Influence of growth hormone on glucose-glucose 6-phosphate cycle and insulin action in normal humans R. D. G. NEELY, D. P. ROONEY, P. M. BELL, N. P. BELL, B. SHERIDAN, A. B. ATKINSON, AND E. R. TRIMBLE Departments of Clinical Biochemistry and Medicine, Queen’s University of Belfast, Regional Endocrine Laboratory, and Sir George E. Clark Metabolic Unit, Royal Victoria Hospital, Belfast BT12 6BA, United Kingdom Neely, R. D. G., D. P. Rooney, P. M. Bell, N. P. Bell, B. Sheridan, A. B. Atkinson, and E. R. Trimble. Influence of growth hormone on glucose-glucose 6-phosphate cycle and insulin action in normal humans. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E980-E987, 1992.-Increased activity of the hepatic glucose-glucose 6-phosphate (G/G-6-P) cycle is associated with hepatic and peripheral insulin resistance in acromegaly. To determine whether a similar association occurs after short-term growth hormone (GH) elevation within the physiological range, two-step euglycemic hyperinsulinemic clamps were performed in normal human males after 12-h GH (2.2 ngekg-l h-l) and control infusions. G/G-6-P cycle activity and endogenous glucose production (EGP) were determined by [2-3H]- and [6-3H]glucose using labeled exogenous glucose infusions and selective enzymatic detritiation. GH increased levels of circulating lipid intermediates despite a twofold increase in basal insulin (P < O.OOS),but plasma glucose, EGP, and G/G-6-P cycle activity were unchanged. GH impaired insulin suppression of EGP and lipid intermediates and impaired insulin stimulation of glucose disposal, but G/G-6-P cycle activity was unchanged. We conclude that increased activity of the G/G-6-P cycle does not contribute to the hepatic insulin resistance induced by GH under these conditions but that changes in fatty acid metabolism may be partly responsible for the impairment in hepatic and peripheral insulin action. endogenous glucose production; euglycemic hyperinsulinemic glucose clamp; free fatty acids; ,&hydroxybutyrate; glycerol; glucose-fatty acid cycle l

GROWTH HORMONE (GH) has profound effects on carbohydrate and lipid metabolism (6). In acromegaly, a disease of chronic GH excess, patients with normal glucose tolerance are typically found to have increased basal endogenous glucose production (EGP) despite fasting hyperinsulinemia (12, 18), and both insulin suppression of EGP and stimulation of glucose uptake are impaired (12). A similiar pattern of hepatic and peripheral insulin resistance may be induced in normal subjects by a sustained elevation of GH within the physiological range; however, basal EGP is restrained by a compensatory increase in insulin levels (4, 25). Although the precise mechanisms remain uncertain, it has been suggested that increased activity of the glucose-glucose 6-phosphate substrate cycle (GIG-63 cycle) may be of importance in GH-induced hepatic insulin resistance (18). In this cycle, which depends on simultaneous activation of glucokinase and glucose6-phosphatase, glucose may be taken up by the liver, phosphorylated, dephosphorylated, and subsequently released from the liver with the net consumption of ATP (19). Increased activity of the G/G-6-P cycle has been reported in association with hepatic insulin resistance in a variety of disease states including type II diabetes (8)) hyperthyroidism (17)) and acromegaly (18). Efendic E980

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et al. (8) proposed that, in the postabsorptive state, increased activity of the G/G-6-P cycle might reduce EGP and limit hyperglycemia by promoting the movement of glucose against the direction of net flux. However, this would be at the expense of an increase in EGP after a glucose load, antagonizing hepatic insulin action and contributing to glucose intolerance (8). In each of the conditions studied the relative increase in G/G-6-P cycle activity appeared much greater than the associated increase in basal EGP. Thus in glucose-tolerant acromegalic patients a 17% increase in basal EGP was accompanied by a 70% increase in cycle activity (18), whereas in hyperthyroidism a 100% increase in cycle activity was associated with an insignificant increase in EGP (17). It was therefore proposed that increased G/G-6-P cycle activity might be a more sensitive marker for the defect in hepatic glucose metabolism than net glucose fluxes (17, 18). Furthermore, in acromegaly the basal G/G-6-P cycle activity was correlated with plasma GH levels, suggesting that this hormone may be directly involved in regulation of the cycle (18). To date there have been no investigations of the effect of GH administration on G/G-6-P cycle activity in normal subjects. The purpose of the present study was to determine the effect of a sustained elevation of GH within the physiological range, sufficient to induce hepatic and peripheral insulin resistance, on the activity of the hepatic G/G-6-P cycle. To do this we have measured glucose kinetics in the basal state and during two-step euglycemic hyperinsulinemic clamps by the simultaneous infusion of [2-3H]- and [6-3H]glucose. In addition, the alterations in insulin action induced by GH were examined in relation to its effects on levels of circulating lipid fuels. MATERIALSAND

METHODS

Subjects

Informed written consent was obtained from eight healthy nonobese (body mass index 24 t 1 kg/m2) male human subjects aged 24-35 yr [28 t 4 (SE) yr]. None had a family history of diabetes mellitus, and none were taking any medications. Approval for the studies was obtained from the Research Ethical Committee of the Queen’s University of Belfast. Protocol Study design. Each subject was studied twice by the euglycemic hyperinsulinemic glucose-clamp technique, once after an overnight infusion of biosynthetic human GH and once after a control infusion containing only saline. Studies were performed single blind, in random order, at least 4 wk apart. Study procedures. On each occasion, subjects were admitted to the metabolic ward of the Royal Victoria Hospital at 1700 h

1992 the American

Physiological

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on the day before the clamp studies. A standard evening meal (10 kcal/kg, 45% CHO, 35% fat, 20% protein) was given at 1730 h, and subjects then remained fasting until the clamp studies were complete. A plastic cannula (21 gauge, Venflon, Viggo, Helsingborg, Sweden) was placed in a right forearm vein, and at 2100 h either GH (Genotropin, Kabivitrum, 2.2 ng=kg-l h-l) in 0.9% NaCl or 0.9% NaCl alone (1 ml/h) was infused using a portable infusion pump (MS 26, Graseby Medical, Watford, UK). The amount of GH required to increase serum GH levels to lo-15 rig/l was initially chosen on the basis of previously published reports (4, 25) and confirmed in a pilot study. A plastic cannula (18 gauge, Venflon, Viggo) was placed in a left antecubital vein, and samples of blood were withdrawn before and at intervals during the infusion for measurement of serum GH and lipid intermediates. The subjects were allowed to remain ambulatory before retiring to bed between 2200 and 2300 h. At 0650 h a sample of blood was taken from the left antecubital cannula to obtain nonradioactive plasma for subsequent use in analysis of plasma glucose specific activities, and a carrier infusion of 0.9% NaCl was connected. Subsequently all infusions were connected to this line. An additional plastic cannula (21 gauge, Venflon, Viggo) was placed retrogradely in a right dorsal hand vein, and the right forearm cannula was removed. The hand was then placed in a thermostatically controlled Perspex box (Northern Ireland Technology Centre, Automation Div., Queen’s University of Belfast) and maintained at 55°C to allow intermittent sampling of arterialized venous blood. At 0700 h a 6-h primed continuous infusion of [2-3H]- and [6-3H]glucose tracers in 0.9% NaCl was commenced. Tracers alone were infused during the next 2 h to allow for isotopic equilibration. At 0900 h a continuous infusion of insulin (Humulin S, Eli Lilly, Basingstoke, UK) was started at a low dose (step 1, 0.2 mU kg-l emin-l) for 2 h and then increased to a high dose (step 2,2.0 mU kg-l min-l) for a further 2 h. The insulin infusion rates were chosen to achieve partial suppression of EGP and lipolysis in step 1 and for maximal stimulation of glucose disposal and maximal supression of EGP and lipolysis in step 2. During the insulin infusion, plasma glucose was maintained at ~5.3 mmol/l using an exogenous infusion of glucose (20% BP, Travenol Laboratories, Thetford, UK) prelabeled with [2-3H]- and [6-3H]glucose tracers to match the predicted basal plasma specific activities (see below). The actual concentration of the infused glucose solution was measured in triplicate on each occasion. Blood samples for determination of plasma glucose specific activities were taken at IO-min intervals from -30 to 0, 90 to 120, and 210 to 240 min relative to the start of the insulin infusion. Preparation of tracer infusions. Because commercially prepared tritiated glucose tracers may contain radioactive nonglucase contaminants, equal quantities (150 &i) of [2-3H]- and [6-3H]glucose tracers (TRK361 and TRK85, Amersham International, Aylesbury, UK) for each study were purified by highperformance liquid chromatography, as previously described (24), on the day before the study. In each case aliquots (10 ~1)of the purified tracers were retained for subsequent use in the measurement of [2-3H]- and [6-3H]glucose specific activities. The tracers were then combined and diluted in a small quantity of 0.9% NaCl. An empirical formula was used to predict the plasma [6-3H]glucose specific activity after 2 h of primed continuous infusion. This was derived by linear regression analysis of the relationship between basal specific activity (SA), body weight (W), and the rate of tracer infusion (F) in earlier clamp studies (24): predicted basal specific activity (dpm/pmol) = F (dpm/min)/[281 + 8.5W (pmol/min)]. The combined tracer solution was then divided between the primed continuous tracer infusate and the exogenous glucose infusate so as to match the [6-3H]glucose specific activity of the latter to the predicted l

l

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basal value. The primed ([6-3H]glucose 8.94 t 1.65 &i; [2-3H]glucose 9.21 t 1.53 &i), continuous ([6-3H]glucose 0.089 t 0.017 &i/min; [2-3H]glucose 0.092 t 0.015 &i/min) tracer infusion was maintained throughout the hyperinsulinemic clamp period in each study. This modified tracer infusion protocol was designed to reduce non-steady-state errors in measurement of glucose turnover and makes no assumptions regarding the time course of EGP or the final rate of glucose disposal. However, a modest rise in plasma glucose specific activity was expected during the hyperinsulinemic period (22). Analytical

Techniques

Arterialized venous blood was used for all analyses. Samples for plasma glucose and glucose specific activity were collected in lithium heparin fluoride tubes, immediately placed on ice, and centrifuged within 30 min. An aliquot was used for duplicate determination of plasma glucose by a glucose oxidase method (Beckman Glucose Analyzer II, Beckman RIIC, High Wycombe, UK). The remaining plasma was stored at -20°C until analysis. Aliquots (100 ~1) of tracer infusate and labeled exogenous glucose infusion were spiked into nonradioactive plasma (900 ~1) before storage at -20°C to prevent radiolytic decomposition of tritiated glucoses. Samples for GH, insulin, and lipid intermediates [free fatty acids (FFA), glycerol, and P-hydroxybutyrate] were collected in plain glass tubes and separated as soon as clotting was complete. The resulting serum was stored at -20°C until required for analysis. Blood samples for lactate and pyruvate were collected into glass tubes containing an equal volume of aqueous perchloric acid solution (8% wt/vol), immediately shaken, and placed on ice. After centrifugation extracts were separated and analyzed immediately or stored at -20°C until analysis. Serum insulin was measured by radioimmunoassay (ll), with a mean between-batch coefficient of variation of 5.6% at 8 mU/l and 8.2% at 36 mu/l; samples were diluted where serum insulin was >50 mu/l. Serum GH was measured using a double-antibody radioimmunoassay (3). Mean betweenbatch coefficients of variation were 6.9,5.0, and 4.9% at 1.0,2.1, and 4.9 rig/l, respectively; samples were diluted where serum GH was >5 rig/l. Commercially available reagent kits were used for the measurement of serum nonesterified fatty acids (Wako Chemicals, Neuss, FRG), P-hydroxybutyrate and glycerol (Randox Laboratories, Crumlin, UK), and blood lactate and pyruvate (Sigma Chemical, Dorset, UK). In each case assays were by automated enzymatic calorimetric methods adapted for use on a centrifugal analyzer (Cobas Farah, Roche Diagnostics, Herts, UK) and gave mean between-batch coefficients of variation of ~5.0% over the range tested. Plasma specific activities of [2-3H] - and [6-3H]glucose were determined by selective enzymatic detritiation using a modification of the method of Issekutz (15) as previously described (26). In this procedure, enzyme treatment results in the loss of tritium from the second carbon of glucose while tritium on the sixth carbon is retained. The amount of [6-3H]glucose present is thus determined as the radioactivity remaining after enzyme treatment. The amount of [2-3H]glucose present was calculated by subtraction of [6-3H]glucose radioactivity from the total radioactivity present before enzyme treatment. Detritiation of [2-3H]glucose was 98.5 t 0.5% and of [6-3H]glucose 0.1 t 0.2%. Calculations

Rates of appearance (R,) and disappearance (Rd) were determined for [2-3H]- and [6-3H]glucose during the periods -30 to 0, 90 to 120, and 210 to 240 min using the non-steady-state equations of Steele et al. (30) as modified by DeBodo et al. (7) to correct for small fluctuations in glucose specific activity. For these calculations, a pool fraction of 0.65 and an extracellular volume of 190 ml/kg were assumed. Infusion rates of [2-3H]and 16-3H1-glucose were calculated as the sum of the tracer

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infused continuously and the tracer in the labeled exogenous glucose infusion. If it is assumed that tritium in C-6 is retained throughout glycolysis (14) and subsequently undergoes negligible recycling into glucose (19), [6-3H]glucose R, and Rd (R,6 and Rd6) are taken to be accurate measures of the true rates of glucose production and disposal, respectively. Rates of EGP were obtained by subtraction of exogenous glucose infusion rates (GIR) from R, determined using [6-3H]glucose (EGP = R,6 - GIR). Because tritium in C-Z is lost in the reversible isomerization between G-6-P and fructose 6-phosphate, glucose passing through the G/G-6-P cycle (i.e., entering and immediately leaving the liver, having been phosphorylated to G-6-P by glucokinase and dephosphorylated back to glucose by glucose6-phosphatase) will contribute to R, determined by [2-3H]glucose (R,2) but not to R, determined by [6-3H]glucose (R,6). Thus the difference in the glucose appearance rates determined by [2-3H]- and [6-3H]glucose (R,2 - R,6) was taken as a measure of the activity of the G/G-6-P cycle. Statistics

I

Unless otherwise stated, all data are given as means t SE. Hormone concentrations and glucose kinetic data in the GH and control studies were compared using two-tailed t tests for paired results. Metabolite concentrations measured overnight and during the hyperinsulinemic clamps were evaluated by twoway analysis of variance (ANOVA) for a repeated measures design with time (min of study) and infusion (GH or control) as independent variables. Where this showed a difference between the GH and control studies, summary measures for the overnight data (time-averaged area under curve of concentration from 2100 to 0830 h) or for the clamp data (means of concentrations during last 30 min of basal period, -30 to 0 min; step 1, 90-120 min; step 2, 210-240 min) were compared using twotailed t tests for paired results. Relationships between glucose disposal rates and concentrations of GH and metabolites were explored using multiple-regression analysis. All statistical analyses were performed using the Solo program (BMDP Statistical Software, Los Angeles, CA). P < 0.05 was considered significant. RESULTS

Overnight GH and Metabolite (Fig. 1, Table 1)

Concentrations

Before the infusions, levels of GH, FFA, glycerol, and P-hydroxybutyrate were similiar in both studies. During the GH infusion, GH levels rose to a steady plateau in the upper physiological range after an early sleep-induced peak. During the control infusions GH levels rose to normal peak levels after the onset of sleep and later fell to the low physiological range. Serum FFA and ,&hydroxybutyrate concentrations increased overnight during the control studies (by ANOVA both P < 0.005); however,



2100

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0100

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0500

time

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Fig. 1. Serum growth hormone (GH), free fatty acids (FFA), glycerol, and P-hydroxybutyrate during overnight GH (solid line) or control (dashed line) infusions (IZ = 8). Values are mean k SE. 12-h

during GH infusion the increases were much larger (by ANOVA both P < 0.005). Only during GH infusion did glycerol concentrations increase significantly (by ANOVA P < 0.02). ~~~~~~~~~~~~~ Studies Hormones, glucose, and glucose infusion rates (Figs. 2 and 4). The stable increase in serum GH levels achieved

overnight during GH infusions was maintained in the basal period and throughout the subsequent clamp studies. In the control studies, GH levels remained for the most part in the low physiological range; however, spontaneous GH peaks were seen during the clamp studies in several subjects (Fig. 4). After overnight GH infusion, basal plasma glucose levels were unchanged compared with control [5.4 t 0.1 vs. 5.3 t 0.1 mmol/l; P = not significant (NS)]. Plasma glucose levels were similiar throughout the hyperinsulinemic clamp periods during GH (5.3 t 0.1 mmol/l; coefficients of variation 5.2 t 0.4%) and control studies (5.2 t 0.1 mmol/l; coefficients of variation 5.4 t 0.4%).Basal serum insulin levels were increased almost twofold by GH infusion (11.0 t 1.3 vs. 5.7+ 0.4mu/l; P < 0.05).In the GH studies insulin levels remained slightly higher (19.0t 1.3vs. 16.2t 0.9 mu/l; P < 0.01) during step 1 (0.2 mU kg-l min-l) insulin l

Table 1. Overnight, basal, and postinsulin serum growth hormone, free fatty acid, glycerol, and P-hydroxybutyrate concentrations Growth GH

Hormone, w/l Control

FFA, mmol/l GH

Overnight (2100-0830 h) 13.2t1.5* 4.6k0.9 0.81t0.05* 1.4kO.2 l.lOt0.12* Basal (-30 to 0 min) 12.0t0.9* 11.6&1.0* 1.6t0.2 Step 1 (90-120 min) 0.76t0.09* 11.9tl.O* 2.7tl.4 Step 2 (210-240 min) 0.14*0.03f Values are means k SE. Overnight data represent time-averaged area fatty acids. * P < 0.005 vs. control. “f P < 0.02 vs. control.

Glycerol, pmol/l Control 0.58t0.05 0.56t0.03 0.18kO.04 0.04t0.01

GH 74.9&6. lt 77.9k11.7.f 58.9t7.1* 29.625.2”

/3-Hydroxybutyrate, mmol/l Control 56.3k4.0 40.1t2.6 17.5t3.3 12.2t2.2

under curve of overnight concentration.

GH 0.38t0.04” 0.85kO.14” 0.39~0.10t 0.03t0.01

Control 0.16kO.02 0.19~0.02 0.04~0.01 O.O1tO.O1

GH, growth hormone; FFA, free

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Fig. 3. Rates of total glucose disposal, endogenous glucose production, and glucose-glucose 6-phosphate (G/G-6-P) cycle activity during basal period, step 1 (0.2 mU kg-’ l rein+), and step 2 (2.0 mU kg-l mix+) hyperinsulinemic clamp periods with GH (filled bars) or control infusions (open bars) (n = 8). Values are mean * SE. * P < 0.05 vs. control. l

Fig. 2. Plasma concentrations of glucose and insulin, [6-3H]glucose specific activity ( [63H]GSA) and glucose infusion rates during 2-step hyperinsulinemic (step 1, 0.2 mU kg-l l rein-l; step 2, 2.0 mU kg-l. min-l) glucose-clamp experiments with GH (0) or control (0) infusions (n = 8). Values are mean * SE.

l

hyperinsulinemia EGP was partly suppressed in both GH (to 8.3 t 0.4 pmol kg-l. min-l; P < 0.0005 vs. basal) and control studies (to 7.2 t 0.8 prnol. kg-l P < 0.001 vs. basal, P = 0.09 vs. GH); however, when expressed as a percentage of basal rates, suppression of EGP was impaired by GH (23 t 3% vs. 36 t 6%; P = O.Ol), despite slightly higher insulin levels. During step 2 hyperinsulinemia EGP was suppressed equally in the GH and control studies (by 75 t 6% vs. 70 t 8%; P = NS) but remained significantly positive (P < 0.01) in both (2.6 t 0.6 and 3.4 t 0.9 prnol. kg-l l rein-l, respectively). During step 1 hyperinsulinemia, glucose disposal rates showed a small increase above basal rates in the control studies (14.1 t 1.0 pmol . kg-l ; min- l; P < 0.02 vs. basal) but not with GH (11.2 t 0.7 prnol. kg-l min-l; P = NS vs. basal, P < 0.005 vs. control). During step 2 hyperinsulinemia glucose disposal rates were markedly reduced (by 38 t 6%) in the GH studies (34.6 t 3.5 vs. 55.7 t 2.8 pmol kg-l min- ‘; P < 0.0005). The decrease in step 2 glucose disposal rate was correlated with the increase in serum GH levels overnight (r = -0.855, P < 0.01;Fig. 5) and during the clamp (r = -0.853, P < 0.01). Activity of the G/G-6-P cycle, measured under isotopic steady-state conditions during the basal period, was not changed by the GH infusion (1.5 t 0.2 vs. 1.5 t 0.1 prnol kg-l min-l; P = NS). Measurements of G/G-6-P cycle activity made during the hyperinsulinemic clamp period, under near-steady-state conditions, showed no change from basal values and did not differ between GH and control studies (Fig. 3) Metabolite concentrations (Fig. 4, Table 1). In parallel with its effect on the overnight levels, GH infusion increased the basal concentrations of FFA, glycerol, and ,&hydroxybutyrate, whereas blood lactate (0.52 t 0.03 vs. 0.52 t 0.02 mmol/l) and pyruvate (0.05 t 0.01 vs. 0.06 t l

l

infusion but were slightly lower (127t 7 vs. 140 t 9 mu/l; P < 0.05) during step 2 (2.0 mU kg-l min-l). Glucose infusion rates required to maintain euglycemia during the GH studies were lower both for step 1 (3.1 t 1.0vs. 6.8 t 1.5 pmol kg-l . min- l; P < 0.001) and step 2 hyperinsulinemia (33.2 t 3.6 vs. 52.3 t 2.5 ~mol*kg-l*min-l; P < 0.0005), consistent with a reduction of insulin-stimulated glucose disposal. The time course of the plasma [6-3H]glucose specific activity is shown in Fig. 2. During the step 1 hyperinsulinemic clamp period there was, as expected, a small rise in specific activity that was similiar in the GH (to 120 t 2% of basal) and control studies (to 121 t 3% of basal, P = NS). During the step 2 hyperinsulinemic clamp period [ 6-3H]glucose specific activities fell back toward basal levels; however, this fall was less during the GH studies (to 114 t 3% of basal) compared with control (103 t 2% of basal; P < 0.05). The rise in [ 2 -3H] glucose specific activities (not shown) was similiar during step 1 (in GH study to 120 t 2% of basal; in control study to 122 t 2% of basal; P = NS). In the GH study, [ 2-3H] glucose specific activities did not change significantly during step 2 (123 t 3% of basal) but in the control study fell back toward basal (to 113 t 2% of basal). In each case plasma tracer specific activities were relatively constant (coefficients of variation of ~5%) during the final 30 min of each clamp period and glucose kinetics were measured under near-steady-state conditions. Glucose kinetics (Fig. 3). Basal glucose turnover rates, measured by [6-3H]glucose under isotopic steady-state conditions, were not changed by GH infusion (10.8 t 0.2 vs. 11.1t 0.4 prnol kg-’ P = NS). During step 1 l

l

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min-l;

l

min-‘;

l

l

l

l

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= -0.836

P = 0.0097

0

al

o z N a Q) G

0.4

A

zi

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FFA

o-

r = -0.855

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A

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-50

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I

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time (mid Fig. 4. Serum GH, FFA, glycerol, and ,&hydroxybutyrate during 2-step hyperinsulinemic (step 1, 0.2 mu. kg-l emin-‘; step 2, 2.0 mU kg-l min-I) glucose-clamp experiments with GH (solid line) or control (dashed line) infusions (n = 8). Values are mean t SE.

A 50vernight d”H (ng.l-‘1 Fig. 5. Relationship between change in step 2 glucose disposal rate and change in overnight FFA concentrations (top) and overnight GH concentrations (bottom) between studies performed with GH and control infusions.

l

0.01 mmol/l) were unchanged (both P = NS). In the GH studies, levels of FFA and glycerol remained higher throughout the clamp (by ANOVA, P < 0.001 and P < 0.002, respectively). During both steps 1 and 2 hyperinsulinemia GH impaired the suppression of FFA and glycerol. Suppression of P-hydroxybutyrate was less markedly affected; however, the complete suppression seen during step 1 hyperinsulinemia in the control studies was seen in the GH studies only during step 2 hyperinsulinemia ,. Blood lactate inc creased during step 2 hyperinsulinemia in both studies (by ANOVA P C 0.05); however, this increase was smaller in the GH studies (to 0.76 t 0.05 vs. 1.01 t 0.05 mmol/l; P < 0.002). Blood pyruvate increased in both studies (by ANOVA P < 0.05) to reach similiar levels during step 2 hyperinsulinemia (0.10 t 0.01 vs. 0.10 t 0.01 mmol/l; P = NS). The increase in overnight FFA concentration due to GH infusion was correlated with the decrease in step 2 glucose disposal rate (r = -0.836, P C 0.01; Fig. 5). This could be attributed only in part to the change in overnight serum GH levels which correlated less strongly with the change in FFA concentration (r = 0.532, P = 0.18). Multiple-regression analysis showed that changes in overnight GH and FFA were more strongly predictive of step 2 glucose disposal rate when taken together (r2= 0.934, P < 0.001) -and that the overnight FFA increase made an independent contribution (P < 0.05). Comparison of standardized multiple-regression coefficients showed that the magnitude of the contribution from FFA was similiar to that from GH. In contrast, no significant relationship was observed between step 2 glucose disposal rate and levels of. lipid intermediates during the clamp period.

DISCUSSION

The present studies were undertaken to examine the effects of sustained GH elevation, within the physiological range, on G/G-6-P cycle activity and levels of lipid intermediates in relation to alterations in hepatic and peripheral insulin action. Our results indicate that increased activity of the G/G-6-P cycle does not contribute to the hepatic insulin resistance induced by GH under these conditions but that changes in fatty acid metabolism caused by GH may be responsible, at least in part, for the impairment in hepatic and peripheral insulin action. In this study normal subjects were infused with GH to produce a sustained elevation of GH levels within the physiological range as seen during sleep (13) and exercise (28). As in previous studies using a similiar experimental protocol (4, 25), basal insulin levels rose to maintain a normal fasting plasma glucose and basal EGP. During the hyperinsulinemic clamp impaired insulin suppression of EGP was found only at lower insulin levels (step 1), whereas impaired insulin stimulation of glucose uptake was evident also at higher insulin levels (step 2). Thus, as expected, GH infusion induced a state of hepatic and peripheral insulin resistance. However, activity of the G/G-6-P cycle, measured in the basal state under isotopic steady-state conditions, was identical in GH and control studies. During the hyperinsulinemic clamp period (steps 1 and 2), G/G-6-P cycle activity remained constant at basal levels regardless of GH elevation. Thus earlier conclusions regarding the role of the cycle as a sensitive marker for alterations of hepatic glucose metabolism in chronic GH excess (18) cannot be extrapolated to shortterm physiological elevation of GH in normal subjects; increased activity of the G/G-6-P cycle does not appear to contribute to hepatic insulin resistance under these conditions. Moreover, our data do not support a role for GH

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in the short-term physiological regulation of G/G-6-P cycle activity but cannot exclude a longer term effect. In our studies, as in those of Karlander et al. (18), G/G-6-P cycle activity was estimated by comparison of the glucose appearance rates measured using [2-3H] - and [ 6-3H] glucose tracers infused simultaneously. Activity of the G/G-6-P cycle is usually equated to the difference in glucose appearance rates measured with these two tracers (R,2 - R,6), assuming that 1) 3H in C-2 of glucose is completely lost in the phosphoglucose isomerase reaction as glucose passes through the G/G-6-P cycle and thus measures total hepatic glucose output; 2) 3H in C-6 of glucose is retained throughout glycolysis but does not reappear in glucose and thus measures true hepatic glucose production. However, it is apparent from in vitro studies that a substantial amount of tritium may be retained in C-2 of glucose 6-phosphate and that the extent to which detritiation occurs may vary according to the prevailing metabolic conditions (19, 2 1). Incomplete detritiation of [2-3H]glucose would lead to an underestimate in the measured total hepatic glucose output, and because a measure of 3H specific activity in C-2 of G-6-P is required to correct for this, accurate quantitation of G/G-6-P cycle activity may be difficult in vivo. Removal of 3H in C-6 during recycling of glucose via pyruvate may also be incomplete (27)) leading to an underestimate in the rates of hepatic glucose production measured by [ 6-3H] glucose; however, if significant losses occur during glycolysis, an overestimate could result (2 1). A recent study performed in humans found evidence for incomplete detritiation of both [2-3H]glucose (minimum 20% 3H retained) and [6-3H]glucose (maximum 10% 3H retained), suggesting that estimates of G/G-6-P cycle activity should be increased by at least 10% of total glucose turnover, or an increase of nearly twofold under basal conditions (32). Although these conclusions may not apply under all conditions, the G/G-6-P cycle activities reported in the present studies should be regarded as minimum estimates rather than absolute values. However, if it is assumed that any underestimate in cycle activity applies equally to both GH and control studies, R,2 - R,6 is a suitable measure of G/G-6-P cycle activity for the purpose of comparison. This would lead to an erroneous conclusion only if GH had induced an increase in cycle activity that was balanced exactly by a decrease in 3H loss from C-2 of G-6-P. Furthermore, our results may be compared directly with those of earlier studies. The enzymatic detritiation method we have used for the in vitro removal of 3H from C-2 of glucose is more specific for C-2 than the chemical (dimedon) method used in the studies of Karlander et al. (18) and is thus less likely to underestimate cycle activity; however, for practical purposes the two methods may be considered equivalent. Indeed, the basal rates of endogenous glucose production ( [6-3H] glucose) and G/G-6-P cycle activity that we have measured in normal subjects (11.1 t 0.4 and 1.5 t 0.2 pmol . kg-l. min-‘) are in close agreement with the values reported for normal controls in the earlier studies (lo.4 t 0.2 and 1.4 t 0.2 pmol kg-l. min-l)( 18). The differences between G/G-6-P cycle activity measured in acromegaly and in the current GH infusion l

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experiments cannot therefore be explained on the basis of methodological discrepancies. Several other explanations may be advanced. First, in glucose-tolerant acromegalic patients hepatic insulin resistance may be more severe than during physiological GH elevation. This is suggested by the increased basal glucose turnover rates found in acromegalic patients (12, 18) and the greater degree by which insulin suppression of endogenous glucose production is impaired in these patients (12) . Second, glucagon is reported to be a potent stimulus to G/G-6-P cycle activity (23). Although hyperglucagonemia is not a consistent finding in acromegalic patients (31), a significant increase in glucagon levels was reported in association with the increase in G/G-6-P cycle activity in those studied by Karlander et al. (18). Finally, long-term tissue changes or other hormonal alterations secondary to the acromegalic state may be responsible. For example, longer (>3 days) GH exposure may stimulate extrathyroidal conversion of thyroxine to triiodothyronine (10) and might thus influence cycle activity indirectly. Even if GH per se does increase G/G-6-P cycle activity on longer exposure, the results of the present study show that such an increase does not occur before the appearance of discernible hepatic insulin resistance. In previous reports which used the euglycemic clamp technique to assess the effect of physiological GH elevation on insulin action in normal subjects, EGP was measured using tritiated glucose tracers in a conventional primed continuous infusion protocol which is prone to negative errors (24). In the present studies, such errors were minimized by the use of highly purified [6-3H]glucose with tracer added to the exogenous glucose infusion to maintain near-steady-state conditions of glucose specific activity during the clamps. During the low insulin-clamp period (step 1, 0.2 mU kg-l amin-l), EGP was suppressed to a lesser extent during GH than control infusions despite slightly higher insulin levels, whereas during the high insulin-clamp period (step 2, 2.0 mU kg -l min-I), EGP was suppressed equally in GH and control studies. Our data therefore provide evidence for a defect of hepatic insulin action that is apparent only at lower insulin levels. Maximal suppression of EGP was to ~25% of basal, and although we cannot exclude a positive error due to a slight rise in plasma glucose specific activity, no “negative values” were seen. Because gluconeogenesis is relatively more resistant to suppression than glycogenolysis (5)) this residual EGP may reflect continued production of new glucose from precursors such as lactate. The slightly lower levels of lactate in the GH studies may be partly offset by increased availability of glycerol for continued production of glucose and stimulation of gluconeogenesis by the increase in circulating FFA (9). The impairment of insulin-stimulated glucose uptake was more striking, particularly at the higher insulin levels in step 2, where a reduction of 38% was seen during GH infusion. We may thus conclude, as did previous authors (4, 25), that sustained physiological elevation of GH induces both hepatic and peripheral insulin resistance but that the latter defect makes a larger contribution to the overall disturbance of glucose metabolism. l

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In the present studies GH infusion caused a marked increase in overnight levels of FFA, glycerol, and /Shydroxybutyrate. This difference persisted in the basal state despite insulin levels that were nearly twofold higher. During the low insulin-clamp period (step 1), FFA, glycerol, and P-hydroxybutyrate were partly suppressed in both GH and control studies; however, in the GH studies the antilipolytic effect of insulin appeared to be transient and incomplete so that levels of all lipid intermediates remained higher than in the control studies. During the high insulin-clamp period (step 2), only P-hydroxybutyrate was completely suppressed, FFA and glycerol remaining higher throughout. Our findings are different from those of Bratusch-Marrain et al. (4), who reported an insignificant increase in the basal FFA levels and normal suppression after infusion of insulin. However, the present data are in keeping with the results of one recent study (1) in which short-term (~4 h) infusion of GH neutralized the antilipolytic effects of a concurrently administered insulin infusion. Our observations indicate that a sustained physiological elevation of GH induces marked resistance to the antilipolytic action of insulin and stimulates hepatic lipid oxidation. Because the basal hyperinsulinemia was apparently sufficient to restrain EGP but insufficient to restrain lipolysis, it appears that under these conditions adipose tissue is relatively more resistant to insulin than liver, which is the reverse of the situation in control subjects (16). Although GH clearly impaired insulin suppression of circulating lipid intermediates, the impairment of insulin-stimulated glucose disposal was not closely related to the circulating levels of FFA measured during the clamp. In contrast, a highly significant correlation was observed between the reduction in the final rate of glucose disposal after GH infusion and the increase in FFA levels overnight (r = -0.836, P < 0.01).This observation is consistent with recent studies (2) demonstrating the timedependent nature of the interaction between lipid and carbohydrate metabolism, so that the inhibitory effect of increased circulating FFA levels on glucose disposal may require some 3-4 h to develop and, once established, may last for several hours after FFA levels have returned to normal. The reduction in step 2 glucose disposal rate was also dependent on the increment in GH levels achieved during GH infusion (r = -0.855, P C O.Ol),and taken together with the increase in overnight FFA levels, these two factors appeared to account for most of the observed impairment in insulin-stimulated glucose disposal (r2 = 0.934, P < 0.001).Because most of the insulinstimulated glucose disposal occurs through the nonoxidative pathway of skeletal muscle glycogen synthesis (29), it seems probable that GH inhibits this pathway directly (1) and also indirectly by increasing the availability of FFA. Inasmuch as the latter effect may require several hours to develop, the contribution of substrate competition (via glucose-fatty acid cycle) to GH-induced insulin resistance may depend on the duration of GH exposure. The increase in fatty acid levels may therefore be of importance in the insulin resistance observed during sustained physiological elevations of GH; however, measurements of substrate oxidation and tissue enzyme activities would be

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necessary to elucidate mechanisms involved. In conclusion, the present studies show that hepatic and peripheral insulin resistance induced by sustained physiological elevation of GH levels is not associated with an increase in the activity of the hepatic G/G-6-P substrate cycle. It is therefore unlikely that abnormal regulation of this cycle contributes to the hepatic insulin resistance induced by GH under these conditions. However, the concentrations of circulating lipid intermediates were increased and showed resistance to suppression by small increments in insulin levels. The relationship between nocturnal elevations of FFA and the subsequent reduction in insulin-stimulated glucose disposal suggests that the glucose-fatty acid cycle may be an important mechanism contributing to the insulin resistance induced by physiological levels of GH. We acknowledge with gratitude the assistance of the nursing staff of the Sir George E. Clark Metabolic Unit and the staff of the Dept. of Pharmacy and the Radioisotope Laboratory, Royal Victoria Hospital, Belfast. We are grateful to Kabi Pharmacia, Stockholm, Sweden, which made a generous donation of recombinant human growth hormone. We also thank C. N. Ennis for expert technical assistance, L. McIlveen for help in preparation of the typescript, and Dr. C. C. Patterson, Dept. of Epidemiology and Public Health Medicine, Queen’s University of Belfast, for advice on statistics. This work was supported by a Department of Health and Social Services, Northern Ireland, Research Grant EB109/4/205. D. P. Rooney is the recipient of a Royal Victoria Hospital Research Fellowship. Address for reprint requests: R. D. G. Neely, Dept. of Clinical Biochemistry, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia. Received 4 October 1991; accepted in final form 1 June 1992. REFERENCES 1.

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Influence of growth hormone on glucose-glucose 6-phosphate cycle and insulin action in normal humans.

Increased activity of the hepatic glucose-glucose 6-phosphate (G/G-6-P) cycle is associated with hepatic and peripheral insulin resistance in acromega...
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