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Defects of Insulin Action and Skeletal Muscle Glucose Metabolism in Growth Hormone-Deficient Adults Persist after 24 Months of Recombinant Human Growth Hormone Therapy¹

The Department of Medicine (M.C., F.L.H., C.R., F.A.), University of Melbourne, Parkville, Melbourne, Victoria 3052; and the Department of Endocrinology and Diabetes (F.L.H., M.O., C.R., F.A.), St. Vincent’s Hospital, Fitzroy, Melbourne, Victoria 3065, Australia.

Address all correspondence and requests for reprints to: M. Christopher, Department of Endocrinology and Diabetes, St. Vincent’s Hospital, Victoria Parade, Fitzroy, Victoria 3065, Australia.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We have previously reported that GH-deficient (GHD) adults are severely insulin resistant. In the present study, we determined the effects of 6 months (n = 7) and 24 months (long-term; n = 11) of recombinant human GH (rhGH) therapy (0.22 IU/kg.week) on body composition and fasting biochemical (including lipid) parameters, and baseline and insulin-stimulated: 1) rates of hepatic glucose production, total glucose disposal (Rd), total glycolysis (GF) and glucose storage (GS); and 2) skeletal muscle glucose processing [using the euglycemic-hyperinsulinemic (60 mU/L) clamp technique with tritiated glucose infusion coupled with skeletal muscle biopsies]. To allow baseline comparison, these measurements were also obtained from 10 control subjects matched to the pretreated GHD adults for age, sex, and body mass index. Long-term rhGH therapy in GHD adults induced significant improvements in fat mass, abdominal fat mass and fat free mass, and reductions in fasting cholesterol and low-density lipoprotein-cholesterol levels (P < 0.05–0.01 vs. pretreatment values). However, there was a significant increase in fasting insulin (13.1 ± 0.9 vs. 8.6 ± 1.1 mU/L; P < 0.01) and connecting peptide (0.56 ± 0.05 vs. 0.41 ± 0.06 nmol/L; P < 0.05). Although rates of baseline hepatic glucose production, GF, and GS were unchanged, the insulin-stimulated increment () in Rd, GF, and GS remained markedly attenuated in the long-term rhGH-treated GHD adults [pretreatment: Rd 16.6 ± 3.4, GF 3.0 ± 1.2, GS 13.6 ± 3.0 vs. 24 months of rhGH: Rd 17.2 ± 3.3, GF 3.1 ± 0.9, GS 14.1 ± 2.5 vs. controls: Rd 42.6 ± 4.3, GF 9.2 ± 1.9, GS 35.9 ± 4.5 µmol/kg fat free mass·min; P < 0.05–0.01 vs. controls]. Additionally, there was a sustained reduction in the insulin-stimulated skeletal muscle glycogen synthase fractional velocity (pretreatment: 0.29 ± 0.03 vs. 24 months of rhGH: 0.24 ± 0.03 vs. controls: 0.48 ± 0.04; both P < 0.05 vs. controls), which was accompanied by a sustained 44% decrease in baseline glycogen content and a 70% increase in baseline im glucose concentrations in the presence of low-to-normal glucose 6-phosphate levels and persisting euglycemia. Stepwise regression analysis revealed that body weight and fasting free fatty acid and high-density lipoprotein (HDL)-cholesterol accounted for 82% of the variance in the insulin sensitivity index in long-term rhGH-treated adults, and that the 24-month fasting insulin-like growth factor 1 was a negative predictor of the change in insulin sensitivity (r = -0.82; P < 0.01). In conclusion, despite improvements in body composition and lipid profiles, the severe defects of in vivo insulin sensitivity and skeletal muscle intracellular glucose phosphorylation and glycogen synthase activity, which are associated with modestly elevated insulin-like growth factor 1 levels, normal free fatty acid levels, and the development of hyperinsulinemia, persist with long-term rhGH therapy.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
RECENT studies have reported a significantly decreased insulin sensitivity in hypopituitary adults with GH deficiency (1, 2, 3, 4, 5). From clamp studies, we have determined that the insulin resistance in these subjects is mainly caused by a defect in the insulin-stimulated glycogen synthase (GlySyn) activity associated with the glucose storage (GS) pathway of skeletal muscle (5). This impaired insulin action is associated with altered body composition, characterized by a decreased fat free mass (FFM) caused mainly by a reduction in extracellular water (ECW) volume (6) and an increased fat mass (FM), predominantly central (abdominal) fat (3, 6). In addition, GH-deficient (GHD) adults have an increased incidence of atheromatous plaque formation (7), which is associated with a higher cardiovascular mortality rate (8) and dyslipidemia (9, 10, 11). The latter feature includes decreased fasting high-density lipoprotein (HDL)-cholesterol levels and low-density lipoprotein (LDL)-particle diameter size (11). It has recently been shown that these body compositional and lipid abnormalities are more evident in adult onset than in childhood onset GHD subjects (12), and in female rather than male GHD adults (13). Thus, GHD adults seem to exhibit the cluster of body compositional and metabolic disturbances associated with the metabolic insulin-resistant syndrome (syndrome X) (3, 5, 11).

Recent trials, investigating the effects of recombinant human GH (rhGH) replacement therapy in GHD adults, have consistently shown a sustained increase in FFM and concomitant reduction in FM (3, 12, 14), particularly a reduction in central obesity (3, 15). In contrast, although insulin sensitivity decreases transiently after 1–6 weeks rhGH treatment (1, 16), whole-body insulin sensitivity seems not to be altered by 3 months (1) or 6 months (16) of low-dose rhGH replacement therapy, compared with pretreatment values. The failure of rhGH therapy to augment insulin sensitivity in GHD adults, despite its favorable impact on body composition, is unexplained but may be caused by the relatively short duration of these studies. The latter concept is supported by the existence of a strong inverse relationship between the degree of insulin sensitivity and the duration of the GHD state (5), indicating that time may be a factor in its reversal. Thus, the impact of long-term rhGH therapy on insulin sensitivity needs to be critically examined in GHD adults.

The intracellular defects of glucose metabolism, present in the skeletal muscle of pretreated vs. rhGH-treated GHD adults, have yet to be studied, but they are of major interest, given our finding of a markedly reduced GlySyn activity in GHD adults (5). Subjects with non-insulin-dependent diabetes mellitus (NIDDM) exhibit a degree of insulin resistance and impairment of GlySyn activity (17, 18) similar to that seen in GHD adults (5), and they also have altered levels of im glucose and/or glucose 6-phosphate (G6P) (18, 19, 20), indicating a significant defect in an early step of intracellular glucose processing (20, 21). This latter conclusion is confirmed by the recent observations of a reduced activity and gene expression of skeletal muscle hexokinase II (HK2) (22) and an impairment of insulin-stimulated glucose phosphorylation and glucose transport in NIDDM subjects (23). Therefore, to further our knowledge of the biochemical basis of the insulin resistance in GHD adults, it is important to measure the im concentrations of these key substrates of glucose metabolism and GlySyn activity during basal and physiologic hyperinsulinemic conditions.

The aims of the present study were, therefore, to determine the effects of 6 and 24 months of rhGH therapy (0.25 IU/kg·week) on insulin sensitivity and muscle glucose processing in GHD adults. More specifically, we employed the euglycemic-hyperinsulinemic clamp technique using a primed-continuous infusion of tritiated glucose ([3-³H]glucose) and exogenous glucose infusion (GINF) prelabeled with [3-³H]glucose (hot GINF), in conjunction with skeletal muscle biopsies (5, 24), to examine the effects of 6 and 24 months of rhGH therapy on basal and insulin-stimulated: 1) in vivo hepatic glucose production (HGP), total glucose disposal (Rd), and the partitioning of glucose metabolism into the pathways of total glycolysis (GF) and GS (5, 24); and 2) in vitro muscle glycogen, glucose and G6P concentrations and GlySyn activity (5, 19). For purposes of baseline comparison, these measurements were also obtained at the outset of the study from a control group carefully matched to the pretreated GHD adults for age, sex, and body mass index (BMI). Additionally, we examined the relationships between the whole body insulin sensitivity index and clinical, body compositional, and fasting biochemical parameters in the GHD adults before and after 24 months of rhGH therapy, and the importance of the fasting insulin-like growth factor 1 (IGF-1) concentrations in improving or worsening insulin action in long-term rhGH-treated GHD adults.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

The 14 GHD adults from our previous study (5), defined by having a peak GH level of less than 5 mU/L, from a standard insulin tolerance test, participated in a randomized double-blind trial, which involved the administration of a sc injection of either rhGH (GENOTROPIN, Pharmacia and Upjohn, Stockholm, Sweden) or placebo, each evening for 6 months (short-term) using a KABIPEN injection device (Pharmacia and Upjohn). The starting dose of rhGH was 0.125 IU/kg·week, increasing to 0.25 IU/kg·week after 4 weeks. Six of the 7 subjects who received rhGH for the first 6 months continued rhGH replacement therapy for a further 18 months, and 5 of the 7 subjects who received placebo for the first 6 months were subsequently treated with rhGH replacement for the next 24 months. Therefore, 11 of the original 14 GHD adults underwent continuous rhGH replacement therapy [average dose 0.222 ± 0.012 IU/kg·week (mean ± SEM)] for 24 months (long-term), with the dose being reduced in 3 of these subjects because of minor side effects such as arthralgia, edema, and/or IGF-1 levels rising above the normal range. Another 3 subjects were withdrawn from the long-term study because of adverse events. In brief, the current study examined the effects of 6 months (n = 7) and 24 months (n = 11) of rhGH replacement on whole-body and regional (abdomen and thigh) body composition, biochemical (including lipid) profiles, and in vivo and in vitro (skeletal muscle) glucose metabolism in GHD adults. The GHD adults had known pituitary pathology (5 nonsecreting adenoma; 2 prolactinoma; 2 Cushing’s disease cured for 4 and 5 yr; and 1 each of craniopharyngioma, idiopathic congenital cyst, and retinoblastoma) and received appropriate stable pituitary hormone (i.e. adrenal, thyroid, sex steroid) replacement for at least 6 months before the baseline (0 months) glucose clamp study (5), as well as throughout the rhGH replacement period. Ten healthy control subjects, carefully matched to the pretreated GHD adults for sex, age, and BMI, were also recruited and studied once only at the outset, to allow baseline comparison. Neither the GHD adults nor the controls had any family history of diabetes mellitus in first-degree relatives nor any clinical evidence of hepatic, renal, or macrovascular disease. Informed written consent was obtained from all subjects, and the study was approved by the Human Research Ethics Committee of St. Vincent’s Hospital.

Whole-body FFM and FM measurements were determined in subjects using the bioelectrical impedance (BIA) method (model BIM 3, SEAC, Boulkham Hills, Australia) and Lukaski’s equation (25) (Tables 1 and 2). These results were compared with those obtained from dual-energy x-ray absorptiometry (DEXA) (model DPX, Lunar Radiation Corp., Madison, WI). Both instruments showed similar FM in the pretreated (BIA 23.2 ± 2.8 vs. DEXA 23.4 ± 2.7 kg; r = 0.98, P < 0.01), 6 months rhGH-treated (BIA 17.8 ± 3.9 vs. DEXA 19.3 ± 2.9 kg; r = 0.91, P < 0.01), and 24-month rhGH-treated (BIA 20.3 ± 2.5 vs. DEXA 22.4 ± 3.1 kg; r = 0.95, P < 0.01) GHD adults. Likewise, when FM was expressed as percent of total body weight [FM(%)], the two instruments produced similar results within each study group (0 months: BIA 30.3 ± 2.7 vs. DEXA 30.4 ± 2.7%, r = 0.94; 6 months: BIA 20.7 ± 3.1 vs. DEXA 22.9 ± 2.0%, r = 0.89; and 24 months: BIA 25.9 ± 2.4 vs. DEXA 28.2 ± 3.1%, r = 0.92; P < 0.01 for all). Abdominal and thigh FFM and FM were determined in GHD adults before (0 months) and after 6 and 24 months of rhGH replacement from DEXA using preselected regions of interest (standard software option), as previously described (26, 27). The mean percent coefficients of variation (CV%) of the measurement of the percent fat of the abdominal and thigh regions were 1.1% and 1.6%, respectively.

All studies were commenced at 0800 h, after each subject had fasted overnight for at least 12 h. Subjects were instructed to ingest at least 150 g carbohydrate daily for 3 days before the study. All GHD adults receiving stable hormone replacement took their usual morning dose on admission to the ward before the study. Infusions were administered via a catheter (Intracath, Becton Dickinson Co., Sandy, UT) placed in an antecubital vein, and blood samples were drawn from a catheter (Angioset, Parke-Davis Co., Sandy, UT) inserted in a contralateral antecubital vein.

After a supine rest period of at least 30 min, fasting blood samples were obtained at -10 and -1 min for measurement of fasting glucose, insulin, connecting peptide (C peptide), free fatty acid (FFA), glucagon, lactate, glycated hemoglobin (HbA_1C), IGF-1, free T₄, cortisol, triglycerides, cholesterol, HDL-cholesterol, LDL-cholesterol, [3-³H]glucose, and tritiated water (³H₂O). A 2-stage clamp study, consisting of a 150-min baseline [3-³H]glucose equilibration period followed by a 150-min euglycemic-hyperinsulinemic (40 mU/kg·h) clamp period, using a variable infusion of exogenous glucose prelabeled with 1.5 µCi/g [3-³H]glucose (hot GINF) (24, 28), was performed as previously detailed (5). Blood glucose levels were determined every 5 min on a portable blood glucose meter (Companion II, Medisense-Abbot, Balwyn, Australia), and the rate of infusion of hot GINF was adjusted accordingly to achieve the subject’s fasting blood glucose level. Steady-state was defined to be reached when both the GINF rate and blood glucose concentration did not alter by more than 10%, over the final 30 min of the hyperinsulinemic clamp, provided that the clamp had proceeded for a minimum of 2 h. Blood samples were obtained at allocated time points throughout both periods for measurement of glucose, insulin, [3-³H]glucose, ³H₂O, C peptide, FFA, glucagon, lactate, and cortisol (5). Blood samples were collected into tubes containing appropriate anticoagulants and preservatives (29), placed on ice, and centrifuged within 2 h; and the separated plasma was stored at -20 C until assayed. Skeletal muscle biopsies were taken from the thigh (vastus lateralis) at the completion of both periods [140–150 min (baseline) and 300–310 min (hyperinsulinemia), respectively] at least 20 min after the sc administration of 1% lignocaine (Xylocaine, Astra Pharmaceuticals, North Ryde, Australia), as previously described (5, 19). Muscle specimens were frozen in liquid nitrogen within 10 sec of biopsy and stored at -70 C until assayed.

Laboratory analyses

Blood biochemistry and in vivo glucose metabolism. Plasma glucose, insulin, C peptide, FFA, glucagon, lactate, cortisol and IGF-1, and serum HbA_1C, free T₄, triglycerides, cholesterol, HDL-cholesterol and LDL-cholesterol levels were measured as previously described (5, 11, 24, 27, 29). The MCR of insulin was calculated from the actual insulin infusion rate and the average plasma insulin and C peptide levels obtained during the final 30 min of both the baseline and hyperinsulinemic clamp periods, as previously described (30).

Rates of in vivo HGP and Rd during the baseline and hyperinsulinemic clamp periods were determined from plasma [3-³H]glucose specific activities at steady-state (31), or, if not at steady-state, by using non-steady-state equations (32). The specific activity of [3-³H]glucose and ³H₂O in plasma samples was determined in our laboratory, as previously described (5, 24). The rate of HGP was calculated as the difference between the rate of Rd and the GINF rate obtained at clamp. In the few studies where the isotopically determined HGP during the hyperinsulinemic clamp was slightly negative, the GINF rate was used as the measure of Rd (24). The measurement of the rate of in vivo GF caused by the generation of plasma ³H₂O from the [3-³H]glucose infusion was estimated from the formula previously described (24, 33). Total body water (TBW) content in each subject was calculated using the BIA method (25). We found that the increment of plasma ³H₂O was linear (defined as r² > 0.95) from 30–140 min of the baseline equilibration period and from 190–300 min of the hyperinsulinemic clamp period, in all GHD adults before and after 6 and 24 months of rhGH therapy, and in the control subjects. The rate of in vivo GS was calculated as the difference between Rd and GF (24). It should be noted that under basal glucose turnover conditions, the rate of GF from extracellularly derived glucose may underestimate the rate of whole-body GF by an amount equal to the residual rate of glycogenolysis (derived endogenously from unlabeled glycogen) (34). This unknown amount of endogenously derived glucose seems to contribute less than 20% towards the rate of whole-body GF (24, 33). Therefore, using this methodology, the calculated rate of GS at baseline would mostly represent glycogen turnover, with no net glycogen formation (35, 36). All isotopically determined glucose metabolic data were normalized to FFM. Rates of GF and GS were determined in all GHD adults before and after 6 and 24 months of rhGH replacement, but in only 6 of the 10 matched control subjects. The insulin sensitivity indexes of GINF, HGP, Rd, GF, and GS were expressed as the increment or decrement () in each glucose metabolic parameter per increment in plasma insulin (Ins) induced by the hyperinsulinemic clamp, compared with baseline values (5).

In vitro skeletal muscle glucose metabolism. Muscle samples, obtained at the completion of the baseline and hyperinsulinemic clamp periods, were analyzed for glycogen content, total glucose and G6P concentrations, and fractional velocity (FV0.1) ratio of GlySyn. Glycogen was determined as glucose residues after enzymatic hydrolysis of the perchloric acid extracts of muscle (20 mg wet wt) with amyloglycosidase (Boehringer Mannheim, Mannheim, Germany) for 2 h at 40 C, pH 4.8, using a modification of the method described by Keppler and Decker (34, 37). Total glucose and G6P concentrations were measured on the same neutralized perchloric acid extracts of muscle (30–40 mg wet wt) using a modification of the method described by Schalin-Jäntti et al. (18). All substrates were measured spectrophotometrically at 340 nm (25 C; 10 mm light path) via the coupled G6P-dehydrogenase and hexokinase reactions, by following the change in absorbance caused by the reduction of NADP. Glycogen content is expressed as mmol glucose residues per kilogram of muscle dry weight. Total glucose and G6P concentrations are expressed as millimoles of substrate per kilogram of muscle dry weight.

The extraction of muscle samples (30 mg wet wt) and subsequent assay of GlySyn, using enzymatic analysis and spectrophotometric detection at 340 nm (pH 7.4; 30 C), was performed as previously described (5, 34, 37). GlySyn activity was measured in the presence of 0, 0.1, and 10.0 mmol/L G6P (Boehringer Mannheim) and 3.0 mmol/L uridine diphosphoglucose (5, 34). GlySyn activity is expressed as micromoles of substrate used per gram of muscle wet weight per minute. The FV0.1 of GlySyn was calculated as the ratio of GlySyn activities determined at 0.1 mmol/L vs. 10 mmol/L G6P. Muscle biopsies were performed at baseline and during the hyperinsulinemic clamp period in 9 of the 11 GHD adults before and after 24 months of rhGH replacement, 6 of the 7 GHD adults after 6 months of rhGH replacement, and 6 of the 10 matched control subjects. However, muscle from the matched control subjects was analyzed for glycogen content and GlySyn activities only.

Statistics

Data are presented as the mean ± SEM. Differences within groups were determined using Wilcoxon’s matched-pairs signed-rank test. Differences between the following groups were also determined using Wilcoxon’s matched-pairs signed-rank test: 1) pretreated (0 months) vs. 24-month rhGH-treated GHD adults (n = 11); and 2) pretreated vs. 6 months rhGH-treated GHD adults (n = 7). Differences between the matched control groups (n = 10) vs. both the pretreated and 24-month rhGH-treated GHD adults were determined using Wilcoxon’s rank sum test. Correlation analyses were performed using Spearman’s rank correlation coefficient (r). P < 0.05 was considered significant. Stepwise (default), best-subsets, and multiple-regression analyses were performed using the statistical analysis program Minitab (Minitab, State College, PA).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The clinical and fasting biochemical characteristics of the GHD adults before and after 24 months (long-term) of rhGH replacement therapy and of sex-, age-, and BMI-matched control subjects are shown in Table 1. Although the pretreated (0 months) GHD adults had a slightly lower FFM and higher waist-to-hip ratio (WHR) than the matched control subjects, these differences did not reach statistical significance. However, both the FM and FM(%) were significantly elevated in the pretreated GHD adults, compared with the control group. There was no statistical difference in HbA_1C or any of the fasting lipid measurements between these two groups, but IGF-1 levels were slightly lower in the pretreated GHD adults [P = NS (not significant)]. After 24 months of rhGH replacement, there was a significant increase in body weight, BMI, and FFM, and a significant decrease in FM, FM(%), and WHR in the GHD adults, compared with their pretreatment values. Interestingly, none of the clinical characteristics at 24 months, including FM and FM(%), were now statistically different from those observed in the control subjects. In addition, TBW, when expressed as percent of body weight (TBW%), as calculated from the BIA method (25), was significantly lower in the pretreated GHD adults, compared with the control subjects (51.3 ± 2.0 vs. 55.3 ± 1.9%; P < 0.05) but returned towards control values after 24 months of rhGH replacement (55.2 ± 2.1%; P = NS vs. controls). There was also a significant 2.6-fold increase in IGF-1 levels and a decrease in both cholesterol and LDL-cholesterol concentrations in GHD adults after 24 months of rhGH replacement, compared with the pretreatment values. However, none of the fasting biochemical parameters at 24 months were statistically different from those obtained in the control subjects. All GHD adults were biochemically and clinically euthyroid both before and after 24 months of rhGH therapy, and free T₄ concentrations were similar at both time points (0 months: 13.5 ± 0.8 vs. 24 months: 14.5 ± 1.3 pmol/L; P = NS). There was no statistical difference in the time-averaged plasma cortisol levels from 0–280 min of the study period (0 months: 347 ± 63 vs. 24 months: 283 ± 34 vs. controls: 203 ± 61 nmol/L·min; P = NS).

In GHD adults, the effects of 6 months (short-term) of rhGH replacement on clinical and fasting biochemical characteristics is shown in Table 2. Similar to the clinical improvements induced by 24 months of rhGH replacement, there was a significant increase in FFM and a decrease in both FM and FM(%) in GHD adults after 6 months of rhGH replacement, compared with the pretreatment values; but body weight, BMI, and WHR were unchanged. TBW% increased significantly after 6 months of rhGH replacement (0 months: 52.8 ± 1.8 vs. 6 months: 58.6 ± 2.3%; P < 0.02). Concerning the fasting biochemical parameters, 6 months of rhGH therapy in the GHD adults induced a significant 2.5-fold rise in IGF-1 levels but failed to elicit any significant changes in HbA_1C levels or any of the simple lipids (data not shown). However, in contrast to the 24-month data, free T₄ levels were significantly reduced by 6 months of rhGH replacement (0 months: 13.9 ± 0.8 vs. 6 months: 9.9 ± 1.3 pmol/L; P < 0.05). There was no difference in time-averaged plasma cortisol levels from 0–280 min of the study period (0 months: 246 ± 32 vs. 6 months: 243 ± 34 nmol/L·min; P = NS).

Regional body composition (abdominal and thigh) was determined in GHD adults before (0 months) and after 6 and 24 months of rhGH replacement. In the abdominal region of interest, FFM was not different at 0 and 24 months (4.05 ± 0.32 vs. 4.14 ± 0.25 kg; P = NS), but both FM and FM(%) were reduced significantly after 24 months of rhGH therapy [0 months: FM 2.75 ± 0.37 kg; FM(%) 39.0 ± 2.6% vs. 24 months: FM 2.15 ± 0.27 kg; FM(%) 33.5 ± 3.3%; P < 0.05 for FM, P < 0.01 for FM(%)]. In the midthigh region of interest, FFM increased significantly after 24 months of rhGH replacement (0 months: 1.13 ± 0.05 vs. 24 months: 1.27 ± 0.06 kg; P < 0.05). Although FM was not different at 0 and 24 months (0.53 ± 0.08 vs. 0.50 ± 0.09 kg; P = NS), FM(%) decreased significantly in the thigh after 24 months of rhGH replacement (31.1 ± 3.4 vs. 27.4 ± 3.6%; P < 0.01). Six months (short-term) of rhGH replacement induced very similar changes in both abdominal and thigh composition (both in absolute and statistical terms) in GHD adults, compared with that seen after 24 months of rhGH replacement (data not shown).

For the clamp studies, the mean CV% of the plasma glucose levels during the final 30 min of both the baseline and hyperinsulinemic clamp periods, in the pretreated GHD adults, was 2.5 ± 0.6% and 5.4 ± 0.9%, respectively. Likewise, these paired mean CV% values were 2.2 ± 0.3% and 4.7 ± 0.6% for the 6 months rhGH-treated GHD adults, 2.6 ± 0.5 and 5.1 ± 1.5% for the 24-month rhGH-treated GHD adults, and 2.0 ± 0.3% and 4.6 ± 1.0% for the control subjects. In addition, the paired mean CV% values of the plasma insulin levels during the final 30 min of both the baseline and hyperinsulinemic clamp periods, in the GHD adults before and after 6 and 24 months of rhGH therapy, and the control subjects were 10.3 ± 1.7% and 5.4 ± 0.9%, 8.3 ± 1.6% and 6.9 ± 1.3%, 11.8 ± 1.7% and 4.9 ± 0.9%, and 10.5 ± 2.8% and 6.1 ± 1.5%, respectively.

The plasma glucose levels during the final 30 min of the hyperinsulinemic clamp were not different from the corresponding values obtained during the final 30 min of the [3-³H]glucose equilibration period (baseline) for any of the groups (Table 3). However, the baseline glucose levels in the GHD adults, both before (0 months) and after 24 months of rhGH replacement, were significantly decreased, compared with the matched control subjects (both P < 0.01). There was no difference in baseline insulin levels in the pretreated GHD adults, compared with the control subjects. However, baseline insulin levels in GHD adults after both 6 and 24 months of rhGH replacement rose significantly (by 50%), compared with pretreatment values. Similarly, the baseline insulin levels obtained in GHD adults after 24 months of rhGH therapy were elevated significantly, compared with the matched control group. Plasma insulin levels rose approximately 5- to 6-fold during the hyperinsulinemic clamp, compared with the baseline period in all groups, and the increment in plasma insulin levels (Ins) induced by the hyperinsulinemic clamp, compared with the baseline period, was not different among any of the groups. Similar to insulin, there was no difference in baseline C peptide levels in the pretreated GHD adults, compared with the control group, and there was a significant increase (by 37%) in baseline C peptide concentrations in GHD adults after 24 months of rhGH replacement, compared with the pretreatment values. In contrast to insulin, however, no difference was observed in the baseline C peptide levels obtained in the 24-month rhGH-treated GHD adults, compared with the matched control subjects. Although the C peptide concentrations decreased slightly at clamp, compared with the corresponding baseline period in all groups, this reduction was found to be significant only in the 24-month rhGH-treated GHD adults. The MCR of insulin did not differ among any of the groups (0 months: 26.1 ± 3.7 vs. 6 months: 21.3 ± 7.5 vs. 24 months: 25.0 ± 3.4 vs. controls: 27.3 ± 3.4 mL/kg FFM·min; P = NS for all).

In vivo glucose metabolism

As shown in Fig. 1A, there was a slight, but significant reduction in the baseline rate of HGP in the 24-month rhGH-treated adults, compared with the pretreated (0 months) GHD adults (13.6 ± 0.4 vs. 15.6 ± 0.8 µmol/kg FFM·min; P < 0.02). However, neither of these baseline values differed from that of the matched control group (14.1 ± 0.9 µmol/kg FFM·min). The hyperinsulinemic clamp significantly suppressed the rate of HGP in all three groups (P < 0.01 vs. corresponding baseline value for each). However, the clamp rate of HGP was significantly lower in the 24-month rhGH-treated GHD adults, compared with the pretreated GHD adults. In contrast, the degree of suppression (HGP) induced by hyperinsulinemia, compared with the corresponding baseline value, was not different among any of the groups (0 months: 10.5 ± 1.1 vs. 24 months: 13.0 ± 0.5 vs. controls: 12.2 ± 1.0 µmol/kg FFM·min; P = NS for all). During the hyperinsulinemic clamp period, the rate of Rd was increased significantly in all groups (P < 0.01 vs. corresponding baseline value for each). (Fig. 1B). However, the rate of Rd at clamp was significantly lower (by 45%) in both the pretreated and 24-month rhGH-treated GHD adults, compared with the control group. Furthermore, the degree of stimulation (Rd) induced by hyperinsulinemia above the corresponding baseline value was markedly attenuated (by 60%) in both the pretreated and 24-month rhGH-treated GHD adults, compared with the matched controls (0 months: 16.6 ± 3.4 vs. 24 months: 17.2 ± 3.3 vs. controls: 42.6 ± 4.3 µmol/kg FFM·min; P < 0.01 and P < 0.05, respectively, vs. controls).

View larger version (30K): [in this window] [in a new window]   Figure 1. Steady-state rates of in vivo HGP (panel A) and Rd (panel B) from baseline (B) samples obtained during the final 30 min of a 150-min [3-3H]glucose equilibration period, and from clamp (+Ins) samples obtained during the final 30 min of a euglycemic-hyperinsulinemic clamp in 11 GHD adults before (0 months) and after 24 months of rhGH replacement therapy, and from 10 matched control subjects. In all groups, there was a significant suppression of HGP and a significant rise in Rd during the hyperinsulinemic clamp, compared with the baseline period (P < 0.01 for all). The histograms represent the means ± SEM.

View larger version (26K): [in this window] [in a new window]   Figure 2. Steady-state rates of in vivo GF (panel A) and GS (panel B) from baseline (B) samples obtained during the final 30 min of a 150-min [3-3H]glucose equilibration period, and from clamp samples (+Ins) obtained during the final 30 min of a euglycemic-hyperinsulinemic clamp in 11 GHD adults before (0 months) and after 24 months of rhGH replacement therapy, and from 6 matched control subjects. In all groups, there was a significant rise in GF (P < 0.05 for 0 months and controls; P < 0.01 for the 24-month rhGH group) and GS (P < 0.05 for controls; P < 0.01 for 0 months and 24-month rhGH groups) during the hyperinsulinemic clamp, compared with the baseline period. The histograms represent the means ± SEM.

In vitro skeletal muscle glucose metabolism

The results of skeletal muscle glycogen content, total glucose and G6P concentrations, and FV0.1 of GlySyn at baseline and during the hyperinsulinemic clamp are shown in Table 5. Baseline muscle glycogen content was reduced significantly by 31% and 44%, respectively, in the pretreated and 24-month rhGH-treated GHD adults, compared with the matched control subjects (P < 0.05 for both). Furthermore, the baseline muscle glycogen content of the 24-month rhGH-treated adults was significantly lower than the corresponding pretreatment (0 months) value. Insulin stimulation did not alter glycogen content in any of the groups. Baseline muscle total glucose levels were markedly elevated (by 63%) in both the pretreated and 24-month rhGH-treated GHD adults, compared with the unmatched control group, despite the presence of normoglycemia in all subjects. The hyperinsulinemic clamp did not induce significant alterations in muscle total glucose levels in the pretreated or 24-month rhGH-treated GHD adults. Baseline muscle G6P concentrations were slightly, but not significantly, lower in the 24-month rhGH-treated GHD adults, compared with the pretreated subjects, and possibly lower than the baseline G6P concentrations obtained in the unmatched control group. Insulin stimulation induced a slight, but not significant, decrease in muscle G6P concentrations in both the pretreated and 24-month rhGH-treated GHD adults, compared with the corresponding baseline values. The baseline FV0.1 of muscle GlySyn clamp was not different among any of the groups. As expected, the hyperinsulinemic clamp induced a striking (100%) increase in the FV0.1 of GlySyn in the matched control subjects. In contrast, insulin stimulation induced only a slight, but nonsignificant, rise in the FV0.1 of GlySyn in both the pretreated and 24-month rhGH-treated GHD adults. In fact, the increment in the FV0.1 of GlySyn (FV0.1) induced by the hyperinsulinemic clamp, compared with the baseline period, was significantly attenuated (by 67% and 79%) in the pretreated and 24-month rhGH-treated GHD adults, respectively, compared with the matched controls.

To identify the mechanisms that may contribute to the alterations in the body compositional, biochemical, and insulin action profiles of the GHD adults induced by 24 months of rhGH replacement, simple correlations were initially performed between: 1) fasting IGF-1 levels vs. the changes in body compositional and fasting biochemical parameters; 2) in vivo vs. in vitro (muscle) glucose metabolism parameters; and 3) clinical/body compositional and fasting biochemical parameters vs. insulin sensitivity, expressed as the in vivo insulin sensitivity index, Rd/Ins. Subsequently, the parameters that were found to correlate significantly with Rd/Ins in the pretreated and 24-month rhGH-treated GHD adults (Table 6) were examined in greater detail by multiple, best-subsets, and stepwise (default) regression analyses to select those independent predictors that could account for the majority of the severe and sustained insulin resistance seen in these subjects.

View this table: [in this window] [in a new window]   Table 6. Correlation analyses of clinical, body compositional and fasting biochemical parameters vs. in vivo index of insulin sensitivity (Rd/Ins) in 11 GHD adults before (0 months) and after 24 months rhGH replacement therapy

Relationships between in vivo vs. in vitro (muscle) glucose metabolism parameters. In the matched control subjects, baseline muscle glycogen content inversely correlated with FV0.1/Ins (r = -0.87; P < 0.05), and GS/Ins positively correlated with FV0.1/Ins (r = 0.98; P < 0.01). In the pretreated (0 months) GHD adults, baseline muscle glycogen content inversely correlated with baseline muscle total glucose (r = -0.68; P < 0.05). However, in contrast to the matched controls, baseline muscle glycogen content did not correlate with FV0.1/Ins, and GS/Ins did not correlate with FV0.1/Ins, suggesting that other factors may be influencing net glycogen synthesis in these subjects. Interestingly, both baseline muscle glycogen content and FV0.1/Ins negatively correlated with duration of GH deficiency. After 24 months of rhGH replacement in the GHD adults, baseline muscle glycogen content remained negatively correlated with baseline muscle total glucose (r = -0.63; P < 0.05), and both baseline muscle glycogen content and GS/Ins still showed no correlation with FV0.1/Ins.

Relationships between clinical/body composition and biochemical parameters vs in vivo insulin sensitivity index, Rd/Ins. Of the clinical and body compositional characteristics shown in Table 1, only body weight correlated significantly with the in vivo insulin sensitivity index, Rd/Ins, in the matched control subjects (r = -0.67; P < 0.05). Likewise, of the fasting biochemical parameters shown in Tables 1 and 3, only FFA correlated significantly with Rd/Ins in the control subjects (r = -0.76; P < 0.05). The relationships between clinical/body compositional and biochemical parameters vs. Rd/Ins in GHD adults before (0 months) and after 24 months of rhGH replacement are summarized in Table 6. In the pretreated GHD adults, only one clinical characteristic, namely duration of GH deficiency, (and importantly, no body composition parameter) showed a significant correlation with Rd/Ins. Of the fasting biochemical parameters, a total of 4 (namely insulin, IGF-1, triglycerides, and cholesterol) significantly (negatively) correlated with Rd/Ins. Importantly, fasting FFA did not correlate with Rd/Ins. After 24 months of rhGH replacement therapy in the same GHD adults, duration of GH deficiency remained negatively correlated with Rd/Ins. Interestingly, however, three body composition parameters (namely body weight, FM, and abdominal FM) now correlated (negatively) with Rd/Ins. Of the fasting biochemical parameters, triglycerides and cholesterol remained negatively correlated with Rd/Ins, and FFA and HDL-cholesterol now correlated with Rd/Ins, but insulin and IGF-1 no longer correlated with Rd/Ins.

To more closely examine these variables and their relationship with Rd/Ins, multiple, best-subsets, and stepwise (default) regression analyses were employed, as previously described (5), to select the independent predictors that could account for the majority of the Rd/Ins value obtained in the GHD adults, both before and after 24 months of rhGH replacement. In the case of the pretreated GHD adults, when the five variables in question (Table 6) were combined against Rd/Ins, using multiple linear regression analysis, an r² (adjusted) value of 78.1% was obtained. When stepwise (default) and best-subsets regression analyses were performed to select the best-fitting models (based on the maximum r² value) incorporating from two to five variables, it was found that duration of GH deficiency, IGF-1 and triglycerides contributed substantially to the overall relationship with Rd/Ins [r² (adjusted) = 82.0%].

Likewise, of the eight variables that correlated significantly with Rd/Ins in the 24-month rhGH-treated GHD adults (Table 6), it was found that FM, triglycerides, and cholesterol each failed to fit the simple linear regression model with Rd/Ins (P > 0.05), thereby eliminating them from further analysis. The remaining five variables were then combined against Rd/Ins, using multiple linear regression analysis, yielding an r² (adjusted) value of 87.3%. Best-subsets regression analysis of these five variables revealed that body weight (rather than abdominal FM) formed the strongest association with the other three independent predictors in accounting for the majority of the variance in the measurement of Rd/Ins. Subsequently, when stepwise (default) regression analysis was performed on these four independent predictors, it was found that body weight and fasting HDL-cholesterol and FFA levels at 24 months combined to produce the best fitting 3-predictor model, yielding an r² (adjusted) value of 82.0%. A marginally less satisfactory fit to the model was achieved by a 2-independent predictor model (fasting HDL-cholesterol and FFA concentrations at 24 months), resulting in an r² (adjusted) value of 79.8%. The regression equation is: = 0.934 + 0.264 (fasting HDL-cholesterol) - 0.900 .

Finally, on inspection of the individual data at 24 months, we noted that, although the mean Rd/Ins remained low in the GHD group, as a whole, after 24 months of rhGH therapy (despite the significant reduction in central obesity), insulin sensitivity did improve in some individuals. We therefore examined which body compositional and biochemical parameters might influence the direction of change in Rd/Ins. There was a striking negative correlation between the change in Rd/Ins induced by 24 months of rhGH therapy with both the 0 and 24 month fasting IGF-1 levels (r = -0.89 and r = -0.82, respectively; P < 0.01 for both) (Fig. 3A). In addition, this change in Rd/Ins was negatively correlated with both the increment in fasting IGF-1 levels (r = -0.66; P < 0.05), and the change in fasting triglyceride levels (r = -0.70; P < 0.05) (Fig. 3B). These correlations indicate that GHD adults should benefit from an improved insulin sensitivity after long-term rhGH therapy, provided that their posttreatment fasting IGF-1 level does not exceed 40 nmol/L and/or their fasting triglyceride level either does not change or decreases.

View larger version (18K): [in this window] [in a new window]   Figure 3. The relationship between the change () in the insulin sensitivity of Rd (Rd/Ins) induced by 24 months of rhGH replacement therapy in 10 GHD adults (compared with the pretreatment values) vs. the fasting IGF-1 concentration at 24 months (r = -0.82; P < 0.01) (panel A), and the in the fasting triglyceride concentration (r = -0.70; P < 0.05) (panel B). The dashed line indicates the intercept point corresponding to a (Rd/Ins) value of zero.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In the present study, and as reported previously (5), our pretreated GHD adults were severely insulin resistant, compared with a carefully matched control group, despite normal fasting levels of glucose, insulin, C peptide, and FFA. Clearly, the pretreated GHD adults had significantly elevated FM and FM(%), compared with the matched control subjects. In insulin resistant states, such as simple obesity and NIDDM, the increase in truncal FM is associated with reduced insulin action, hyperinsulinemia, and increased FFA levels (40, 41). However, as pointed out recently by Ludvik et al. (42), obesity accounts only for approximately 40% of the insulin resistance of NIDDM, implying that other mechanisms must be responsible for the overall insulin resistance of these subjects. In our GHD adults, none of the clinical obesity measurements correlated with the in vivo index of insulin sensitivity (Rd/Ins), and fasting insulin and FFA levels were not raised. In fact, 82% of the variance in Rd/Ins was attributable to the duration of GH deficiency and to fasting IGF-1 and triglyceride levels. Supportive of these data, it has been shown that the reduced insulin sensitivity in GHD adults is associated with a significant reduction in LDL-particle diameter (11, 27), and that attenuated GH secretion and IGF-1 levels are seen in insulin resistant nondiabetic obese subjects (43, 44). Moreover, the duration of GH deficiency in our subjects was inversely correlated to the baseline muscle glycogen content and insulin-stimulated GlySyn activity, suggesting a possible link between the duration of GH deficiency and/or IGF-1 levels and the defects associated with skeletal muscle glucose metabolism.

After 24 months of rhGH therapy (0.222 ± 0.012 IU/kg·week) in the GHD adults, a significant increase (5 kg) in whole-body FFM and a corresponding decrease (3 kg) in FM occurred. This 12.5% reduction in FM was mirrored by a significant decrease (21.8%) in abdominal FM and was associated with a significant reduction in both fasting cholesterol and LDL-cholesterol levels. In agreement with previous studies (1, 15, 16, 27), fasting HbA_1C and glucose levels did not change with long-term rhGH therapy, and this is consistent with the data of Beshyah et al. (45), which showed that glucose tolerance was unchanged in GHD adults after 6 months of rhGH therapy and that both fasting glucose levels and the glucose under the curve during an oral glucose tolerance test was unchanged after 18 months of rhGH therapy. This apparently unaltered glucose tolerance may have been maintained by the associated ß cell hyperfunction (1, 3, 45). Fasting insulin and C peptide levels significantly increased after 24 months of rhGH therapy, which is consistent with most previous reports (1, 3, 14, 27, 45), and probably represents true hypersecretion, because the MCR of insulin was not altered by rhGH therapy. Interestingly, similar to an earlier study (14), we found a greater percent increase in fasting insulin levels than C peptide levels with long-term rhGH therapy, suggesting a possible GH-induced alteration in the metabolic clearance kinetics of C peptide, relative to insulin. In vivo whole-body insulin sensitivity remained significantly decreased by approximately 43% in our 24-month rhGH-treated GHD adults, compared with the matched control subjects, and the severe defect in the insulin-stimulated GS rate persisted. Additionally, there was a deterioration in the insulin sensitivity of the GF pathway after 24 months of rhGH therapy. This unchanged (or slight worsening of) insulin resistance after rhGH therapy was similar to the result obtained by Weaver et al., who used the Homeostatic Model of Assessment to measure insulin sensitivity (3). Similar to that 12-month study, we found that the insulin resistance was related to the subject’s obesity after treatment with rhGH, which suggests that GH alters the degree of adiposity to match the prevailing insulin sensitivity and lipid composition of these subjects, in accord with the relationships that are expected between these measurements in the general population (3).

From the simple and multiple-regression analyses, the degree of general obesity (expressed as body weight), and fasting FFA and HDL-cholesterol levels, were strong, independent predictors of the sustained insulin resistance in our 24-month rhGH-treated GHD adults. This is consistent with the relationships observed in NIDDM and simple obesity, whereby the insulin resistance correlates with abdominal fat (40, 41) and involves predominantly (but not exclusively) the GS-GlySyn pathway of skeletal muscle (17, 18, 19), and in which abnormal FFA metabolism has been implicated as a major factor in the genesis of the insulin resistance (46). Additionally, the fasting IGF-1 level after 24 months of rhGH therapy was found to be a negative predictor of the change in insulin sensitivity. This observation supports the recent findings that a lower replacement dose of rhGH (0.10 IU/kg·week) is required if optimal body compositional, lipid profile, and IGF-1 responses are to be achieved (13, 47). Thus, the combination of ongoing hyperinsulinemia and raised fasting IGF-1 levels and normal FFA levels are implicated in the genesis of the persisting insulin-resistant state in the long-term rhGH-treated GHD adults.

In contrast to other insulin-resistant states, however, our pre- and post-rhGH-treated GHD adults displayed a unique combination of defects associated with in vitro skeletal muscle glucose metabolism. These defects included an ongoing severe inhibition of insulin-stimulated GlySyn activity, which was accompanied by a reduced baseline glycogen content, low-to-normal G6P levels, and high total (and, therefore, by inference) high intracellular glucose concentrations in the presence of persisting euglycemia. This combination of abnormalities is not seen in other insulin-resistant states associated with reduced insulin-stimulated GlySyn activity, such as obesity and NIDDM (17, 18, 48). Additionally, the expected relationships that exist in controls and in obese and NIDDM subjects, between the insulin-stimulated muscle GlySyn activity vs. both baseline glycogen content and the rate of GS (17, 19, 34, 48), were not seen in our GHD adults before or after rhGH therapy. The combination of high intracellular glucose concentrations and low-to-normal G6P levels suggests a prime defect in skeletal muscle glucose processing at the level of glucose phosphorylation (via reduced HK2 activity), which simultaneously and proportionally limits the rate of the key intracellular pathways of glucose metabolism, namely GS and GF.

The baseline muscle total glucose concentrations in our pre- and post-rhGH-treated GHD adults were at least 50% higher than those values obtained in muscle biopsies from our control subjects and from other control groups (18, 49, 50, 51, 52, 53). To calculate the concentration of muscle intracellular glucose, it was assumed that: 1) the concentration of interstitial glucose is equivalent to the plasma glucose concentration; 2) there is 0.3 L of ECW per kilogram of dry weight of muscle (54); and 3) the ECW-to-intracellular water ratio is 0.91 in healthy subjects (6). However, TBW was reduced by 3.1 L in the pretreated GHD adults, compared with the matched controls, and one would therefore predict a reduction of approximately 15% in the ECW content in our GHD adults (from 0.3 to 0.25 L/kg dry wt muscle). Thus, the calculated average baseline muscle intracellular glucose concentration in the pretreated GHD adults would be approximately 3.7 mmol/kg dry wt, far exceeding any values obtained in comparable resting control subjects (19, 49, 51). After 24 months of rhGH therapy, ECW content is normalized in the GHD adults (15), and the calculated baseline muscle intracellular glucose concentration would remain at approximately 3.7 mmol/kg dry wt muscle, still far exceeding the calculated value of approximately 1.4 mmol/kg dry wt for our control group. Hence, by inference, the increase in im total glucose concentration found in the GHD adults, compared with control subjects, is attributable to an increase in muscle intracellular glucose, with little change in the muscle extracellular or plasma glucose concentrations. Furthermore, despite the severe reductions in the insulin-stimulated GS and GF rates with the presumed subsequent decreased use of G6P, the low-to-normal baseline G6P levels (18, 19, 50, 51, 52, 53) were not raised during the hyperinsulinemic clamp, which underscores the intensity of the glucose phosphorylation defect in our GHD adults.

The activity of HK2, the enzyme which regulates phosphorylation of glucose in human skeletal muscle (55), is known to be reduced in impaired glucose-tolerant prediabetic individuals (56) and in muscle biopsy samples from NIDDM subjects (22). Additionally, studies employing dynamic position emission tomography (23) or glucose modeling techniques (21) have found an impaired rate of glucose phosphorylation in NIDDM subjects. Thus, impaired activity of HK2 in muscle of insulin-resistant subjects is not unique to GHD adults, but whether it is secondary to a direct effect of GH and/or enhanced FFA availability (22, 57) is not known. Interestingly, in our study, the baseline muscle total glucose concentration was inversely correlated to the baseline im glycogen content, which implies that the severity of the glucose phosphorylation defect was related to the severity of the impairments in both the GS and GF rates.

The apparent accumulation of muscle intracellular glucose in our pre- and post-rhGH-treated GHD adults suggests that glucose phosphorylation, and not glucose transport, is rate limiting for glucose disposal under conditions of physiologic hyperinsulinemia. However, from the available data, we cannot discard the possibility that a defect exists in muscle glucose transport but is masked by the severity of the glucose phosphorylation defect. It is known that both glucose transport and intracellular phosphorylation of glucose are impaired in NIDDM subjects (21, 23). An inhibitory effect of GH on insulin-mediated (58) and glucose-mediated (59) glucose transport has been claimed, but proof of the mechanism(s) involved is lacking. GH does not influence the insulin-regulatable glucose transporter, GLUT 4 (60), but it may reduce the in vitro activity of insulin-sensitive hexose transport (61).

Recent studies have shown that increased FFA availability, secondary to direct GH-induced lipolysis (62) or via GH-enhanced sensitivity of adipocytes to lipolysis by ß adrenergic stimulation or cortisol (63), may inhibit insulin activation of GlySyn (46, 57, 64, 65). A role for ongoing lipolysis with increased FFA availability in our long-term treated GHD adults was supported by the results of stepwise regression analysis, and by the fact that fasting FFA levels were related to abdominal FM (r = 0.63; P < 0.05) and to the duration of the GH deficiency (r = 0.70; P < 0.05) after 24 months of rhGH treatment. The clinical evidence of a sustained reduction of visceral and total body fat during rhGH therapy, despite basal hyperinsulinemia, further supports the view that the ongoing GH-induced lipolysis in these subjects exceeds the lipotrophic effect of insulin (14, 66) and may lead to impaired peripheral glucose metabolism (67), resulting in sustained insulin resistance. In addition, inhibition of muscle GlySyn may be mediated by small changes in muscle membrane FFA content (68) and/or by a direct influence of membrane-located FFA on an early step in the insulin signal activation cascade via the phosphatidylinositol 3-kinase (PI 3-kinase) activation pathway (69). Enhanced FFA availability is also known to inhibit glucose oxidation (67) and to directly limit glucose phosphorylation (22, 57). Therefore, increased availability of FFA may be important in the development of the insulin resistance in GHD adults.

However, other studies question whether the development of insulin resistance and decreased GlySyn activity subsequent to GH therapy is actually mediated by the increased lipolysis, lipid availability, and lipid oxidation (70, 71, 72). Although Hettiarachchi and co-workers (72) demonstrated an early GH-induced lipolysis and raised FFA-induced inhibition of muscle glucose uptake in normal rats infused with a modest physiologic dose of GH for 5 h, muscle glycogen synthesis and insulin action remained markedly reduced after 3 days of GH infusion, despite the presence of lower plasma FFA and triglyceride levels and reduced hepatic total long-chain acetyl CoA levels, compared with control rats. Moreover, Bak et al. found markedly reduced insulin-stimulated muscle GlySyn activity before a rise in FFA levels in healthy controls exposed to 6 h of rhGH, thereby postulating a direct (non-FFA-mediated) GH-induced inhibition of GlySyn (70). Although these latter studies show that GH-induced elevation of FFA levels is not always necessary to produce GH-associated insulin resistance in control groups, they do not exclude a role for FFA in the insulin resistance of rhGH-treated GHD adults. Interestingly, in acromegalic subjects with chronic GH excess, severe insulin resistance occurs, despite the presence of a normal lipid supply and a normal or slightly decreased lipid oxidation, compared with matched control subjects, after a glucose load (73).

Among the more striking metabolic changes induced by long-term rhGH treatment in the GHD adults are the raised IGF-1 levels and the onset of chronic fasting hyperinsulinemia induced by the pharmacologic surge of nocturnal GH after the evening injection of rhGH. Despite the chronic hyperinsulinemia, fasting FFA levels were not suppressed. Apart from acromegaly, in which insulin resistance is also present (74), this is a unique metabolic model, which defies the usual reciprocal relationships that occur between IGF-1 and insulin secretion (75) and between insulin and FFA levels (46). IGF-1 has its own specific receptors in muscle, whereas insulin receptors are present in muscle and adipocyte cell membranes (75). Although IGF-1 acts both through its own receptor and the insulin receptor, and insulin acts via its own receptor only (76), both hormones jointly activate insulin receptor substrate 1 tyrosine phosphorylation, with its subsequent activation of phosphatidylinositol (PI) 3-kinase pathway (76, 77). It is the activation of the PI 3-kinase pathway that activates GlySyn (69) and GLUT 4 mobilization (78). However, although high levels of insulin and IGF-1 initially stimulate the insulin receptor substrate 1-PI 3-kinase pathway in the soleus muscle of mice, chronic exposure of soleus muscle to insulin and IGF-1 leads to a rapid decline in the activity of PI 3-kinase (76). Similarly, chronic hyperinsulinemia down-regulates in vitro glucose transport in human muscle cultures (79) and impairs the nonoxidative (glycogen synthetic) pathway in man (80). Therefore, it is tempting to postulate that chronic hyperinsulinemia and slightly raised IGF-1 levels lead to down-regulation of the PI 3-kinase pathway, with a subsequent reduction in GlySyn activation, whereas the ongoing availability of FFA inhibits glucose oxidation (67) and hexokinase activity (22, 57). Together, these defects would be expressed as a severe and proportional reduction in the insulin-stimulated rates of GS and GF, as seen in our 24-month rhGH-treated GHD adults. Importantly, this study has shown that the defects in skeletal muscle glucose metabolism and insulin sensitivity were almost identical in our GHD adults, both before and after the prescribed trial dose of GH.

Another factor that may have contributed to the development and persistence of the insulin resistance in our rhGH-treated GHD adults was the standard replacement of other pituitary hormones. The thyroid hormone replacement used in this study maintained euthyroidism throughout the study period and should not have influenced insulin action (81). Moreover, stable cortisone replacement resulted in time-averaged plasma cortisol levels on the study day similar to that seen in our control subjects and, therefore, was unlikely to have been a major factor in the development of insulin resistance (5). In a recent study in hypopituitary subjects, reproduction of the physiological diurnal cortisol rhythm, using a variable iv infusion of hydrocortisone, resulted in glucose and insulin levels, after an oral glucose load, identical to those obtained in the same subjects treated with standard twice-daily oral hydrocortisone (82). Testosterone and estrogen replacement were also stable throughout the 24-month study. Long-term testosterone replacement therapy in hypogonadal men is known to increase FFM but not to alter FM (83), and to increase IGF-1 levels (84). Nevertheless, testosterone therapy is not known to alter insulin action. In contrast, long-term estrogen replacement in hypogonadal women decreases visceral FM (85) and produces a normo-IGF-1-GH resistant state (13, 86) but does not affect their glucose metabolism or insulin action (87).

In conclusion, the severe impairments in the insulin-stimulated GS and GF pathways of glucose metabolism in GHD adults is attributable to elevated skeletal muscle intracellular glucose levels, reduced glycogen levels, and impaired GlySyn activity. This marked insulin resistance is related to the duration of GH deficiency and to fasting IGF-1 and triglyceride levels (but not obesity), and it occurs despite the presence of normal glucose, insulin, C peptide, and FFA concentrations. After 24 months of rhGH therapy, using a conventional dose of rhGH, these subjects experienced a significant reduction in total body fat, abdominal fat, WHR, cholesterol and LDL-cholesterol, thereby reducing the risk of cardiovascular morbidity and mortality (3, 12, 14). However, the severe defects of both in vivo insulin action and in vitro skeletal muscle glucose metabolism persisted. This sustained insulin resistance was now related to the subject’s obesity and to fasting FFA and lipid levels and was associated with the development of chronic hyperinsulinemia. Importantly, we found that the higher the absolute or increment in fasting IGF-1 levels obtained with rhGH replacement therapy, the greater the likelihood that insulin sensitivity actually worsened in these subjects. Given our observations of the ongoing severe insulin resistance and chronic hyperinsulinemia in these long-term rhGH-treated GHD adults, there is an urgent need to lower the replacement dose of rhGH to optimize the improvements in body composition, lipid profiles, biochemical profiles, and insulin action.

	Acknowledgments

 
We are grateful to Pharmacia and Upjohn (Australia) for supplying each GHD subject with Genotropin (rhGH) and a Kabipen injection device; Gillian Jones and Colleen Fietz, Department of Medicine, St. Vincent’s Hospital, for performing the DEXA scans; Dr. Margaret Zacharin, Department of Endocrinology, and Associate Professor Jim Best, Department of Medicine, St. Vincent’s Hospital, for allowing us to study some of their patients; and to all the subjects who participated in the study. We also thank Cathryn Walker for typing assistance.

	Footnotes

 
¹ This work was supported by the National Health and Medical Research Council of Australia and Pharmacia and Upjohn (Australia).

² Sponsored as a Doctoral Research Fellow by Novo-Nordisk (Australia).

Received September 12, 1997.

Revised February 6, 1998.

Accepted February 13, 1998.

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