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.     

Decreased insulin sensitivity and muscle enzyme activity in elderly subjects

Skeletal muscle glycogen deposition, and the activation of muscle glycogen synthase and pyruvate dehydrogenase during a hyerinsulinaemic euglycaemic clamp have been measured in six young and six elderly males matched for body mass index, physical activity and diet. Clamp glucose requirement (insulin, 0.1 U kg-1 h-1) was significantly lower in the older subjects (8.0 +/- 0.4 mg kg-1 min-1) than in younger subjects (10.5 +/- 0.6 mg kg-1 min-1, P less than 0.02). Although the older subjects had a 6.5% decrease in lean body mass, clamp glucose requirement expressed per unit of lean body mass was also significantly decreased in the older subjects (10.2 +/- 0.5 vs. 12.4 +/- 0.6 mg kg-1 min-1, P less than 0.05). The increase in muscle glycogen with the clamp was decreased by 33% in the older subjects (elderly: 13.1 +/- 1.3 mg g-1 protein, young: 19.6 +/- 2.2 mg g-1 protein; P less than 0.05), and was strongly correlated with clamp glucose requirement (r = 0.72, P less than 0.01). Glucose-6-phosphate independent glycogen synthase activity increased significantly between fasting and the end of the clamps in both groups (P less than 0.001), but was lower at the end of the clamp in the older subjects (P less than 0.05). Glycogen synthase activity at the end of the clamp correlated with both clamp glucose requirement (r = 0.83, P less than 0.01) and muscle glycogen deposition (r = 0.73, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determinants of insulin-stimulated skeletal muscle glycogen metabolism in man

Abstract. To examine factors which influence skeletal muscle glycogen synthesis in man, we related insulin sensitivity measured by euglycaemic insulin clamp in 43 healthy males to muscle glycogen synthase (GS) activity, GS protein content (Western blot), glycogen concentrations and fibre composition. Insulin increased muscle glycogen content (P<0.05) and the change in glycogen content correlated with the GS protein content (r=0.90, P=0.01). GS protein concentration correlated inversely with age (r=-0.69, P=0.04). Non-oxidative glucose disposal was inversely related to per cent type 2B fibres (r=-0.52, P< 005). The influence of age on these relationships was separately studied in young (n=12, age=26 ± 2 years) and elderly (n=15, age = 56 ± 2 years) males. Insulin increased GS activity significantly only in young subjects (from 17.8 ± 30 to 25.3 ± 3.2 nmol mg protein ' min^-1; P=0.015). GS activity and non-oxidative glucose disposal correlated in young (r=0.69, P=001) but not in the elderly (r=0.064, P = 0.82) males, and this relationship was not influenced by the degree of obesity. In conclusion, muscle fibre type and GS activity are both determinants of muscle glycogen metabolism in healthy, normoglycae-mic males. The close relationship between non-oxidative glucose metabolism and GS activity in young males is altered in ageing.     

Skeletal muscle glucose uptake, glycogen synthase activity and GLUT 4 content during hypoglycaemia in type 1 diabetic subjects

In healthy subjects, hypoglycaemia induces a profound 80% reduction in skeletal muscle glucose uptake and a similar suppression of glycogen synthase activity. The aim of this study was to examine the efficacy of this counterregulatory mechanism in type 1 diabetic subjects, who are especially prone to hypoglycaemic incidents. Nine type 1 diabetic male subjects were examined twice; during 120 min of hyperinsulinaemic (1.5 mU x kg(-1) x min(-1)) euglycaemia followed by (i) 240 min of graded hypoglycaemia (glucose nadir 2.8 mM) or (ii) 240 min of euglycaemia. At 345-360 min a muscle biopsy was taken and indirect calorimetry was performed at 210-240 and 320-340 min. The sensitivity of glycogen synthase to glucose-6-P was reduced by hypoglycaemia, as shown by an increase in A0.5 for glucose-6-P (at 0.07 mmol/L) from 0.21+/-0.02 to 0.28+/-0.03 mM (p=0.06). Likewise, the fractional velocity for glycogen synthase was reduced by 25%; i.e. from 20.8+/-2.0 to 15.5+/-1.4% (p<0.05). Total glucose disposal was decreased during hypoglycaemia (5.3+/-0.6 vs. 8.3+/-0.7 mg x kg(-1) x min(-1) (euglycaemia), n = 9; p<0.05), primarily due to a reduction of non-oxidative glucose disposal (2.7+/-0.3 vs. 5.1+/-0.6 mg x kg(-1) x min(-1) (euglycaemia), n=7; p<0.05). Forearm arteriovenous glucose differences were decreased by 50% in the hypoglycaemic situation (0.7+/-0.1 vs. 1.4+/-0.3 mmol/L (320-340 min)), and counterregulatory hormonal responses seemed less conspicuous than described in healthy subjects. We conclude that hypoglycaemia induces decrements of forearm glucose uptake and glycogen synthase activity in type 1 diabetic subjects. The study indicates a decreased magnitude of these responses, but this remains to be confirmed.     

Relative contribution of glycogen synthesis and glycolysis to insulin-mediated glucose uptake. A dose-response euglycemic clamp study in normal and diabetic rats.

To examine the relationship between plasma insulin concentration and intracellular glucose metabolism in control and diabetic rats, we measured endogenous glucose production, glucose uptake, whole body glycolysis, muscle and liver glycogen synthesis, and rectus muscle glucose-6-phosphate (G-6-P) concentration basally and during the infusion of 2, 3, 4, 12, and 18 mU/kg.min of insulin. The contribution of glycolysis decreased and that of muscle glycogen synthesis increased as the insulin levels rose. Insulin-mediated glucose disposal was decreased by 20-30% throughout the insulin dose-response curve in diabetics compared with controls. While at low insulin infusions (2 and 3 mU/kg.min) reductions in both the glycolytic and glycogenic fluxes contributed to the defective tissue glucose uptake in diabetic rats, at the three higher insulin doses the impairment in muscle glycogen repletion accounted for all of the difference between diabetic and control rats. The muscle G-6-P concentration was decreased (208 +/- 11 vs. 267 +/- 18 nmol/g wet wt; P less than 0.01) compared with saline at the lower insulin infusion, but was gradually increased twofold (530 +/- 16; P less than 0.01 vs. basal) as the insulin concentration rose. The G-6-P concentration in diabetic rats was similar to control despite the reduction in glucose uptake. These data suggest that (a) glucose transport is the major determinant of glucose disposal at low insulin concentration, while the rate-limiting step shifts to an intracellular site at high physiological insulin concentration; and (b) prolonged moderate hyperglycemia and hypoinsulinemia determine two distinct cellular defects in skeletal muscle at the levels of glucose transport/phosphorylation and glycogen synthesis.     

Decreased insulin activation of glycogen synthase in skeletal muscles in young nonobese Caucasian first-degree relatives of patients with non-insulin-dependent diabetes mellitus.

Insulin resistance in non-insulin-dependent diabetes is associated with a defective insulin activation of the enzyme glycogen synthase in skeletal muscles. To investigate whether this may be a primary defect, we studied 20 young (25 +/- 1 yr) Caucasian first-degree relatives (children) of patients with non-insulin-dependent diabetes, and 20 matched controls without a family history of diabetes. Relatives and controls had a normal oral glucose tolerance, and were studied by means of the euglycemic hyperinsulinemic clamp technique, which included performance of indirect calorimetry and muscle biopsies. Insulin-stimulated glucose disposal was decreased in the relatives (9.2 +/- 0.6 vs 11.5 +/- 0.5 mg/kg fat-free mass per (FFM) min, P less than 0.02), and was due to a decreased rate of insulin-stimulated nonoxidative glucose metabolism (5.0 +/- 0.5 vs 7.5 +/- 0.4 mg/kg fat-free mass per min, P less than 0.001). The insulin-stimulated, fractional glycogen synthase activity (0.1/10 mmol liter glucose-6-phosphate) was decreased in the relatives (46.9 +/- 2.3 vs 56.4 +/- 3.2%, P less than 0.01), and there was a significant correlation between insulin-stimulated, fractional glycogen synthase activity and nonoxidative glucose metabolism in relatives (r = 0.76, P less than 0.001) and controls (r = 0.63, P less than 0.01). Furthermore, the insulin-stimulated increase in muscle glycogen content over basal values was lower in the relatives (13 +/- 25 vs 46 +/- 9 mmol/kg dry wt, P = 0.05). We conclude that the defect in insulin activation of muscle glycogen synthase may be a primary, possibly genetically determined, defect that contributes to the development of non-insulin-dependent diabetes.     

Impaired insulin-stimulated muscle glycogen synthase activation in vivo in man is related to low fasting glycogen synthase phosphatase activity.

Insulin-mediated glycogen synthase activity in skeletal muscle correlates with the rate of insulin-mediated glycogen deposition and is reduced in human subjects with insulin resistance. To assess the role of glycogen synthase phosphatase as a possible mediator of reduced glycogen synthase activity, we studied 30 Southwestern American Indians with a broad range of insulin action in vivo. Percutaneous biopsies of the vastus lateralis muscle were performed before and during a 440-min euglycemic clamp at plasma insulin concentrations of 89 +/- 5 and 1,470 +/- 49 microU/ml (mean +/- SEM); simultaneous glucose oxidation was determined by indirect calorimetry. After insulin stimulation, glycogen synthase activity was correlated with the total and nonoxidative glucose disposal at both low (r = 0.73, P less than 0.0001; r = 0.68, P less than 0.0001) and high (r = 0.75, P less than 0.0001; r = 0.74, P less than 0.0001) plasma insulin concentrations. Fasting muscle glycogen synthase phosphatase activity was correlated with both total and nonoxidative glucose disposal rates at the low (r = 0.48, P less than 0.005; r = 0.41, P less than 0.05) and high (r = 0.47, P less than 0.05; r = 0.43, P less than 0.05) plasma insulin concentrations. In addition, fasting glycogen synthase phosphatase activity was correlated with glycogen synthase activity after low- (r = 0.47, P less than 0.05) and high- (r = 0.50, P less than 0.01) dose insulin stimulations. These data suggest that the decreased insulin-stimulated glucose disposal and reduced glycogen synthase activation observed in insulin resistance could be secondary to a low fasting glycogen synthase phosphatase activity.     

Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulin-dependent diabetes mellitus.

In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limiting enzymes in glycogen synthesis and glycolysis, glycogen synthase (GS) and phosphofructokinase (PFK), respectively. Analysis of biopsies of quadriceps muscle from 19 NIDDM patients and 19 control subjects showed in the basal state a 30% decrease (P < 0.005) in total GS activity and a 38% decrease (P < 0.001) in GS mRNA/microgram DNA in NIDDM patients, whereas the GS protein level was normal. The enzymatic activity and protein and mRNA levels of PFK were all normal in diabetic patients. In subgroups of NIDDM patients and control subjects an insulin-glucose clamp in combination with indirect calorimetry was performed. The rate of insulin-stimulated nonoxidative glucose metabolism was decreased by 47% (P < 0.005) in NIDDM patients, whereas the glucose oxidation rate was normal. The PFK activity, protein level, and mRNA/microgram DNA remained unchanged. The relative activation of GS by glucose-6-phosphate was 33% lower (P < 0.02), whereas GS mRNA/micrograms DNA was 37% lower (P < 0.05) in the diabetic patients after 4 h of hyperinsulinemia. Total GS immunoreactive mass remained normal. In conclusion, qualitative but not quantitative posttranslational abnormalities of the GS protein in muscle determine the reduced insulin-stimulated nonoxidative glucose metabolism in NIDDM.     

Insulin resistance in skeletal muscles in patients with NIDDM.

Skeletal muscles in patients with non-insulin-dependent diabetes mellitus (NIDDM) are resistant to insulin; i.e., the effect of insulin on glucose disposal is reduced compared with the effect in control subjects. This defect has been found to be localized to the nonoxidative pathway of glucose disposal; hence, the deposition of glucose, as glycogen, is abnormally low. This defect may be inherited, because it is present in first-degree relatives to NIDDM patients two to three decades before they develop frank diabetes mellitus. The cellular defects responsible for the abnormal insulin action in NIDDM patients is reviewed in this article. The paper focuses mainly on convalent insulin signaling. Insulin is postulated to stimulate glucose storage by initiating a cascade of phosphorylation and dephosphorylation events, which results in dephosphorylation and hence activation of the enzyme glycogen synthase. Glycogen synthase is the key enzyme in regulation of glycogen synthesis in the skeletal muscles of humans. This enzyme is sensitive to insulin, but in NIDDM patients it has been shown to be completely resistant to insulin stimulation when measured at euglycemia. The enzyme seems to be locked in the glucose-6-phosphate (G-6-P)-dependent inactive D-form. This hypothesis is favored by the finding of reduced activity of the glycogen synthase phosphatase and increased activity of the respective kinase cAMP-dependent protein kinase. A reduced glycogen synthase activity has also been found in normoglycemic first-degree relatives of NIDDM patients, indicating that this abnormality precedes development of hyperglycemia in subjects prone to develop NIDDM. Therefore, this defect may be of primary genetic origin. However, it does not appear to be a defect in the enzyme itself, but rather a defect in the covalent activation of the enzyme system. Glycogen synthase is resistant to insulin but may be activated allosterically by G-6-P. This means that the defect in insulin activation can be compensated for by increased intracellular concentrations of G-6-P. In fact, we found that both hyperinsulinemia and hyperglycemia are able to increase the G-6-P level in skeletal muscles. Thus, insulin resistance in the nonoxidative pathway of glucose processing can be overcomed (compensated) by hyperinsulinemia and hyperglycemia. In conclusion, we hypothesize that insulin resistance in skeletal muscles may be a primary genetic defect preceding the diabetic state. The cellular abnormality responsible for that may be a reduced covalent insulin activation of the enzyme glycogen synthase.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Impact of a glycogen phosphorylase inhibitor and metformin on basal and glucagon-stimulated hepatic glucose flux in conscious dogs

The effects of a glycogen phosphorylase inhibitor (GPI) and metformin (MT) on hepatic glucose fluxes (μmol · kg(-1) · min(-1)) in the presence of basal and 4-fold basal levels of plasma glucagon were investigated in 18-h fasted conscious dogs. Compared with the vehicle treatment, GPI infusion suppressed net hepatic glucose output (NHGO) completely (-3.8 ± 1.3 versus 9.9 ± 2.8) despite increased glucose 6-phosphate (G-6-P) neogenesis from gluconeogenic precursors (8.1 ± 1.1 versus 5.5 ± 1.1). MT infusion did not alter those parameters. In response to a 4-fold rise in plasma glucagon levels, in the vehicle group, plasma glucose levels were increased 2-fold, and NHGO was increased (43.9 ± 5.7 at 10 min and 22.7 ± 3.4 at steady state) without altering G-6-P neogenesis (3.7 ± 1.5 and 5.5 ± 0.5, respectively). In the GPI group, there was no increase in NHGO due to decreased glucose-6-phosphatase flux associated with reduced G-6-P concentration. A lower G-6-P concentration was the result of increased net glycogenesis without altering G-6-P neogenesis. In the MT group, the increment in NHGO (22.2 ± 4.4 at 10 min and 12.1 ± 3.6 at steady state) was approximately half of that of the vehicle group. The lesser NHGO was associated with reduced glucose-6-phosphatase flux but a rise in G-6-P concentration and only a small incorporation of plasma glucose into glycogen. In conclusion, the inhibition of glycogen phosphorylase a activity decreases basal and glucagon-induced NHGO via redirecting glucose 6-phosphate flux from glucose toward glycogen, and MT decreases glucagon-induced NHGO by inhibiting glucose-6-phosphatase flux and thereby reducing glycogen breakdown.     

Multiple defects in muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus.

To define the mechanisms of impaired muscle glycogen synthase and reduced glycogen formation in non-insulin dependent diabetes mellitus (NIDDM), glycogen synthase activity was kinetically analyzed during the basal state and three glucose clamp studies (insulin approximately equal to 300, 700, and 33,400 pmol/liter) in eight matched nonobese NIDDM and eight control subjects. Muscle glycogen content was measured in the basal state and following clamps at insulin levels of 33,400 pmol/liter. NIDDM subjects had glucose uptake matched to controls in each clamp by raising serum glucose to 15-20 mmol/liter. The insulin concentration required to half-maximally activate glycogen synthase (ED50) was approximately fourfold greater for NIDDM than control subjects (1,004 +/- 264 vs. 257 +/- 110 pmol/liter, P less than 0.02) but the maximal insulin effect was similar. Total glycogen synthase activity was reduced approximately 38% and glycogen content was approximately 30% lower in NIDDM. A positive correlation was present between glycogen content and glycogen synthase activity (r = 0.51, P less than 0.01). In summary, defects in muscle glycogen synthase activity and reduced glycogen content are present in NIDDM. NIDDM subjects also have less total glycogen synthase activity consistent with reduced functional mass of the enzyme. These findings and the correlation between glycogen synthase activity and glycogen content support the theory that multiple defects in glycogen synthase activity combine to cause reduced glycogen formation in NIDDM.     

Regulation of glycogen synthase and phosphorylase activities by glucose and insulin in human skeletal muscle.

We examined the insulin dose-response characteristics of human muscle glycogen synthase and phosphorylase activation. We also determined whether increasing the rate of glucose disposal by hyperglycemia at a fixed insulin concentration activates glycogen synthase. Physiological increments in plasma insulin but not glucose increased the fractional activity of glycogen synthase. The ED50: s for insulin stimulation of whole body and forearm glucose disposal were similar and unaffected by glycemia. Glycogen synthase activation was exponentially related to the insulin-mediated component of whole body and forearm glucose disposal at each glucose concentration. Neither insulin nor glucose changed glycogen phosphorylase activity. These results suggest that insulin but not the rate of glucose disposal per se regulates glycogen synthesis by a mechanism that involves dephosphorylation of glycogen synthase but not phosphorylase. This implies that the low glycogen synthase activities found in insulin-resistant states are a consequence of impaired insulin action rather than reduced glucose disposal.     

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.     

Correlation between muscle glycogen synthase activity and in vivo insulin action in man.

We have studied the relationship between in vivo insulin-mediated glucose disposal rates, muscle glycogen content, and muscle glycogen synthase activity in 25 southwest American Indians with normal glucose tolerance and with varying degrees of glucose intolerance. Insulin-mediated glucose disposal (M) was measured by using the hyperinsulinemic euglycemic clamp technique at plasma insulin concentrations of 134 +/- 7 and 1709 +/- 72 microU/ml, with simultaneous indirect calorimetry to assess glucose oxidation and storage rates. Muscle glycogen content and glycogen synthase activity were measured in percutaneous muscle biopsy samples obtained from the vastus lateralis muscle before and after the euglycemic clamp procedure. The results showed that muscle glycogen synthase activity at the end of the euglycemic clamp was well correlated with insulin-mediated glucose storage rates at both low (r = 0.50, P less than 0.02) and high (r = 0.78, P less than 0.0001) insulin concentrations; and also correlated with M (r = 0.66, P less than 0.001 and r = 0.76, P less than 0.0001). Similar correlations were observed between the change in muscle glycogen synthase activity and glucose storage rates and M. The change in muscle glycogen synthase activity correlated with the change in muscle glycogen content (r = 0.46, P less than 0.03) measured before and after the insulin infusions. The change in muscle glycogen content did not correlate with glucose storage rates or M. The data suggest the possible importance of glycogen synthesis in muscle in determining in vivo insulin-mediated glucose disposal rates in man.     

葡萄糖-6-磷酸脱氢酶活性浓度检测法的初步研究

目的建立一种简单、快速、可自动检测葡萄糖-6-磷酸脱氢酶（G-6-PD）在血红蛋白（Hb）中活性的方法。方法用自配的检测试剂，在全自动生化分析仪上检测抗凝血标本的G-6-PD活性，同时检测Hb浓度，并计算出G-6-PD在Hb中的活性浓度（U／gHb），对同一标本用简易快速高铁血红蛋白还原试验、G-6-P／6-GP比值法进行对比试验。结果G-6-PD活性浓度的参考范围为≥5．0U／gHb，G-6-PD活性浓度检测法与G-6-P／6-GP比值法及简易快速高铁血红蛋白还原试验检测法的检测结果具有高度一致性。G-6-PD活性浓度检测法可用不洗涤红细胞，其检测试剂及检测方法具有良好的重复性。制备溶血液时吸样量从7～13μl的G-6-PD活性浓度检测结果差异无统计学意义（P〉0．05）。直接用酸性枸橼酸右旋糖（ACD）抗凝全血比用压积红细胞检测G-6-PD活性浓度的结果高。结论G-6-PD活性浓度检测法是一种简单、快速、重复性好、操作简便的检测方法。     

Correction of chronic hyperglycemia with vanadate, but not with phlorizin, normalizes in vivo glycogen repletion and in vitro glycogen synthase activity in diabetic skeletal muscle.

Vanadate has insulin-like activity in vitro and in vivo. To characterize the in vivo mechanism of action of vanadate, we examined meal tolerance, insulin-mediated glucose disposal, in vivo liver and muscle glycogen synthesis, and in vitro glycogen synthase activity in 90% partially pancreatectomized rats. Four groups were studied: group I, sham-operated controls; group II, diabetic rats; group III, diabetic rats treated with vanadate; and group IV, diabetic rats treated with phlorizin. Insulin sensitivity, assessed with the euglycemic hyperinsulinemic clamp technique in awake, unstressed rats, was reduced by approximately 28% in diabetic rats. Both vanadate and phlorizin treatment completely normalized meal tolerance and insulin-mediated glucose disposal. Muscle glycogen synthesis was reduced by approximately 80% in diabetic rats (P less than 0.01) and was completely restored to normal by vanadate, but not by phlorizin treatment. Glycogen synthase activity was reduced in skeletal muscle of diabetic rats (P less than 0.05) compared with controls and was increased to supranormal levels by vanadate treatment (P less than 0.01). Phlorizin therapy did not reverse the defect in muscle glycogen synthase. These results suggest that (a) the defect in muscle glycogen synthesis is the major determinant of insulin resistance in diabetic rats; (b) both vanadate and phlorizin treatment normalize meal tolerance and insulin sensitivity in diabetic rats; (c) vanadate treatment specifically reverses the defect in muscle glycogen synthesis in diabetic rats. This effect cannot be attributed to the correction of hyperglycemia because phlorizin therapy had no direct influence on the glycogenic pathway.     

Glucose-6-phosphatase activity in liver and blood platelets of two patients with glycogen storage disease type I.

Glucose-6-phosphatase (G-6-Pase) activity in liver and blood platelets of two patients with glycogen storage disease (GSD) type I is described. Both patients had a reduced activity of G-6-Pase in liver. The km value for glucose 6-phosphate (G-6-P) of residual activity in liver of both patients was similar to that of control liver. We could not demonstrate any reduced activity of platelet G-6-Pase in the patients. Platelet G-6-Pase with our assay method seems to represent a nonspecific phosphatase activity. Our observation suggests that it is necessary to examine platelet G-6-Pase of many other patients with GSD type I to confirm that G-6-Pase deficiency can be diagnosed by enzyme assay performed on blood platelets.     

Relationship between muscle morphology and metabolism in obese women: the effects of long-term physical training

To evaluate the relationships between changes in muscle morphology and metabolic adaptation to physical training in obesity, twenty obese women were subjected to a physical training programme with three sessions a week for 3 months. Physical training resulted in lowering of plasma insulin and improved glucose tolerance. Neither body weight nor body fat changed. With physical training the percentage distribution of fast twitch oxidative (FTa) muscle fibres (m vastus lateralis) increased (from 30.3 +/- 5.1% to 35.2 +/- 4.8%, P less than 0.05) and that of fast twitch glycolytic fibres decreased (from 18.3 +/- 6.6 to 5.8 +/- 4.8%, P less than 0.05). The number of capillaries increased, mainly around slow twitch (ST) fibres (from 4.5 +/- 0.6 to 5.8 +/- 0.8, P less than 0.01) and fast twitch oxidative (FTa) fibres (from 3.9 +/- 0.7 to 4.7 +/- 0.8, P less than 0.01). The activities of oxidative enzymes (cytochrome-c-oxidase and citrate synthase) increased (P less than 0.05) while those of glycolytic enzymes (phosphofructokinase and hexokinase) decreased after physical training (P less than 0.01). Significant negative correlations between plasma insulin and number of capillaries in contact with ST fibres (r = 0.80, P less than 0.001) and FTa fibres (r = 0.62, P less than 0.001) were found before training. The capillary density around those fibres could predict 80% of the explained variance of plasma insulin levels (P less than 0.001). The changes of glucose concentration after training could be predicted by observed changes in enzyme activities. The strong associations between muscle morphology and capillarization and enzyme activities and glucose and insulin concentrations and their changes after training suggest an important regulatory role of muscle which warrants further studies.