patient group. Low-insulin

high-glucose, LIHG (variable insulin with BG of 7 – 9mmol/L; n=7), Low-insulin

low-glucose, LILG (variable insulin with BG of 4 – 6mmol/L; n=6), High-insulin

high-glucose, HIHG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin

requirement from study one with BG of 7 – 9mmol/L; n=6), High-insulin low-

glucose, HILG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin

requirement from study one with BG of 4 – 6mmol/L; n=6). Low-dose insulin (0.5

mU kg-1min-1), LI. High-dose insulin (2.0 mU kg-1min-1), HI. Mean ± SEM.

Figure 4. Calculated net protein balance over a 24 hour period. Study one (black

boxes) and study two (hatched boxes). Low-insulin high-glucose, LIHG (variable

insulin with BG of 7 – 9mmol/L; n=7), Low-insulin low-glucose, LILG (variable

insulin with BG of 4 – 6mmol/L; n=6), High-insulin high-glucose, HIHG (high dose

36

insulin infusion [2.0 mU kg-1min-1] plus the insulin requirement from study one with

BG of 7 – 9mmol/L; n=6), High-insulin low-glucose, HILG (high dose insulin

infusion [2.0 mU kg-1min-1] plus the insulin requirement from study one with BG of 4

– 6mmol/L; n=6). Mean ± SEM.

37

Table 1. Group Characteristics

LILG

(n=6)

LIHG

(n=7)

HILG

(n=6)

HIHG

(n=6)

Gender (M/F) 4 / 2 6 / 1 3 / 3 4 / 2

Age (yr) 70.8 ± 5.1 66.0 ± 4.4 68.5 ± 2.6 58.2 ± 7.4

BMI (kg/m2) 24.8 ± 2.4 25.3 ± 1.8 27.1 ± 3.0 27.3 ± 2.7

APACHE II Study 1 17.2 ± 2.8 15.9 ± 1.4 15.0 ± 2.6 19.2 ± 2.9

Study 2 15.4 ± 1.5 17.0 ± 1.8 18.3 ± 3.6 18.3 ± 2.8

Plasma glucose

(mmol/L)

Study 1 7.94 ± 0.92 7.41 ± 0.44 8.02 ± 0.47 8.46 ± 0.69

Study 2 5.60 ± 0.43 a 8.81 ± 0.41 4.76 ± 0.39 a 8.09 ± 0.47

Insulin infusion

(U/h)

Study 1 5.28 ± 1.26 3.97 ± 0.7 4.02 ± 0.85 5.73 ± 1.93

Study 2 5.03 ± 1.76 2.09 ± 0.73 13.57 ± 1.61 a b 15.48 ± 2.67 a b

Inotropes (n) Study 1 5 4 3 2

Study 2 0 1 2 0

Glucocorticoids

(n)

Study 1 5 6 4 2

Study 2 3 6 3 2

Propofol (n) Study 1 4 5 3 6

Study 2 2 4 3 6

Heparin (n) Study 1 1 1 2 3

Study 2 1 1 3 3

CRP (mg/L) Study 1 118.3 ± 28.7 150.0 ± 48.8 180.8 ± 50.6 72.8 ± 26.0

Study 2 79.3 ± 24.1 83.7 ± 29.7 129.8 ± 29.6 45.5 ± 16.1

Mean daily caloric intake (kcal/day)

1725 ± 107 1779 ± 222 2885 ± 344 4217 ± 678 b

Data are mean ± SEM. a P ≤ 0.01 v study 1, b P < 0.001 v low-insulin groups.

Low-insulin high-glucose, LIHG (variable insulin with BG of 7 – 9mmol/L), Low-

insulin low-glucose, LILG (variable insulin with BG of 4 – 6mmol/L), High-insulin

38

high-glucose, HIHG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin

requirement from study one with BG of 7 – 9mmol/L), High-insulin low-glucose,

HILG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin requirement

from study one with BG of 4 – 6mmol/L). Acute Physiology and Chronic Health

Evaluation score II (APACHE II), C-reactive protein (CRP).

39

Table 2. Hormone Concentration

Insulin

(pmol/L)

C-peptide

(nmol/L)

Cortisol

(nmol/L)

LILG

(n=6)

Study 1 287.24 ± 75.70 3.19 ± 1.04 920.55 ± 410.67

Study 2 293.18 ± 81.73 0.53 ± 0.13 a 825.05 ± 222.21

LIHG

(n=7)

Study 1 221.69 ± 28.80 2.56 ± 0.34 1276.58 ± 416.07

Study 2 193.58 ± 27.24 2.50 ± 0.45 840.51 ± 129.65

HILG

(n=6)

Study 1 224.54 ± 28.45 2.43 ± 0.79 1122.27 ± 303.49

Study 2 1435.79 ± 204.05 a 0.28 ± 0.053 a 924.43 ± 211.96

HIHG

(n=6)

Study 1 340.04 ± 94.82 1.60 ± 0.49 1796.04 ± 626.68

Study 2 1769.12 ± 480.99 a 1.51 ± 0.69 1563.18 ± 632.37

Control

subjects

(n=5)

Basal 24.93 ± 5.60 0.70 ± 0.11 268.94 ± 22.08

LI 318.63 ± 26.20 b 0.42 ± 0.036 c 344.80 ± 40.73

HI 1922.85 ± 192.50 b 0.37 ± 0.066 c 365.25 ± 98.93

Data are mean ± SEM. a P < 0.05 v study 1, b P < 0.001 v basal control, c P < 0.05 v

basal control. Low-insulin high-glucose, LIHG (variable insulin with BG of 7 –

9mmol/L), Low-insulin low-glucose, LILG (variable insulin with BG of 4 –

6mmol/L), High-insulin high-glucose, HIHG (high dose insulin infusion [2.0 mU kg-

1min-1] plus the insulin requirement from study one with BG of 7 – 9mmol/L), High-

insulin low-glucose, HILG (high dose insulin infusion [2.0 mU kg-1min-1] plus the

insulin requirement from study one). Low-dose insulin (0.5 mU kg-1min-1), LD.

High-dose insulin (2.0 mU kg-1min-1), HD.

40

Table 3. Plasma Metabolite Concentration

Lactate

(mmol/L)

NEFA

(mmol/L)

Glycerol

(µmol/L)

Total

Cholesterol

(mmol/L)

LILG

(n=6)

Study 1 1.63 ± 0.36 0.19 ± 0.07 75.68 ± 19.70 2.16 ± 0.39 a

Study 2 1.67 ± 0.16 0.42 ± 0.27 68.72 ± 18.15 b 2.56 ± 0.31 a

LIHG

(n=7)

Study 1 1.65 ± 0.34 0.25 ± 0.09 73.87 ± 12.38 2.42 ± 0.29 a

Study 2 1.53 ± 0.34 0.22 ± 0.05 c 58.14 ± 7.45 b 2.68 ± 0.39 a

HILG

(n=6)

Study 1 1.84 ± 0.43 0.27 ± 0.13 68.30 ± 24.32 2.34 ± 0.33 a

Study 2 1.70 ± 0.22 0.07 ± 0.02 c 63.26 ± 10.96 b 2.34 ± 0.35 a

HIHG

(n=6)

Study 1 1.86 ± 0.32 0.19 ± 0.05 74.14 ± 18.02 2.88 ± 0.46 a

Study 2 2.15 ± 0.17 0.15 ± 0.05 c 101.88 ± 12.93 c 2.92 ± 0.35 a

Control

subjects

(n=5)

Basal - 0.67 ± 0.11 62.20 ± 5.30 4.88 ± 0.38

LI - 0.04 ± 0.005 d 8.42 ± 2.06 d -

HI - 0.02 ± 0.006 d 7.74 ± 1.48 d -

Data are mean ± SEM. a P ≤ 0.01 v basal control, b P < 0.001 v LD and HD control, c P < 0.05 v

HD controls, d P < 0.001 v basal control.

Low-insulin high-glucose, LIHG (variable insulin with BG of 7 – 9mmol/L), Low-

insulin low-glucose, LILG (variable insulin with BG of 4 – 6mmol/L), High-insulin

high-glucose, HIHG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin

requirement from study one with BG of 7 – 9mmol/L), High-insulin low-glucose,

HILG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin requirement

41

from study one with BG of 4 – 6mmol/L). Low-dose insulin (0.5 mU kg-1min-1), LI.

High-dose insulin (2.0 mU kg-1min-1), HI.

42

Table 4 Glycerol production rate, leucine oxidation rate and NOLD

Glycerol Ra

(µmol kg-1min-1)

Leucine

oxidation

(µmol kg-1min-1)

NOLD

(µmol kg-1min-1)

LILG

(n=6)

Study 1 1.74 ± 0.28 0.40 ± 0.08 1.57 ± 0.20

Study 2 1.66 ± 0.24 0.44 ± 0.10 1.61 ± 0.18

LIHG

(n=7)

Study 1 1.37 ± 0.21 0.48 ± 0.09 1.69 ± 0.23

Study 2 1.30 ± 0.21 0.48 ± 0.05 1.63 ± 0.19

HILG

(n=6)

Study 1 1.94 ± 0.71 0.24 ± 0.03 1.65 ± 0.08

Study 2 1.90 ± 0.28 0.35 ± 0.03 1.66 ± 0.12

HIHG

(n=6)

Study 1 1.45 ± 0.11 0.29 ± 0.04 1.27 ± 0.08

Study 2 2.67 ± 0.71 0.34 ± 0.05 1.98 ± 0.31

Control

subjects

(n=5)

Basal 4.07 ± 0.45 - -

LI 1.33 ± 0.17 a - -

HI 1.22 ± 0.16 a - -

Data are mean ± SEM. a P < 0.05 v basal control.

Low-insulin high-glucose, LIHG (variable insulin with BG of 7 – 9mmol/L), Low-

insulin low-glucose, LILG (variable insulin with BG of 4 – 6mmol/L), High-insulin

high-glucose, HIHG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin

43

requirement from study one with BG of 7 – 9mmol/L), High-insulin low-glucose,

HILG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin requirement

from study one). Low-dose insulin (0.5 mU kg-1min-1), LI. High-dose insulin (2.0

mU kg-1min-1), HI. Endogenous glucose rate of appearance (Endog glucose Ra),

glucose rate of disappearance (glucose Rd), glycerol rate of appearance (glycerol Ra),

non-oxidative leucine disposal (NOLD).

Table 5. Glucose turnover data and RQ

44

Endog Glucose Ra

(mg kg-1min-1)

Glucose Rd

(mg kg-1min-1)

RQ

LILG

(n=6)

Study 1 0.069 ± 0.54 3.80 ± 0.69 0.83 ± 0.03

Study 2 0.61 ± 0.40 3.89 ± 0.65 0.88 ± 0.02

LIHG

(n=7)

Study 1 0.61 ± 0.45 4.09 ± 0.34 0.80 ± 0.04

Study 2 0.87 ± 0.24 4.36 ± 0.20 0.79 ± 0.03

HILG

(n=6)

Study 1 0.51 ± 0.32 4.27 ± 0.68 0.81 ± 0.02

Study 2 -1.10 ± 0.57 a h 7.04 ± 0.80 b c d 1.02 ± 0.03 a b

HIHG

(n=6)

Study 1 0.67 ± 0.32 4.07 ± 0.46 0.84 ± 0.05

Study 2 0.20 ± 0.33 h 7.46 ± 0.87 a b c e 1.02 ± 0.02 a b

Control

subjects

(n=5)

Basal 1.89 ± 0.058 1.89 ± 0.058 0.82 ± 0.02 c

LI 0.46 ± 0.59 4.58 ± 0.32 f 0.88 ± 0.01 c

HI -0.92 ± 0.41 f h 10.15 ± 0.57 f g 0.99 ± 0.03

Data are mean ± SEM. a P < 0.007 v HILG group at study 1, b P < 0.02 v LILG group at

study 2, c P < 0.03 v HI control, d P = 0.05 v LIHG at study 2, e P < 0.02 v LIHG group

at study 2, f P ≤ 0.05 v basal control, g P < 0.003 v LI control, h NS different from zero

Low-insulin high-glucose, LIHG (variable insulin with BG of 7 – 9mmol/L), Low-

insulin low-glucose, LILG (variable insulin with BG of 4 – 6 mmol/L), High-insulin

high-glucose, HIHG (high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin

requirement from study one with BG of 7 – 9mmol/L), High-insulin low-glucose, HILG

(high dose insulin infusion [2.0 mU kg-1min-1] plus the insulin requirement from study

one with BG of 4 – 6mmol/L). Low-dose insulin (0.5 mU kg-1min-1), LI. High-dose

insulin (2.0 mU kg-1min-1), HI. Endogenous glucose rate of appearance (Endog glucose

45

Ra), glucose rate of disappearance (glucose Rd), glycerol rate of appearance (glycerol

Ra), non-oxidative leucine disposal (NOLD).

46