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Department of Endocrinology, University Hospital MAS, S-205 02 Malmo, Sweden
Address all correspondence and requests for reprints to: Dr. Mikael Segerlantz, Department of Endocrinology, University Hospital MAS, S-205 02 Malmo, Sweden. E-mail: mikael.segerlantz{at}skane.se
Abstract
To test the hypothesis that GH-induced insulin resistance is mediated by an increase in FFA levels we assessed insulin sensitivity after inhibiting the increase in FFA by a nicotine acid derivative, Acipimox, in nine GH-deficient adults receiving GH replacement therapy. The patients received in a double blind fashion either Acipimox (500 mg) or placebo before a 2-h euglycemic (plasma glucose, 5.5 ± 0.2 mmol/liter) hyperinsulinemic (serum insulin, 28.7 ± 6.3 mU/liter) clamp in combination with indirect calorimetry and infusion of [3-3H]glucose.
Acipimox decreased fasting FFA by 88% (P = 0.012) and basal lipid oxidation by 39% (P = 0.015) compared with placebo. In addition, the insulin-stimulated lipid oxidation was 31% (P = 0.0077) lower during Acipimox than during placebo. Acipimox increased insulin-stimulated total glucose uptake by 36% (P = 0.021) compared with placebo, which mainly was due to a 47% (P = 0.015) increase in glucose oxidation.
GH induced insulin resistance is partially prevented by inhibition of lipolysis by Acipimox.
GH REPLACEMENT therapy of adult GH-deficient patients is often associated with impaired insulin sensitivity (1, 2, 3, 4, 5). GH is a lipolytic hormone and has been reported to increase FFA (6, 7). It has been speculated that worsening of insulin sensitivity is a consequence of an activated fatty acid-glucose cycle, i.e. increased FFA supply leads to substrate competition with increased lipid and decreased glucose oxidation (8). If this hypothesis is correct, prevention of lipolysis by an antilipolytic agent such as Acipimox should ameliorate the deterioration of insulin sensitivity (9, 10, 11). To test this hypothesis we inhibited the rise in FFA by prior administration of Acipimox in GH-deficient adults receiving long-term GH replacement therapy. Insulin sensitivity was assessed by a euglycemic insulin clamp combined with infusion [3-3H]glucose.
Subjects and Methods
Subjects
Nine GH-deficient patients, receiving chronic GH replacement
therapy (mean age, 45 ± 12.8 yr; body mass index, 27.8 ±
2.1 kg/m2), participated in the study (Tables 1
and 2). Patients with diabetes
mellitus, lipid disorders, or medication
interfering with glucose/lipid metabolism were excluded. The patients
gave their written consent before participating. The study protocol was
approved by the ethics committee of the Medical Faculty of Lund
University, the isotope committee at Malmo University Hospital MAS, and
the Swedish Medical Product Agency.
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Experimental design
The patients were studied on two occasions with at least a 1-wk interval. The prescribed GH dose (Genotropin, Pharmacia & Upjohn, Inc., Stockholm, Sweden) was administered sc at 22 h the night before the study. All subjects were admitted to a metabolic ward at 0700 h after a 10-h overnight fast. All studies were performed with the patient in a supine position. Before the study a catheter was inserted anterogradely into an antecubital vein for infusions, another catheter was positioned retrogradely into a wrist vein for blood sampling. This hand was then enclosed in a heated box (70 C) to achieve arterialization of venous blood, and the wrist catheter was kept open by a saline infusion. At the start of the study and again after 105 min the patients were in a double blind manner given a capsule of either 250 mg Acipimox (Farmitalia Carlo Erba, Milan, Italy) or placebo. All subjects participated in the following experiments: Exp 1, a 2-h euglycemic hyperinsulinemic insulin clamp with a variable infusion of 20% glucose labeled with [3-3H]glucose in combination with indirect calorimetry and constant infusion of [3-3H]glucose; and Exp 2, measurement of lean body mass with a bioelectrical impedance method.
Methods
Glucose metabolism was assessed by euglycemic hyperinsulinemic clamp (12), and substrate oxidation by indirect calorimetry (13). Hepatic glucose production (HGP) was measured by infusion of [3-3H]glucose. A primed continuous infusion of human insulin (Actrapid Human, Novo Industri, Copenhagen, Denmark), at a dose of 160 mU/m2 during a 10-min period was administered to acutely raise the plasma insulin concentration. To maintain plasma insulin concentrations at a desired level throughout the rest of the insulin clamp the patient received a continuous insulin infusion of 10 mU/m2·min. A variable infusion of 20% glucose was begun and periodically adjusted (clamped) to maintain the plasma glucose concentration constant at 5.5 mmol/liter. Plasma glucose was measured at 5-min intervals.
HGP was measured by the isotope dilution technique using [3-3H]glucose (Amersham International, Little Chalfont, UK), administrated as a priming dose of 8.3 µCi/m2 and a constant infusion of 0.083 µCi/m2·min for 270 min. The basal plasma tracer specific activity is linearly related to the rate of tracer infusion per m2 body surface area (14). To achieve a similar basal plasma specific activity in all subjects, the tracer amount was adjusted for body surface area (14). To achieve tracer equilibrium in the plasma glucose pool we allowed 150 min to proceed before the insulin infusion was started. Plasma glucose was clamped by a variable labeled glucose infusion. The glucose infusion was labeled with [3-3H]glucose to maintain plasma glucose specific activity at baseline levels (15). Labeling of the glucose infusion was based upon previous estimates of the expected glucose infusion and the HGP rate during the clamp (15). The radiochemical purity of the tracer was 99% as reported by the manufacturer.
Indirect calorimetry was performed in the basal state and during the last 45 min of the insulin clamp to estimate net rates of carbohydrate and lipid oxidation (13). A computerized open circuit system was used to measure gas exchange through a transparent plastic canopy (Deltatrac, Datex, Helsinki, Finland). The monitor calculates the carbon dioxide production and oxygen consumption rates from the differences in gas concentrations measured between upstream and downstream flow in the plastic canopy. The air flow is measured by an air dilution method, carbon dioxide concentrations are determined by a conventional infrared detector, and oxygen concentration is measured by a fast differential paramagnetic oxygen sensor. The carbon dioxide concentration in the room is automatically remeasured every 30 min to avoid errors. The monitor has a precision of 2.6% for oxygen consumption and 1.0% for carbon dioxide production.
The bioelectrical impedance technique estimates total body water using a two-terminal portable impedance analyzer (BIA 101, RJL, Akern, Copenhagen, Denmark). Electrodes were placed on the dorsal surface of the left hand and foot. Measurements were made in the supine position in the morning after voiding (16).
Blood samples for measurements of plasma glucose, serum insulin, FFA,
and [3-3H]glucose specific activity were drawn
in accordance with the flowchart (Fig. 1). To prevent in vitro
lipolysis, the samples for FFA measurements were collected in
prechilled tubes (17). The FFA samples from the entire
group were analyzed at the same time.
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Calculation of glucose metabolism
Basal HGP was calculated by dividing the [3-3H]glucose infusion rate by the steady state plateau of [3-3H]glucose specific activity in plasma during the last 30 min of the basal tracer infusion period. During administration of insulin and glucose a nonsteady state condition in plasma [3-3H]glucose specific activity exists. At high rates of glucose uptake the classical model of Steele is known to produce negative estimates of HGP. When adding [3-3H]glucose to the variable exogenous glucose infusion, the plasma [3-3H]glucose specific activity was maintained constant, and only a few negative numbers of HGP were adapted as zero in calculations. The infusion rate of exogenous glucose was integrated over 20-min intervals and subtracted from the total rate of glucose appearance to obtain the rate of residual HGP during the clamp. Total body glucose metabolism was calculated by adding the mean rate of HGP (if it is a positive number) during the last 60 min of the insulin clamp to the mean glucose infusion rate during the same period. Nonoxidative glucose metabolism, mainly storage of glucose as glycogen, was calculated as the difference between total body glucose metabolism and glucose oxidation, as determined by indirect calorimetry.
Net rates of glucose and lipid oxidation were calculated from indirect calorimetric measurements in the basal state and during the last 60 min of the insulin clamp.
Protein oxidation was calculated from the overnight urinary urea nitrogen excretion collected by the patient and urinary urea nitrogen excretion determined during the insulin clamp. Corrections were made for urea clearance (18).
Statistical analysis
Values are presented as the mean ± SD or the median (interquartile range), if the variable is not normally distributed. The significance of difference between changes during the Acipimox and placebo periods were tested by Wilcoxon signed rank test. All statistical analyses were performed with StatView software (version 4.5 for Windows, Abacus Concepts, Inc., Berkeley, CA). P < 0.05 was considered statistically significant.
Results
Plasma glucose and serum insulin (Fig. 2)
There were no differences in the plasma glucose concentrations between the Acipimox and placebo experiments in either the basal (4.9 ± 0.3 vs. 5.2 ± 0.6 mmol/liter) or insulin-stimulated (5.5 ± 0.2 vs. 5.4 ± 0.2 mmol/liter) state.
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FFA concentration and lipid oxidation (Figs. 2
and 3)
The basal FFA concentrations were not significantly different before Acipimox and placebo treatments (460 ± 247 vs. 324 ± 153 nmol/liter). After Acipimox, FFA levels decreased to 58 ± 48 nmol/liter, whereas they remained unchanged after placebo. During the clamp the FFA concentrations were suppressed to the same extent in both the Acipimox and placebo periods (30 ± 30 vs. 31 ± 42 nmol/liter). Both basal (0.49 ± 0.21 vs. 0.80 ± 0.21 mg/kg·min; P = 0.015) and insulin-suppressed (0.49 ± 0.19 vs. 0.71 ± 0.24 mg/kg·min; P = 0.0077) rates of lipid oxidation were significantly lower after Acipimox than after placebo.
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)Insulin-stimulated glucose uptake was significantly greater during Acipimox than during placebo (4.94 ± 2.3 vs. 3.63 ± 0.86 mg/kg·min; P = 0.02); this was mainly due to an increased rate of glucose oxidation (2.37 ± 0.60 vs. 1.61 ± 0.61 mg/kg·min; P = 0.01). Glucose oxidation was significantly greater in the basal state during Acipimox treatment (1.92 ± 0.37 vs. 1.22 ± 0.44 mg/kg·min; P = 0.01), whereas no significant difference was observed in nonoxidative glucose metabolism between Acipimox and placebo (2.58 ± 1.82 vs. 2.02 ± 0.53 mg/kg·min; P = NS). The basal rate of HGP was similar in the Acipimox and placebo periods (2.21 ± 0.32 vs. 2.37 ± 0.34 mg/kg·min; P = NS). Although HGP was slightly more suppressed by insulin during Acipimox than during placebo, this difference did not reach statistical significance (0.49 ± 0.54 vs.1.01 ± 0.53 mg/kg·min; P = 0.07).
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)The basal rate of energy expenditure was similar in the Acipimox and placebo periods (1728 ± 241 vs. 1754 ± 232 kcal/24 h; P = NS). However, there was a blunted thermogenic response to insulin during placebo (18 ± 132 kcal/24 h), which was restored by Acipimox (108 ± 113 kcal/24 h).
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The basal rate of protein oxidation did not differ between the Acipimox and placebo periods basally (0.63 ± 0.27 vs. 0.62 ± 0.20 mg/kg·min; P = NS) or during insulin-stimulated conditions (0.47 ± 0.31 vs. 0.53 ± 0.20 mg/kg·min; P = NS).
Discussion
Administration of the antilipolytic agent Acipimox before the euglycemic clamp resulted in a 36% improvement in insulin-stimulated glucose uptake compared with the effect of placebo in GH-treated patients with GH deficiency. The improvement in glucose uptake was almost entirely explained by an increase in glucose oxidation and a concomitant decrease in lipid oxidation, whereas there was no significant effect on nonoxidative glucose uptake. The data thus provide support for our hypothesis that 1) GH-induced insulin resistance is mediated by GH-induced lipolysis; and 2) inhibition of lipolysis will result in improved insulin sensitivity. In keeping with some previous reports in other patient groups, oxidative substrate competition was mainly responsible for the effect (19, 20, 21). This could partially be a consequence of the study design, as the Km for insulin-stimulated glucose uptake is lower than that for nonoxidative glucose uptake; the insulin levels attained during the clamp were submaximal for stimulation of nonoxidative glucose metabolism (22).
Total glucose uptake is the sum of glucose infused during the clamp and residual HGP. During this low dose insulin clamp we could not assume that HGP would be completely suppressed, particularly not in insulin-resistant individuals such as patients with GH deficiency receiving chronic GH replacement therapy (4, 23). We therefore considered it important to assess HGP by the hot glucose method to avoid problems from negative values of HGP. As predicted, HGP was not completely suppressed by insulin during the placebo experiment. As HGP was slightly more suppressed during Acipimox than during placebo, this could to some extent attenuate the difference in nonoxidative glucose uptake between the two experiments. The slightly higher rate of HGP during the placebo clamp could result from more FFA driving gluconeogenesis and thereby HGP (24). Similarly, lowering of FFA with Acipimox resulted in enhanced suppression of HGP in a previous study (25).
One caveat of the study could be how to express rates of glucose metabolism. We expressed them per kg total body mass, but we could also have expressed them per fat-free mass measured with the bioimpedance method. It is, however, known that bioimpedance, which measures conductance in the water space, can be influenced by water retention induced by short-term GH therapy. These patients had been on moderate doses of GH replacement therapy (median dose, 0.094 IU/kg·wk) for a mean of 48 months, which makes it unlikely that they would encounter marked water retention. If expressed per fat-free mass, the findings were virtually unchanged, i.e. there was a significant increase in glucose uptake by 36%, which was almost completely accounted for by the increase in glucose oxidation.
In conclusion, the study provides direct evidence that lowering of FFA at least partially prevents GH-induced insulin resistance by stimulating glucose oxidation. The lipolytic effect of GH thus seems to explain most of the insulin resistance observed during GH therapy.
Acknowledgments
We are indebted to Gertrud Ahlqvist, Marianne Lundberg, and Ronnie Thomasson for their skillful assistance.
Footnotes
This work was supported by a grant from Pharmacia & Upjohn, Inc. (Stockholm, Sweden), the Skane County Council Research and Development Foundation (to M.S.), the Novo Nordisk Foundation, and the Swedish Medical Research Council (to L.G.).
Abbreviation: HGP, Hepatic glucose production.
Received March 5, 2001.
Accepted August 29, 2001.
References
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fat; 
, protein. The SD is calculated on the total energy
expenditure.