Glycogen synthase activity is reduced in cultured skeletal muscle cells of non-insulin-dependent diabetes mellitus subjects. Biochemical and molecular mechanisms.

To determine whether glycogen synthase (GS) activity remains impaired in skeletal muscle of non-insulin-dependent diabetes mellitus (NIDDM) patients or can be normalized after prolonged culture, needle biopsies of vastus lateralis were obtained from 8 healthy nondiabetic control (ND) and 11 NIDDM subjects. After 4-6 wk growth and 4 d fusion in media containing normal physiologic concentrations of insulin (22 pM) and glucose (5.5 mM), both basal (5.21 +/- 0.79 vs 9.01 +/- 1.25%, P < 0.05) and acute insulin-stimulated (9.35 +/- 1.81 vs 16.31 +/- 2.39, P < 0.05) GS fractional velocity were reduced in NIDDM compared to ND cells. Determination of GS kinetic constants from muscle cells of NIDDM revealed an increased basal and insulin-stimulated Km(0.1) for UDP-glucose, a decreased insulin-stimulated Vmax(0.1) and an increased insulin-stimulated activation constant (A(0.5)) for glucose-6-phosphate. GS protein expression, determined by Western blotting, was decreased in NIDDM compared to ND cells (1.57 +/- 0.29 vs 3.30 +/- 0.41 arbitrary U/mg protein, P < 0.05). GS mRNA abundance also tended to be lower, but not significantly so (0.168 +/- 0.017 vs 0.243 +/- 0.035 arbitrary U, P = 0.08), in myotubes of NIDDM subjects. These results indicate that skeletal muscle cells of NIDDM subjects grown and fused in normal culture conditions retain defects of basal and insulin-stimulated GS activity that involve altered kinetic behavior and possibly reduced GS protein expression. We conclude that impaired regulation of skeletal muscle GS in NIDDM patients is not completely reversible in normal culture conditions and involves mechanisms that may be genetic in origin.     

Cyclic guanosine monophosphate is the mediator of platelet-activating factor inhibition on transport by the mouse kidney thick ascending limb.

Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal muscle from 15 insulin-sensitive subjects and 5 insulin-resistant nondiabetic subjects, as well as 18 subjects with non-insulin-dependent diabetes mellitus (NIDDM). Approximately 90% of total PTPase activity resided in the particulate fraction. In comparison with lean nondiabetic subjects, particulate PTPase activity was reduced 21% (P < 0.05) and 22% (P < 0.005) in obese nondiabetic and NIDDM subjects, respectively. PTPase1B protein levels were likewise decreased by 38% in NIDDM subjects (P < 0.05). During hyperinsulinemic glucose clamps, glucose disposal rates (GDR) increased approximately sixfold in lean control and twofold in NIDDM subjects, while particulate PTPase activity did not change. However, a strong positive correlation (r = 0.64, P < 0.001) existed between particulate PTPase activity and insulin-stimulated GDR. In five obese NIDDM subjects, weight loss of approximately 10% body wt resulted in a significant and corresponding increase in both particulate PTPase activity and insulin-stimulated GDR. These findings indicate that skeletal muscle particulate PTPase activity and PTPase1B protein content reflect in vivo insulin sensitivity and are reduced in insulin resistant states. We conclude that skeletal muscle PTPase activity is involved in the chronic, but not acute regulation of insulin action, and that the decreased enzyme activity may have a role in the insulin resistance of obesity and NIDDM.     

Impaired activity and gene expression of hexokinase II in muscle from non-insulin-dependent diabetes mellitus patients.

After entering the muscle cell, glucose is immediately and irreversibly phosphorylated to glucose-6-phosphate by hexokinases (HK) I and II. Previous studies in rodents have shown that HKII may be the dominant HK in skeletal muscle. Reduced insulin-stimulated glucose uptake and reduced glucose-6-phosphate concentrations in muscle have been found in non-insulin-dependent diabetes mellitus (NIDDM) patients when examined during a hyperglycemic hyperinsulinemic clamp. These findings [correction of finding] are consistent with a defect in glucose transport and/or phosphorylation. In the present study comprising 29 NIDDM patients and 25 matched controls, we tested the hypothesis that HKII activity and gene expression are impaired in vastus lateralis muscle of NIDDM patients when examined in the fasting state. HKII activity in a supernatant of muscle extract accounted for 28 +/- 5% in NIDDM patients and 40 +/- 5% in controls (P = 0.08) of total muscle HK activity when measured at a glucose media of 0.11 mmol/liter and 31 +/- 4 and 47 +/- 7% (P = 0.02) when measured at 0.11 mmol/liter of glucose. HKII mRNA, HKII immunoreactive protein level, and HKII activity were significantly decreased in NIDDM patients (P < 0.0001, P = 0.03, and P = 0.02, respectively) together with significantly decreased glycogen synthase mRNA level and total glycogen synthase activity (P = 0.02 and P = 0.02, respectively). In the entire study population HKII activity estimated at 0.11 and 11.0 mM glucose was inversely correlated with fasting plasma glucose concentrations (r = -0.45, P = 0.004; r = -0.54, P < 0.0001, respectively) and fasting plasma nonesterified fatty acid concentrations (r = -0.46, P = 0.003; r = -0.37, P = 0.02, respectively). In conclusion, NIDDM patients are characterized by a reduced activity and a reduced gene expression of HKII in muscle which may be secondary to the metabolic peturbations. HKII contributes with about one-third of total HK activity in a supernatant of human vastus lateralis muscle.     

Insulin resistance in liver cirrhosis. Positron-emission tomography scan analysis of skeletal muscle glucose metabolism.

BACKGROUND. Insulin resistance and glucose intolerance are a major feature of patients with liver cirrhosis. However, site and mechanism of insulin resistance in cirrhosis are unknown. We investigated insulin-induced glucose metabolism of skeletal muscle by positron-emission tomography to identify possible defects of muscle glucose metabolism in these patients. METHODS. Whole body glucose disposal and oxidation were determined by the combined use of the euglycemic-hyperinsulinemic clamp technique (insulin infusion rate: 1 mU/kg body wt per min) and indirect calorimetry in seven patients with biopsy-proven liver cirrhosis (Child: 1A, 5B, and 1C) and five healthy volunteers. Muscle glucose uptake of the thighs was measured simultaneously by dynamic [18F]fluorodeoxyglucose positron-emission tomography scan. RESULTS. Both whole body and nonoxidative glucose disposal were significantly reduced in patients with liver cirrhosis (by 48%, P < 0.001, and 79%, P < 0.0001, respectively), whereas glucose oxidation and the increase in plasma lactate were normal. Concomitantly, skeletal muscle glucose uptake was reduced by 69% in liver cirrhosis (P < 0.003) and explained 55 or 92% of whole body glucose disposal in cirrhotics and controls, respectively. Analysis of kinetic constants using a three-compartment model further indicated reduced glucose transport (P < 0.05) but unchanged phosphorylation of glucose in patients with liver cirrhosis. CONCLUSIONS. Patients with liver cirrhosis show significant insulin resistance that is characterized by both decreased glucose transport and decreased nonoxidative glucose metabolism in skeletal muscle.     

Characterization of cellular defects of insulin action in type 2 (non-insulin-dependent) diabetes mellitus.

Seven non-insulin-dependent diabetes mellitus (NIDDM) patients participated in three clamp studies performed with [3-3H]- and [U-14C]glucose and indirect calorimetry: study I, euglycemic (5.2 +/- 0.1 mM) insulin (269 +/- 39 pM) clamp; study II, hyperglycemic (14.9 +/- 1.2 mM) insulin (259 +/- 19 pM) clamp; study III, euglycemic (5.5 +/- 0.3 mM) hyperinsulinemic (1650 +/- 529 pM) clamp. Seven control subjects received a euglycemic (5.1 +/- 0.2 mM) insulin (258 +/- 24 pM) clamp. Glycolysis and glucose oxidation were quantitated from the rate of appearance of 3H2O and 14CO2; glycogen synthesis was calculated as the difference between body glucose disposal and glycolysis. In study I, glucose uptake was decreased by 54% in NIDDM vs. controls. Glycolysis, glycogen synthesis, and glucose oxidation were reduced in NIDDM patients (P < 0.05-0.001). Nonoxidative glycolysis and lipid oxidation were higher. In studies II and III, glucose uptake in NIDDM was equal to controls (40.7 +/- 2.1 and 40.7 +/- 1.7 mumol/min.kg fat-free mass, respectively). In study II, glycolysis, but not glucose oxidation, was normal (P < 0.01 vs. controls). Nonoxidative glycolysis remained higher (P < 0.05). Glycogen deposition increased (P < 0.05 vs. study I), and lipid oxidation remained higher (P < 0.01). In study III, hyperinsulinemia normalized glycogen formation, glycolysis, and lipid oxidation but did not normalize the elevated nonoxidative glycolysis or the decreased glucose oxidation. Lipid oxidation and glycolysis (r = -0.65; P < 0.01), and glucose oxidation (r = -0.75; P < 0.01) were inversely correlated. In conclusion, in NIDDM: (a) insulin resistance involves glycolysis, glycogen synthesis, and glucose oxidation; (b) hyperglycemia and hyperinsulinemia can normalize total body glucose uptake; (c) marked hyperinsulinemia normalizes glycogen synthesis and total flux through glycolysis, but does not restore a normal distribution between oxidation and nonoxidative glycolysis; (d) hyperglycemia cannot overcome the defects in glucose oxidation and nonoxidative glycolysis; (e) lipid oxidation is elevated and is suppressed only with hyperinsulinemia.     

Intact insulin stimulation of skeletal muscle blood flow, its heterogeneity and redistribution, but not of glucose uptake in non-insulin-dependent diabetes mellitus.

We tested the hypothesis that defects in insulin stimulation of skeletal muscle blood flow, flow dispersion, and coupling between flow and glucose uptake contribute to insulin resistance of glucose uptake in non-insulin-dependent diabetes mellitus (NIDDM). We used positron emission tomography combined with [15O]H2O and [18F]-2-deoxy--glucose and a Bayesian iterative reconstruction algorithm to quantitate mean muscle blood flow, flow heterogeneity, and their relationship to glucose uptake under normoglycemic hyperinsulinemic conditions in 10 men with NIDDM (HbA1c 8.1+/-0.5%, age 43+/-2 yr, BMI 27.3+/-0.7 kg/m2) and in 7 matched normal men. In patients with NIDDM, rates of whole body (35+/-3 vs. 44+/-3 micromol/kg body weight.min, P < 0.05) and femoral muscle (71+/-6 vs. 96+/-7 micromol/kg muscle.min, P < 0.02) glucose uptake were significantly decreased. Insulin increased mean muscle blood flow similarly in both groups, from 1.9+/-0.3 to 2.8+/-0.4 ml/100 g muscle.min in the patients with NIDDM, P < 0.01, and from 2.3+/-0.3 to 3.0+/-0.3 ml/100 g muscle.min in the normal subjects, P < 0.02. Pixel-by-pixel analysis of flow images revealed marked spatial heterogeneity of blood flow. In both groups, insulin increased absolute but not relative dispersion of flow, and insulin-stimulated but not basal blood flow colocalized with glucose uptake. These data provide the first evidence for physiological flow heterogeneity in human skeletal muscle, and demonstrate that insulin increases absolute but not relative dispersion of flow. Furthermore, insulin redirects flow to areas where it stimulates glucose uptake. In patients with NIDDM, these novel actions of insulin are intact, implying that muscle insulin resistance can be attributed to impaired cellular glucose uptake.     

Effects of insulin infusion on human skeletal muscle pyruvate dehydrogenase, phosphofructokinase, and glycogen synthase. Evidence for their role in oxidative and nonoxidative glucose metabolism.

To determine whether activation by insulin of glycogen synthase (GS), phosphofructokinase (PFK), or pyruvate dehydrogenase (PDH) in skeletal muscle regulates intracellular glucose metabolism, subjects were studied basally and during euglycemic insulin infusions of 12, 30, and 240 mU/m2 X min. Glucose disposal, oxidative and nonoxidative glucose metabolism were determined. GS, PFK, and PDH were assayed in skeletal muscle under each condition. Glucose disposal rates were 2.37 +/- 0.11, 3.15 +/- 0.19, 6.71 +/- 0.44, and 11.7 +/- 1.73 mg/kg X min; glucose oxidation rates were 1.96 +/- 0.18, 2.81 +/- 0.28, 4.43 +/- 0.32, and 5.22 +/- 0.52. Nonoxidative glucose metabolism was 0.39 +/- 0.13, 0.34 +/- 0.26, 2.28 +/- 0.40, and 6.52 +/- 1.21 mg/kg X min. Both the proportion of active GS and the proportion of active PDH were increased by hyperinsulinemia. PFK activity was unaffected. Activation of GS was correlated with nonoxidative glucose metabolism, while activation of PDH was correlated with glucose oxidation. Sensitivity to insulin of GS was similar to that of nonoxidative glucose metabolism, while the sensitivity to insulin of PDH was similar to that of glucose oxidation. Therefore, the activation of these enzymes in muscle may regulate nonoxidative and oxidative glucose metabolism.     

Increased epinephrine and skeletal muscle responses to hypoglycemia in non-insulin-dependent diabetes mellitus.

We evaluated skeletal muscle counterregulation during hypoglycemia in nine subjects with non-insulin-dependent diabetes mellitus (NIDDM) (HbA1c 9.4 +/- 0.5%, nl < 6.2%) compared with six normal controls, matched for age (51 +/- 3 and 49 +/- 5 yr, respectively) and body mass index (27.3 +/- 1.2 and 27.0 +/- 2.1 kg/m2). After 60 min of euglycemia (plasma insulin approximately 140 microU/ml), plasma glucose was lowered to 62 +/- 2 mg/dl by 120 min. Hypoglycemia induced a 2.2-fold greater increase in plasma epinephrine in NIDDM (P < 0.001), while the plasma glucagon response was blunted (P < 0.01). Hepatic glucose output ([3H-3]glucose) suppressed similarly during euglycemia, but during hypoglycemia was greater in NIDDM (P < 0.005). Conversely, glucose uptake during euglycemia was 150% greater in controls (P < 0.01) and remained persistently higher than in NIDDM during hypoglycemia. In NIDDM, plasma FFA concentrations were approximately fivefold greater (P < 0.001), and plasma lactate levels were approximately 40% higher than in controls during hypoglycemia (P < 0.01); the rates of glycolysis from plasma glucose were similar in the two groups despite a 49% lower rate of glucose uptake in NIDDM (3.4 +/- 0.9 vs. 6.9 +/- 1.3 mg/kg per minute, P < 0.001). Muscle glycogen synthase activity fell by 42% with hypoglycemia (P < 0.01) in NIDDM but not in controls. In addition, glycogen phosphorylase was activated by 56% during hypoglycemia in NIDDM only (P < 0.01). Muscle glucose-6-phosphate concentrations rose during hypoglycemia by a twofold greater increment in NIDDM (P < 0.01). Thus, skeletal muscle participates in hypoglycemia counterregulation in NIDDM, directly by decreased removal of plasma glucose and, indirectly, by providing lactate for hepatic gluconeogenesis. Consequently, in addition to inherent insulin resistance in NIDDM, the enhanced plasma epinephrine response during hypoglycemia may partially offset impaired glucagon secretion and counteract the effects of hyperinsulinemia on liver, fat, and skeletal muscle.     

Effect of the antilipolytic nicotinic acid analogue acipimox on whole-body and skeletal muscle glucose metabolism in patients with non-insulin-dependent diabetes mellitus.

Increased nonesterified fatty acid (NEFA) levels may be important in causing insulin resistance in skeletal muscles in patients with non-insulin-dependent diabetes mellitus (NIDDM). The acute effect of the antilipolytic nicotinic acid analogue Acipimox (2 X 250 mg) on basal and insulin-stimulated (3 h, 40 mU/m2 per min) glucose metabolism was therefore studied in 12 patients with NIDDM. Whole-body glucose metabolism was assessed using [3-3H]glucose and indirect calorimetry. Biopsies were taken from the vastus lateralis muscle during basal and insulin-stimulated steady-state periods. Acipimox reduced NEFA in the basal state and during insulin stimulation. Lipid oxidation was inhibited by Acipimox in all patients in the basal state (20 +/- 2 vs. 33 +/- 3 mg/m2 per min, P less than 0.01) and during insulin infusion (8 +/- 2 vs. 17 +/- 2 mg/m2 per min, P less than 0.01). Acipimox increased the insulin-stimulated glucose disposal rate (369 +/- 49 vs. 262 +/- 31 mg/m2 per min, P less than 0.01), whereas the glucose disposal rate was unaffected by Acipimox in the basal state. Acipimox increased glucose oxidation in the basal state (76 +/- 4 vs. 50 +/- 4 mg/m2 per min, P less than 0.01). During insulin infusion Acipimox increased both glucose oxidation (121 +/- 7 vs. 95 +/- 4 mg/m2 per min, P less than 0.01) and nonoxidative glucose disposal (248 +/- 47 vs. 167 +/- 29 mg/m2 per min, P less than 0.01). Acipimox enhanced basal and insulin-stimulated muscle fractional glycogen synthase activities (32 +/- 2 vs. 25 +/- 3%, P less than 0.05, and 50 +/- 5 vs. 41 +/- 4%, P less than 0.05). Activities of muscle pyruvate dehydrogenase and phosphofructokinase were unaffected by Acipimox. In conclusion, Acipimox acutely improved insulin action in patients with NIDDM by increasing both glucose oxidation and nonoxidative glucose disposal. This supports the hypothesis that elevated NEFA concentrations may be important for the insulin resistance in NIDDM. The mechanism responsible for the increased insulin-stimulated nonoxidative glucose disposal may be a stimulatory effect of Acipimox on glycogen synthase activity in skeletal muscles.     

Interaction between glucose and free fatty acid metabolism in human skeletal muscle.

The mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.     

Mechanism of impaired insulin-stimulated muscle glucose metabolism in subjects with insulin-dependent diabetes mellitus.

To determine the mechanism of impaired insulin-stimulated muscle glycogen metabolism in patients with poorly controlled insulin-dependent diabetes mellitus (IDDM), we used 13C-NMR spectroscopy to monitor the peak intensity of the C1 resonance of the glucosyl units in muscle glycogen during a 6-h hyperglycemic-hyperinsulinemic clamp using [1-(13)C]glucose-enriched infusate followed by nonenriched glucose. Under similar steady state (t = 3-6 h) plasma glucose (approximately 9.0 mM) and insulin concentrations (approximately 400 pM), nonoxidative glucose metabolism was significantly less in the IDDM subjects compared with age-weight-matched control subjects (37+/-6 vs. 73+/-11 micromol/kg of body wt per minute, P < 0.05), which could be attributed to an approximately 45% reduction in the net rate of muscle glycogen synthesis in the IDDM subjects compared with the control subjects (108+/-16 vs. 195+/-6 micromol/liter of muscle per minute, P < 0.001). Muscle glycogen turnover in the IDDM subjects was significantly less than that of the controls (16+/-4 vs. 33+/-5%, P < 0.05), indicating that a marked reduction in flux through glycogen synthase was responsible for the reduced rate of net glycogen synthesis in the IDDM subjects. 31P-NMR spectroscopy was used to determine the intramuscular concentration of glucose-6-phosphate (G-6-P) under the same hyperglycemic-hyperinsulinemic conditions. Basal G-6-P concentration was similar between the two groups (approximately 0.10 mmol/kg of muscle) but the increment in G-6-P concentration in response to the glucose-insulin infusion was approximately 50% less in the IDDM subjects compared with the control subjects (0.07+/-0.02 vs. 0.13+/-0.02 mmol/kg of muscle, P < 0.05). When nonoxidative glucose metabolic rates in the control subjects were matched to the IDDM subjects, the increment in the G-6-P concentration (0.06+/-0.02 mmol/kg of muscle) was no different than that in the IDDM subjects. Together, these data indicate that defective glucose transport/phosphorylation is the major factor responsible for the lower rate of muscle glycogen synthesis in the poorly controlled insulin-dependent diabetic subjects.     

Mechanism of enhanced insulin sensitivity in athletes. Increased blood flow, muscle glucose transport protein (GLUT-4) concentration, and glycogen synthase activity.

We examined the mechanisms of enhanced insulin sensitivity in 9 male healthy athletes (age, 25 +/- 1 yr; maximal aerobic power [VO2max], 57.6 +/- 1.0 ml/kg per min) as compared with 10 sedentary control subjects (age, 28 +/- 2 yr; VO2max, 44.1 +/- 2.3 ml/kg per min). In the athletes, whole body glucose disposal (240-min insulin clamp) was 32% (P < 0.01) and nonoxidative glucose disposal (indirect calorimetry) was 62% higher (P < 0.01) than in the controls. Muscle glycogen content increased by 39% in the athletes (P < 0.05) but did not change in the controls during insulin clamp. VO2max correlated with whole body (r = 0.60, P < 0.01) and nonoxidative glucose disposal (r = 0.64, P < 0.001). In the athletes forearm blood flow was 64% greater (P < 0.05) than in the controls, whereas their muscle capillary density was normal. Basal blood flow was related to VO2max (r = 0.63, P < 0.05) and glucose disposal during insulin infusion (r = 0.65, P < 0.05). The forearm glucose uptake in the athletes was increased by 3.3-fold (P < 0.01) in the basal state and by 73% (P < 0.05) during insulin infusion. Muscle glucose transport protein (GLUT-4) concentration was 93% greater in the athletes than controls (P < 0.01) and it was related to VO2max (r = 0.61, P < 0.01) and to whole body glucose disposal (r = 0.60, P < 0.01). Muscle glycogen synthase activity was 33% greater in the athletes than in the controls (P < 0.05), and the basal glycogen synthase fractional activity was closely related to blood flow (r = 0.88, P < 0.001). In conclusion: (a) athletes are characterized by enhanced muscle blood flow and glucose uptake. (b) The cellular mechanisms of glucose uptake are increased GLUT-4 protein content, glycogen synthase activity, and glucose storage as glycogen. (c) A close correlation between glycogen synthase fractional activity and blood flow suggests that they are causally related in promoting glucose disposal.     

Oral vanadyl sulfate improves hepatic and peripheral insulin sensitivity in patients with non-insulin-dependent diabetes mellitus.

We examined the in vivo metabolic effects of vanadyl sulfate (VS) in non-insulin-dependent diabetes mellitus (NIDDM). Six NIDDM subjects treated with diet and/or sulfonylureas were examined at the end of three consecutive periods: placebo for 2 wk, VS (100 mg/d) for 3 wk, and placebo for 2 wk. Euglycemic hyperinsulinemic (30 mU/m2.min) clamps and oral glucose tolerance tests were performed at the end of each study period. Glycemic control at baseline was poor (fasting plasma glucose 210 +/- 19 mg/dl; HbA1c 9.6 +/- 0.6%) and improved after treatment (181 +/- 14 mg/dl [P < 0.05], 8.8 +/- 0.6%, [P < 0.002]); fasting and post-glucose tolerance test plasma insulin concentrations were unchanged. After VS, the glucose infusion rate during the clamp was increased (by approximately 88%, from 1.80 to 3.38 mg/kg.min, P < 0.0001). This improvement was due to both enhanced insulin-mediated stimulation of glucose uptake (rate of glucose disposal [Rd], +0.89 mg/kg.min) and increased inhibition of HGP (-0.74 mg/kg.min) (P < 0.0001 for both). Increased insulin-stimulated glycogen synthesis (+0.74 mg/kg.min, P < 0.0003) accounted for > 80% of the increased Rd after VS, and the improvement in insulin sensitivity was maintained after the second placebo period. The Km of skeletal muscle glycogen synthase was lowered by approximately 30% after VS treatment (P < 0.05). These results indicate that 3 wk of treatment with VS improves hepatic and peripheral insulin sensitivity in insulin-resistant NIDDM humans. These effects were sustained for up to 2 wk after discontinuation of VS.     

Transmembrane glucose transport in skeletal muscle of patients with non-insulin-dependent diabetes.

Insulin resistance for glucose metabolism in skeletal muscle is a key feature in non-insulin-dependent diabetes mellitus (NIDDM). Which cellular effectors of glucose metabolism are involved is still unknown. We investigated whether transmembrane glucose transport in vivo is impaired in skeletal muscle in nonobese NIDDM patients. We performed euglycemic insulin clamp studies in combination with the forearm balance technique (brachial artery and deep forearm vein catheterization) in six nonobese NIDDM patients and five age- and weight-matched controls. Unlabeled D-mannitol (a nontransportable molecule) and radioactive 3-O-methyl-D-glucose (the reference molecular probe to assess glucose transport activity) were simultaneously injected into the brachial artery, and the washout curves were measured in the deep venous effluent blood. In vivo transmembrane transport of 3-O-methyl-D-glucose in forearm muscle was determined by computerized analysis of the washout curves. At similar steady-state plasma concentrations of insulin (approximately 500 pmol/liter) and glucose (approximately 5.15 mmol/liter), transmembrane inward transport of 3-O-methyl-D-glucose in skeletal muscle was markedly reduced in the NIDDM patients (6.5 x 10(-2) +/- 0.56 x 10(-2).min-1) compared with controls (12.5 x 10(-2) +/- 1.5 x 10(-2).min-1, P < 0.005). Mean glucose uptake was also reduced in the diabetics both at the whole body level (9.25 +/- 1.84 vs. 28.3 +/- 2.44 mumol/min per kg, P < 0.02) and in the forearm tissues (5.84 +/- 1.51 vs. 37.5 +/- 7.95 mumol/min per kg, P < 0.02). When the latter rates were extrapolated to the whole body level, skeletal muscle accounted for approximately 80% of the defect in insulin action seen in NIDDM patients. We conclude that transmembrane glucose transport, when assessed in vivo in skeletal muscle, is insensitive to insulin in nonobese NIDDM patients, and plays a major role in determining whole body insulin resistance.     

Defects in insulin secretion and insulin action in non-insulin-dependent diabetes mellitus are inherited. Metabolic studies on offspring of diabetic probands.

No studies are available that have compared early defects in glucose metabolism in the offspring of insulin-deficient and insulin-resistant probands with non-insulin-dependent diabetes mellitus (NIDDM). To investigate this issue, we evaluated insulin secretion capacity with oral and intravenous glucose tolerance tests and with the hyperglycemic clamp, and insulin action with the euglycemic insulin clamp in 20 offspring of NIDDM patients with low fasting C-peptide (+/-450 pmol/liter), reflecting deficient insulin secretion (IS-group), 18 offspring of NIDDM patients with high fasting C-peptide (>/= 880 pmol/liter), reflecting insulin resistance (IR-group), and 14 healthy control subjects without a family history of NIDDM. The frequency of impaired glucose tolerance was 45.0% in the IS-group and 50% in the IR-group. The IS-group had lower insulin-glucose response at 30 min in the oral glucose tolerance test (85.2+/-10.0 pmol insulin per mmol glucose) than the control group (136.4+/-23.1 pmol insulin per mmol glucose; P < 0.05) and the IR-group (115.6+/-11.8 pmol insulin per mmol glucose; P = 0.05). Furthermore, the acute insulin response during the first 10 min of an intravenous glucose tolerance test was lower in the IS-group than in the IR-group. Maximal insulin secretion capacity evaluated by C-peptide levels during the hyperglycemic clamp did not differ between the groups. The IR-group had lower rates of whole body glucose uptake (60.1+/-4.6 micromol per lean body mass per minute) than did the control group (84.2+/-5.0 micromol per lean body mass per minute; P < 0.001) or the IS-group (82.6+/-5.9 micromol per lean body mass per minute; P < 0.01) and this was due to reduced glucose nonoxidation. To conclude, both impaired insulin secretion and insulin action seem to be inherited and could represent the primary defects in glucose metabolism in the offspring of NIDDM probands.     

Evidence for decreased splanchnic glucose uptake after oral glucose administration in non-insulin-dependent diabetes mellitus.

The role of splanchnic glucose uptake (SGU) after oral glucose administration as a potential factor contributing to postprandial hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM) has not been established conclusively. Therefore, we investigated SGU in six patients with NIDDM and six weight-matched control subjects by means of the hepatic vein catheterization (HVC) technique. In a second part, we examined the applicability of the recently developed OG-CLAMP technique in NIDDM by comparing SGU and first-pass SGU during HVC with SGU during the OG-CLAMP experiment. The OG-CLAMP method combines a euglycemic, hyperinsulinemic clamp and an oral glucose tolerance test (75 g) during steady state glucose infusion (GINF). During HVC, SGU equals the splanchnic fractional extraction times the total (oral and arterial) glucose load presented to the liver. For OG-CLAMP, SGU was calculated as first-pass SGU by subtracting the integrated decrease in GINF over 180 min from 75 g. Cumulative splanchnic glucose output after oral glucose correlated significantly between both methods and was increased significantly in NIDDM patients (73.1+/-5.1 g for HVC, 76.5+/-5.5 for OG-CLAMP) compared with nondiabetic patients (46.7+/-4.4 g for HVC, 57.5+/-1.9 for OG-CLAMP). Thus, in NIDDM patients, SGU (7.4+/-2.1 vs. 37.8+/-5.9% in nondiabetic patients, P < 0.001) and first-pass SGU (4.7+/-1.7 vs. 26.5+/-5.1% in nondiabetic patients, P < 0.01) were decreased significantly during HVC, as was SGU during OG-CLAMP (3.9+/-1.7 vs. 23.4+/-2.5% in nondiabetic patients, P < 0.0001). SGU measured during OG-CLAMP correlated significantly with SGU (r = 0.87, P < 0.05 for NIDDM patients; r = 0.94, P < 0.01 for nondiabetic patients) and first-pass SGU (r = 0.87, P < 0.05 for NIDDM patients; r = 0.84, P < 0.05 for nondiabetic patients) during HVC. In conclusion, (a) SGU after oral glucose administration is decreased in NIDDM as measured by both methods, and (b) SGU during the OG-CLAMP is well-correlated to SGU and first-pass SGU during HVC in NIDDM. The decrease in SGU in NIDDM might contribute to postprandial hyperglycemia in diabetic subjects.     

Impaired free fatty acid utilization by skeletal muscle in non-insulin-dependent diabetes mellitus.

This study was undertaken to assess utilization of FFA by skeletal muscle in patients with non-insulin-dependent diabetes mellitus (NIDDM). 11 NIDDM and 9 nondiabetic subjects were studied using leg balance methods to measure the fractional extraction of [3H]oleate. Limb indirect calorimetry was used to estimate RQ. Percutaneous muscle biopsy samples of vastus lateralis were analyzed for muscle fiber type distribution, capillary density, and metabolic potential as reflected by measurements of the activity of seven muscle enzyme markers of glycolytic and aerobic-oxidative pathways. During postabsorptive conditions, fractional extraction of oleate across the leg was lower in NIDDM subjects (0.31 +/- 0.08 vs. 0.43 +/- 0.10, P < 0.01), and there was reduced oleate uptake across the leg (66 +/- 8 vs. 82 +/- 13 nmol/min, P < 0.01). Postabsorptive leg RQ was increased in NIDDM (0.85 +/- 0.03 vs. 0.77 +/- 0.02, P < 0.01), and rates of lipid oxidation by skeletal muscle were lower while glucose oxidation was increased (P < 0.05). In subjects with NIDDM, proportions of type I, IIa, and IIb fibers were 37 +/- 2, 37 +/- 6, and 26 +/- 5%, respectively, which did not differ from nondiabetics; and capillary density, glycolytic, and aerobic-oxidative potentials were similar. During 6 h after ingestion of a mixed meal, arterial FFA remained greater in NIDDM subjects. Therefore, despite persistent reduced fractional extraction of oleate across the leg in NIDDM (0.34 +/- 0.04 vs. 0.38 +/- 0.03, P < 0.05), rates of oleate uptake across the leg were greater in NIDDM (54 +/- 7 vs. 45 +/- 8 nmol/min, P < 0.01). In summary, during postabsorptive conditions there is reduced utilization of FFA by muscle, while during postprandial conditions there is impaired suppression of FFA uptake across the leg in NIDDM. During both fasting and postprandial conditions, NIDDM subjects have reduced rates of lipid oxidation by muscle.     

31P nuclear magnetic resonance measurements of muscle glucose-6-phosphate. Evidence for reduced insulin-dependent muscle glucose transport or phosphorylation activity in non-insulin-dependent diabetes mellitus.

To assess the rate-limiting step in muscle glycogen synthesis in non-insulin-dependent diabetes mellitus (NIDDM), the concentration of glucose-6-phosphate (G6P) was measured by 31P nuclear magnetic resonance (NMR) during a hyperglycemic-hyperinsulinemic clamp. Six subjects with NIDDM and six age weight-matched controls were studied at similar steady-state plasma concentrations of insulin (approximately 450 pmol/liter) and glucose (11 mmol/liter). The concentration of G6P in the gastrocnemius muscle was measured by 31P NMR. Whole-body oxidative and nonoxidative glucose metabolism was determined by the insulin-glucose clamp technique in conjunction with indirect calorimetry. Nonoxidative glucose metabolism which under these conditions is a measure of muscle glycogen synthesis (1990. N. Engl. J. Med. 322:223-228), was 31 +/- 7 mumol/(kg body wt-min) in the normal subjects and 13 +/- 3 mumol/(kg body wt-min) in the NIDDM subjects (P less than 0.05). The concentration of G6P was higher (0.24 +/- 0.02 mmol/kg muscle) in the normal subjects than in the NIDDM subjects (0.17 +/- 0.02, P less than 0.01). Increasing insulin concentrations to insulin 8,500 pmol/liter in four NIDDM subjects restored the glucose uptake rate and G6P concentrations to normal levels. In conclusion, the lower concentration of G6P in the diabetic subjects despite a decreased rate of nonoxidative glucose metabolism is consistent with a defect in muscle glucose transport or phosphorylation reducing the rate of muscle glycogen synthesis.     

Metabolic effects of liver transplantation in cirrhotic patients.

To assess whether liver transplantation (LTx) can correct the metabolic alterations of chronic liver disease, 14 patients (LTx-5) were studied 5+/-1 mo after LTx, 9 patients (LTx-13) 13+/-1 mo after LTx, and 10 patients (LTx-26) 26+/-2 months after LTx. Subjects with chronic uveitis (CU) and healthy volunteers (CON) were also studied. Basal plasma leucine and branched-chain amino acids were reduced in LTx-5, LTx-13, and LTx-26 when compared with CU and CON (P < 0.01). The basal free fatty acids (FFA) were reduced in LTx-26 with respect to CON (P < 0.01). To assess protein metabolism, LTx-5, LTx-13, and LTx-26 were studied with the [1-14C]leucine turnover combined with a 40-mU/m2 per min insulin clamp. To relate changes in FFA metabolism to glucose metabolism, eight LTx-26 were studied with the [1-14C]palmitate and [3-3H]glucose turnovers combined with a two-step (8 and 40 mU/m2 per min) euglycemic insulin clamp. In the postabsorptive state, LTx-5 had lower endogenous leucine flux (ELF) (P < 0.005), lower leucine oxidation (LO) (P < 0.004), and lower non-oxidative leucine disposal (NOLD) (P < 0.03) with respect to CON (primary pool model). At 2 yr (LTx-26) both ELF (P < 0.001 vs. LTx-5) and NOLD (P < 0.01 vs. LTx-5) were normalized, but not LO (P < 0.001 vs. CON) (primary and reciprocal pool models). Suppression of ELF by insulin (delta-reduction) was impaired in LTx-5 and LTx-13 when compared with CU and CON (P < 0.01), but normalized in LTx-26 (P < 0.004 vs. LTx-5 and P = 0.3 vs. CON). The basal FFA turnover rate was decreased in LTx-26 (P < 0.01) and CU (P < 0.02) vs. CON. LTx-26 showed a lower FFA oxidation rate than CON (P < 0.02). Tissue glucose disposal was impaired in LTx-5 (P < 0.005) and LTx-13 (P < 0.03), but not in LTx-26 when compared to CON. LTx-26 had normal basal and insulin-modulated endogenous glucose production. In conclusion, LTx have impaired insulin-stimulated glucose, FFA, and protein metabolism 5 mo after surgery. Follow-up at 26 mo results in (a) normalization of insulin-dependent glucose metabolism, most likely related to the reduction of prednisone dose, and, (b) maintenance of some alterations in leucine and FFA metabolism, probably related to the functional denervation of the graft and to the immunosuppressive treatment.     

Severe insulin-resistant diabetes mellitus in patients with congenital muscle fiber type disproportion myopathy.

Congenital muscle fiber type disproportion myopathy (CFTDM) is a chronic, nonprogressive muscle disorder characterized by universal muscle hypotrophy and growth retardation. Histomorphometric examination of muscle shows a preponderance of smaller than normal type 1 fibers and overall fiber size heterogeneity. Concomitant endocrine dysfunctions have not been described. We report the findings of altered insulin secretion and insulin action in two brothers affected with CFTDM and glucose intolerance as well as in their nonconsanguineous glucose-tolerant parents. Results are compared with those of six normoglycemic control subjects. All study participants underwent an oral glucose tolerance test to estimate insulin secretion. The oldest boy and his parents volunteered for studies of whole-body insulin sensitivity consisting of a 4-h euglycemic hyperinsulinemic clamp in combination with indirect calorimetry. Insulin receptor function and glycogen synthase (GS) activity and expression were examined in biopsies of vastus lateralis muscle. Despite a 45-90-fold increase in both fasting and postprandial serum insulin levels, both CFTDM patients had diabetes mellitus. Clamp studies revealed that the oldest boy had severe insulin resistance of both liver and peripheral tissues. The impaired insulin-stimulated glucose disposal to peripheral tissues was primarily due to reduced nonoxidative glucose metabolism. These changes were paralleled by reduced basal values of muscle GS total activity, allosterical activation of GS by glucose-6-phosphate, GS protein, and GS mRNA. The father expressed a lesser degree of insulin resistance, and studies of muscle insulin receptor function showed a severe impairment of receptor kinase activity. In conclusion, CFTDM is a novel form of severe hyperinsulinemia and insulin resistance. Whether insulin resistance is causally related to the muscle disorder awaits to be clarified.