Hyperglycaemia compensates for the defects in insulin-mediated glucose metabolism and in the activation of glycogen synthase in the skeletal muscle of patients with Type 2 (non-insulin-dependent) diabetes mellitus

Diabetologia, Jan 1992

A. Vaag, P. Damsbo, O. Hother-Nielsen, H. Beck-Nielsen

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Hyperglycaemia compensates for the defects in insulin-mediated glucose metabolism and in the activation of glycogen synthase in the skeletal muscle of patients with Type 2 (non-insulin-dependent) diabetes mellitus

Diabetologia Hyperglycaemia compensates for the defects in insulin-mediated glucose metabolism and in the activation o f glycogen synthase in the skeletal muscle o f patients with Type 2 (non-insulin-dependent) diabetes mellitus A. Vaag 0 1 e. Damsbo 0 O. HotherlNielsen 1 H. Beck-Nielsen 0 1 0 Hvid6re Hospital , Klampenborg , Denmark 1 Department ofEndocrinologyand Internal MedicineM , Odense UniversityHospital, Odense Summary. Insulin resistance and a defective insulin activation of the enzyme glycogen synthase in skeletal muscle during euglycaemia may have important pathophysiological implications in Type2 (non-insulin-dependent) diabetes mellitus. Hyperglycaemia may serve to compensate for these defects in Type 2 diabetes by increasing glucose disposal through a mass action effect. In the present study, rates of whole-body glucose oxidation and glucose storage were measured during fasting hyperglycaemia and isoglycaemic insulin infusion (40 mU. m -2. min-1, 3 h) in 12 patients with Type 2 diabetes. Eleven control subjects were studied during euglycaemia. Biopsies were taken from the vastus lateralis muscle. Fasting and insulin-stimulated glucose oxidation, glucose storage and muscle glycogen synthase activation were all fully compensated (normalized) during hyperglycaemia in the diabetic patients. The insulin-stimulated increase in muscle glycogen content was the same in the Glycogen synthase; skeletal muscle; Type 2 (noninsulin-dependent) diabetes mellitus; hyperglycaemia; insulin resistance - 9 Springer-Verlag1992 Patients with Type 2 (non-insulin-dependent) diabetes mellitus are characterized by insulin resistance when studied by means of the euglycaemia hyperinsulinaemic clamp technique [ 1-4 ]. The insulin resistance occurs in both the oxidative and non-oxidative pathways, although the predominant defect is on non-oxidative glucose metabolism [ 3-7 ]. The main site of the insulin resistance is considered to be in skeletal muscle [ 3, 8 ], and insulinstimulated non-oxidative glucose metabolism is considered primarily to represent muscle glycogen synthesis. The rate limiting key enzyme in this pathway is glycogen synthase [ 9, 10 ]. Glycogen synthase is stimulated covalently (dephosphorylated) by insulin [ 10, 11 ], and a close correlation between in vivo insulin-stimulated muscle glycogen synthase activity and whole-body non-oxidative glucose disposal has been demonstrated during euglycaemia in subjects with and without Type 2 diabetes [ 4, 10, 12 ]. In addition, a defective activation of glycogen synthase in skeletal muscle during a euglycaemic insulin diabetic patients and in the control subjects. Besides hyperglycaemia, the diabetic patients had elevated muscle free glucose and glucose 6-phosphate concentrations. A positive correlation was demonstrated between intracellular free glucose concentration and muscle glycogen synthase fractional velocity insulin activation (0.1 mmolfl glucose 6-phosphate: r = 0.65,p < 0.02 and 0.0 mmol/1 glucose 6-phosphate: r = 0.91, p < 0.0001). In conclusion, this study indicates an important role for hyperglycaemia and elevated muscle free glucose and glucose 6-phosphate concentrations in compensating (normalizing) intracellular glucose metabolism and skeletal muscle glycogen synthase activation in Type 2 diabetes. infusion is a consistent finding in Type 2 diabetic patients [ 4, 8, 13 ]. Hyperglycaemia increases glucose uptake in peripheral tissues by a mass action effect, and it has been previously demonstrated that when glucose clamp studies are performed at the patients' ambient fasting glucose levels (isoglycaemic hyperinsulinaemic clamp procedure), Type 2 diabetic patients exhibit overall glucose disposal rates that are similar to those observed in normal subjects studied during euglycaemia at similar insulin concentrations [ 14 ]. This suggests that the ambient level of fasting hyperglycaemia may compensate for the insulin resistance in Type 2 diabetes. The aim of the present study was to elucidate the extent of, and the mechanisms behind, a potentially compensated (or normalized) intracellular muscle glucose metabolism and glycogen synthase activation in patients with Type 2 diabetes during fasting hyperglycaemia and isoglycaemic insulin infusion. Twelve patients with Type 2 diabetes were compared with a group of age- and sex-matched normal subjects (Table 1). The control subjects were non-obese with normal oral glucose tolerance on testing. None of the control subjects had any familial history of diabetes. The diabetic patients had significantly higher fasting plasma glucose, insulin and C-peptide concentrations according to the nature of their disease. Furthermore, the diabetic patients were more obese than the control subjects. None of the diabetic patients were treated with insulin, and none of the diabetic or control subjects were exercising regularly. Seven patients were being treated with diet alone and five patients were treated with diet and oral hypoglycaemic drugs (sulfonylurea or metformin). Oral hypoglycaemic drugs were withdrawn at least I week prior to the studies. The patients with Type 2 diabetes were on a diet consisting of approximately 55 % carbohydrate, 15 % protein and 30 % fat. None of the study subjects had clinical evidence of cardiac, hepatic or renal disease or endocrine disorders other than diabetes. Furthermore, the diabetic patients had no evidence of diabetic neuropathy, nephropathy or proliferative retinopathy. Informed consent was obtained and the study was approved by the regional ethical committee. The procedure was performed according to the principles of the Helsinki Declaration. In vivo methods Isoglycaemic insulin clamp. All studies were started at 07.30 hours after a 10-h overnight fast. A polyethylene catheter was inserted into an antecubital vein for infusion of test substances. A n o t h e r polyethylene catheter was inserted into a contralateral wrist vein for blood sampling. This hand was placed and maintained in a heated plexiglas box to obtain arterialized venous blood [ 15 ]. After a 120min basal equilibration period for basal measurements (Fig. 1), insulin (Actrapid, Novo-Nordisk, Bagsvaerd, Denmark) was infused for 180 rain at a constant rate of 40 m U . m - 2 . m i n t in both Type 2 diabetic patients and control subjects. The plasma glucose concentration was maintained using a variable glucose infusion (1 mol/1) (isogIycaemic clamp procedure). Thus, the Type 2 diabetic patients were studied at their ambient levels of fasting hyperglycaemia, whereas the control subjects were studied at euglycaemic levels. The plasma glucose concentration was monitored in arterialized blood every 5-10 rain using an automated glucose oxidase method (Glucose Analyser 2, Beckman Instruments, Fullerton, Calif., USA). Steady-state periods were defined as the last 30 min during basal measurements ( - 30-0 min) and the last 30 min of insulin-stimulated measurements (150-180 min). Urinary glucose excretion was measured during each clamp study in order to correct for this when calculating the total peripheral glucose utilization Tritiatedglucose. The glucose clamp studies were combined with a primed continous infusion of 3-3H-glucose (New England Nuclear, Boston, Mass., USA) (Fig. 1). In order to ensure isotope equilibrium, the continuous infusion of 3-3H-glucose (0.22 gCi/min) was begun 90.120 min before measurements of glucose turnover were performed, and the priming dose of 3-3H-glucose was increased in proportion to fasting hyperglycaemia in the Type 2 diabetic patients according to the formula: priming dose = 22 gCi x plasma glucose concentration (mmol/1) / 5 (mmol/1) [ 16 ]. During steady-state periods, blood samples were drawn in fluoride-treated tubes at 10-min intervals for determination of plasma glucose and plasma 3-~H-glucose activitY. During the remainder of the study period, plasma glucose and 3-3H-glucose activity was measured every 30 min. Indirect calorimetry. Indirect calorimetry was performed using a computerized flowthrough canopy gas analyser system (Deltatrac, Datex, Helsinki, Finland). Briefly, air is suctioned at a rate of 40 litres per min through a canopy placed over the head of the subject. Samples of inspired and expired air are analysed for oxygen concentration using a paramagnetic differential oxygen sensor and for carbon dioxide using an infra-red carbon dioxide sensor. Signals from the gas analysers are processed by the computer and oxygen consumption and carbon dioxide production are calculated and recorded once per min. After an equilibration period of 10 min, the average gas exchange rates recorded over the two 30-rain steadystate periods (Fig. 1) were used to calculate rates of glucose oxidation, lipid oxidation and energy expenditure as previously described [ 17,18 ]. The protein oxidation rate was estimated from urinary urea nitrogen excretion (1 g nitrogen = 6.25 g protein) and corrected for changes in pool size [19]. Muscle biopsy. Muscle biopsies were performed using a modified BergstrOm needle (including suction) under local anaesthesia. The t 150 180 Time (min) -150 - 20 biopsies were rapidly (within 10-15 s) frozen and stored in liquid nitrogen for later analysis. Before the biochemical analysis the muscle samples were freeze-dried and dissected free of visible connective tissue, fat and blood. Calculations. During the steady-state periods, glucose turnover rates (hepatic glucose output (HGO) and total peripheral glucose disposal) were calculated at 10-min intervals using Steele's non-steadystate equations [ 20 ]. Non-steady-state equations were used during basal measurements because of spontaneously decliningplasma glucose concentrations in the Type 2 diabetic patients. In these calculations, the distribution volume of glucose was taken as 200 ml/kg body weight and the pool fraction as 0.65 [ 21 ]. During the insulin infusion periods negative rates of H G O were calculated in all subjects. Such underestimation of glucose turnover by the tracer method is largely accounted for by a model error emerging at high rates of glucose metabolism [ 22 ]. We took the negative numbers to indicate a nil HGO. Thus, because H G O was negative during the insulin infusion in all of the subjects studied, the infusion rate of exogenous glucose was equal to total peripheral glucose disposal. Total peripheral glucose disposal was corrected for urinary glucose excretion. Non-oxidative glucose metabolism (glucose storage) was calculated as the difference between total body glucose utilization and glucose oxidation, as determined by indirect calorimetry. Glucose and lipid metabolism data were expressed as mg per kg fat free mass (FFM) per min. Total body fat and thus FFM was measured using the bioimpedance method [ 23 ]. In vitro methods Glycogen and metabolite concentrations in muscle biopsies. Glycogen was measured as glucose residues after hydrolysis of the muscle samples, in i mol/1 HCL at 100 ~ for 2 h [ 24 ]. Glucose, glucose 6-phosphate (G6P) and lactate were measured fluorometrically on neutralized perchloric acid extracts [ 24 ]. Intracellular concentrations of free glucose, G6P and lactate were calculated as millimoles per litre of intracellular water, assuming an extracellular water content in the biopsies of 0.3 1/kg dry weight and an intracellular water content of 2.81/kg dry weight [ 25, 26 ]. Intracellular concentrations of free glucose and lactate were corrected for extracellular concentrations using the above assumptions of intracellular and extracellular water content in the biopsies. Glycogen synthase activity. Extraction of muscle samples and assays for glycogen synthase were performed as we previously described [ 4, 10 ] by a modification of the method of Thomas et al. [27]. Glycogen synthase activitywas assayed without adding its allostericmodulator G6R in the presence of a near physiological concentration of G6P (0.1 mmol/1) and in the presence of a high G6P concentration (10 mmol/1). This concentration was used to determine maximal enzyme activity. The total concentration of uridine diphosphate glucose (14C-UDPG + cold UDPG) in the reaction mixture was 0.31 mmol/1. Glycogen synthase activitywas expressed as nanomoles of U D P G incorporated into glycogen per rain per milligram extract protein. Fractional velocities (FV) of the enzyme were calculated as the ratio between glycogen synthase activities assayed at 0 mmol/1 G6P and 10 mmol/1 G6P (FV 0.0) and at 0.1 mmol/1 G6P and 10 mmol/1 G6P (FV 0.1). The fractional velocities of glycogen synthase in muscle biopsies have previously been demonstrated to correlate with in vivo non-oxidative glucose metabolism during a euglycaemic insulin infusion [ 4, 10, 12 ], and fractional velocities are therefore thought to merely reflect the in vivo covalent activation of the enzyme. Protein content of the extracts was determined by the method of Lowry et al. [ 28 ]. Analytical determinations. Glucose in plasma and urine was determined by a hexokinase method [ 29 ]. Tritiated glucose activity was measured as described by Hother-Nielsen and Beck-Nielsen [ 16 ]. Plasma insulin [ 30 ] and C-peptide [ 31 ] concentrations were measured with radioimmunological methods. Non-esterified fatty A. Vaag et al.: Hyperglycaemiaand muscle glucose metabolism in Type 2 diabetes acids (NEFA) in plasma were determined by the method of Itaya et al. [ 32 ] and lactate by the method of Passonneau [ 33 ]. Plasma concentrations of glucose, insulin, C-peptide, NEFA and lactate were measured at 10-rain intervals during both steady-state periods. HbAlc was measured by isoelectric focussing [ 34 ] (normal range 4.1~6.1%). Statistical analysis Non-parametric statistical methods (Wilcoxon test for paired data, Mann-Whitney test for unpaired data, and Spearmans rho (r) for correlation analysis) were employed in analysis of data.p values less than 0.05 were considered significant. Data in text and figures are presented as the mean + SEM. R e s u l t s P l a s m a g l u c o s e , insulin, C - p e p t i d e , N E F A a n d l a c t a t e c o n c e n t r a t i o n s d u r i n g c l a m p s t u d i e s a r e g i v e n in T a b l e 2. P l a s m a g l u c o s e c o n c e n t r a t i o n s w e r e s i g n i f i c a n t l y h i g h e r in t h e T y p e 2 d i a b e t i c p a t i e n t s d u r i n g t h e b a s a l p o s t p r a n d i a l s t a t e a n d d u r i n g i n s u l i n infusion. M e a n c o e f f i c i e n t s o f v a r i a t i o n o f p l a s m a g l u c o s e c o n c e n t r a t i o n s d u r i n g ins u l i n - s t i m u l a t e d s t e a d y - s t a t e p e r i o d s w e r e s m a l l a n d s i m i l a r in d i a b e t i c p a t i e n t s a n d c o n t r o l s u b j e c t s (3 + 1 vs 5 + 1 % , N S ) . P l a s m a insulin a n d C - p e p t i d e c o n c e n t r a t i o n s w e r e s i g n i f i c a n t l y h i g h e r in t h e d i a b e t i c p a t i e n t s in t h e b a s a l s t a t e . T h e h i g h e r i n s u l i n a n d C - p e p t i d e c o n c e n t r a t i o n s d u r i n g insulin i n f u s i o n d i d n o t r e a c h statist i c a l s i g n i f i c a n c e . I n c r e m e n t s in p l a s m a i n s u l i n c o n c e n t r a t i o n s d u r i n g i n s u l i n i n f u s i o n s w e r e i d e n t i c a l in d i a b e t i c p a t i e n t s a n d c o n t r o l s u b j e c t s (0.41 vs 0.37 nmol/1, N S ) . P l a s m a l a c t a t e c o n c e n t r a t i o n s w e r e e l e v a t e d in t h e d i a b e t i c p a t i e n t s in b o t h t h e b a s a l s t a t e a n d d u r i n g i n s u l i n i n f u s i o n , w h e r e a s p l a s m a N E F A c o n c e n t r a t i o n s w e r e n o r m a l . I s o t o p i c s t e a d y - s t a t e ( c o n s t a n t p l a s m a c o n c e n t r a t i o n o f 3 - 3 H - g l u c o s e ) w a s o b t a i n e d d u r i n g t h e p r e - d e f i n e d b a s a l s t e a d y - s t a t e p e r i o d in b o t h d i a b e t i c p a t i e n t s a n d Mean + SEM. ~ p < 0.02 vs control subjects, b p < 0.01 vs control subjects c o n t r o l subjects. I n s u l i n - s t i m u l a t e d r a t e s o f t o t a l p e r i p h e r a l g l u c o s e utilization, g l u c o s e o x i d a t i o n a n d n o n - o x i d a t i v e g l u c o s e m e t a b o l i s m w e r e s i m i l a r in t h e T y p e 2 d i a b e t i c p a t i e n t s d u r i n g h y p e r g i y c a e m i a a n d in t h e c o n trol subjects d u r i n g e u g l y c a e m i a ( T a b l e 3). I n t h e b a s a l state, t h e t o t a l g l u c o s e u t i l i z a t i o n r a t e was slightly e l e v a t e d in t h e d i a b e t i c p a t i e n t s ( T a b l e 3). T h i s was d u e t o e l e v a t e d r a t e s o f b o t h g l u c o s e o x i d a t i o n a n d g l u c o s e s t o r a g e , a l t h o u g h n e i t h e r o f t h e s e d i f f e r e n c e s r e a c h e d statistical significance w h e n t e s t e d s e p a r a t e l y . B a s a l H G O w a s slightly e l e v a t e d in t h e d i a b e t i c p a t i e n t s , w h e n e x p r e s s e d b o t h as m g g l u c o s e - m - 2 - rain - ~(86 + 2 vs 78 + 3, p < 0.05), o r as m g g l u c o s e . ( k g F F M ) - 1. r a i n - 1 (3.1 + 0.1 vs 2.7 + 0.2, p < 0.05). I n t h e d i a b e t i c p a t i e n t s , t h e b a s a l g l u c o s e u t i l i z a t i o n r a t e e x c e e d e d t h e b a s a l H G O (3.3 + 0.2 vs 3.1 + 0.1 m g . (kg F F M - ~ ) . m i n - ~, p < 0.05). T h u s , a s p o n t a n e o u s l y falling p l a s m a g l u c o s e c o n c e n t r a t i o n w a s o b s e r v e d d u r i n g t h e 2-h b a s a l s t a t e in t h e d i a b e t i c p a t i e n t s . N o d i f f e r e n c e in lipid o x i d a t i o n ( T a b l e 3) w a s f o u n d in t h e b a s a l s t a t e b e t w e e n d i a b e t i c p a t i e n t s (1.13 + 0.08 m g . ( k g F F M ) - 1. m i n - 1) a n d c o n t r o l subjects (1.45 m g . (kg F F M ) - 1. m i n - 1). F u r t h e r m o r e , t h e lipid oxid a t i o n w a s e q u a l l y s u p p r e s s e d d u r i n g insulin i n f u s i o n in t h e d i a b e t i c p a t i e n t s (0.50 + 0.02 m g . (kg F F M ) - ~. m i n - ~) a n d in t h e c o n t r o l subjects (0.71 + 0.13 m g - ( k g F F M ) -1. m i n - 1 ) . M u s c l e g l y c o g e n s y n t h a s e activities, e x p r e s s e d as fract i o n a l velocities, w e r e s t i m u l a t e d b y insulin in all t h e T y p e 2 d i a b e t i c p a t i e n t s a n d c o n t r o l subjects ( T a b l e 4). N o d i f f e r e n c e in insulin sensitivity o f m u s c l e g l y c o g e n synt h a s e activity w a s f o u n d b e t w e e n p a t i e n t s w i t h T y p e 2 d i a b e t e s o r t h e c o n t r o l subjects. M u s c l e g l y c o g e n c o n t e n t i n c r e a s e d significantly in t h e d i a b e t i c p a t i e n t s d u r i n g t h e i s o g l y c a e m i c insulin i n f u s i o n ( T a b l e 5). T h e i n s u l i n - s t i m u l a t e d i n c r e a s e in m u s c l e glyc o g e n c o n t e n t in the n o r m a l subjects w a s similar t o t h e d i a b e t i c (i. e. 28 m m o l / k g d r y w e i g h t ) , a l t h o u g h this inc r e a s e w a s n o t statistically significant. A significantly h i g h e r m u s c l e f r e e g l u c o s e c o n c e n t r a t i o n w a s f o u n d in t h e p a t i e n t s w i t h T y p e 2 d i a b e t e s in t h e b a s a l s t a t e a n d d u r i n g insulin i n f u s i o n ( T a b l e 5). N o c o r r e l a t i o n was f o u n d b e t w e e n t h e p l a s m a g l u c o s e c o n c e n t r a t i o n a n d t h e i n t r a c e l l u l a r c o n c e n t r a t i o n o f f r e e g l u c o s e in the d i a b e t i c p a t i e n t s (r -- 0.08, NS), indicating t h a t t h e f r e e g l u c o s e c o n c e n t r a t i o n in the d i a b e t i c p a t i e n t s w a s n o t o v e r e s t i m a t e d . T h e p a t i e n t s w i t h T y p e 2 d i a b e t e s h a d a h i g h e r m u s c l e G 6 P c o n c e n t r a t i o n in t h e b a s a l s t a t e a n d d u r i n g insulin infusion. T h e d i f f e r e n c e in G 6 P c o n c e n t r a t i o n s , h o w ever, was o n l y statistically significant d u r i n g insulin infusion. T h e h i g h e r m u s c l e l a c t a t e c o n c e n t r a t i o n in t h e d i a b e t i c p a t i e n t s w a s n o t statistically significant (p < 0.07 d u r i n g insulin infusion). M u s c l e f r e e glucose, G 6 P a n d lact a t e c o n c e n t r a t i o n s r e m a i n e d u n c h a n g e d d u r i n g insulin inf u s i o n in b o t h d i a b e t i c p a t i e n t s a n d c o n t r o l subjects. I n t h e p a t i e n t s w i t h T y p e 2 d i a b e t e s , t h e i n s u l i n - s t i m u l a t e d i n c r e a s e in f r a c t i o n a l g l y c o g e n s y n t h a s e activity at 0.1 mmol/1 G 6 P ( F V 0.1) c o r r e l a t e d significantly w i t h t h e i n s u l i n - s t i m u l a t e d i n c r e a s e in n o n - o x i d a t i v e g l u c o s e disp o s a l ( r = 0 . 6 5 , p < 0.03, Fig.2). N o significant c o r r e l a t i o n was f o u n d b e t w e e n i n s u l i n - s t i m u l a t e d i n c r e a s e in fracData are mg glucose. (kg fat free mass) -1.min -1, mean + SEM, p < 0.01 vs control subjects Mean + SEM. a p < 0.05 vs control subjects, b p < 0.02 vs control subjects, c p < 0.01 vs basal measurements t i o n a l velocities at 0.0 retool/1 G 6 P ( F V 0.0) a n d t h e inc r e a s e in n o n - o x i d a t i v e g l u c o s e d i s p o s a l in t h e d i a b e t i c p a t i e n t s (r = 0.14, NS). I n t h e n o r m a l subjects, h o w e v e r , a ~ 7 >= o Z "~ ~ E oO x _=~" "o~ o Z lO significant c o r r e l a t i o n w a s f o u n d b e t w e e n t h e i n c r e a s e in F V 0.0 a n d t h e i n c r e a s e in n o n - o x i d a t i v e g l u c o s e d i s p o sal d u r i n g insulin i n f u s i o n (r = 0.69, p < 0.02), while t h e c o r r e l a t i o n b e t w e e n i n c r e a s e in F V 0.1 a n d i n c r e a s e in n o n - o x i d a t i v e g l u c o s e d i s p o s a l w a s n o t statistically signific a n t (r = 0 . 4 4 , p = 0.19). W h e n c o r r e l a t i o n a n a l y s e s w e r e m a d e f o r t h e t w o g r o u p s t o g e t h e r (n -- 23), t h e i n c r e a s e in F V 0.1 c o r r e l a t e d w i t h the i n c r e a s e in n o n - o x i d a t i v e gluc o s e m e t a b o l i s m (r = 0.58, p < 0.005), while t h e c o r r e l a t i o n b e t w e e n i n c r e a s e in F V 0.0 a n d i n c r e a s e in n o n o x i d a t i v e g l u c o s e d i s p o s a l did n o t r e a c h statistical significance (r = 0 . 3 5 , p = 0.1). T h e i n t r a c e l l u l a r c o n c e n t r a t i o n o f f r e e g l u c o s e ( m e a n o f b a s a l + i n s u l i n - s t i m u l a t e d ) w a s significantty c o r r e l a t e d with t h e i n s u l i n - s t i m u l a t e d i n c r e a s e in f r a c t i o n a l g l y c o g e n s y n t h a s e activity ( F V 0.1: r = 0.65, p < 0.02 a n d F V 0.0: r = 0.91, p < 0.001, F i g . 3 ) in the p a t i e n t s w i t h T y p e 2 d i a b e t e s . A w e a k e r a n d n o n - s i g n i f i c a n t c o r r e l a t i o n was f o u n d b e t w e e n t h e p l a s m a g l u c o s e c o n c e n t r a t i o n a n d t h e c o eO"o 0.8 2 = =o~o 0.6 -=E _= o -= T h e e x t e n t t o w h i c h h y p e r g l y c a e m i a c o m p e n s a t e s f o r t h e v a r i o u s d e f e c t s in i n t r a c e l l u l a r g l u c o s e m e t a b o l i s m in T y p e 2 d i a b e t i c p a t i e n t s w a s t h e s u b j e c t o f r e c e n t p u b l i c a A. Vaaget al.:Hyperglycaemiaand muscleglucosemetabolismin Type 2 diabetes 1.5 tions from two different groups, in which contradictory results were reported. Thus, Thorburn et al. found that hyperglycaemia in Type 2 diabetes does not compensate for the defects in glucose oxidation and in muscle glycogen synthase activation [ 35, 36 ], whereas the defect in whole-body non-oxidative glucose metabolism was overcompensated during hyperglycaemia in their diabetic patients in the basal state and during insulin infusion. On the other hand, Kelley and Mandarino recently reported that the ambient level of fasting hyperglycaemia in Type 2 diabetic patients completely normalized the defects in both of the major pathways of intracellular glucose metabolism, including the activation of glycogen synthase in skeletal muscle [ 8, 37 ]. Furthermore, using the limb balance technique, Kelley and Mandarino demonstrated an increased muscle glucose oxidation in the Type 2 diabetic patients during fasting hyperglycaemia [ 8 ]. Clearly, the finding in the present study of normal rates of basal and insulin-stimulated whole-body glucose oxidation and non-oxidative glucose metabolism during the ambient level of fasting hyperglycaemia in Type 2 diabetic patients supports the findings of Kelley and Mandarino [ 8, 37 ]. The explanation for the differences between this data and that from Thorburn et al. are not clear, however, but may be due to differences in selection of study subjects or in study design. Specifically, Thorburn et al. had to increase the plasma glucose concentration markedly in the Type 2 diabetic patients above their ambient fasting blood glucose to about 20 mmol/1 in order to match the glucose flux in these subjects to the flux in their normal control subjects. This indicates that the normal control subjects in the study of Thorburn et aL may have been extremely insulin sensitive, which may also explain the lack of normalization of muscle glycogen synthase activity in their diabetic patients during the matched glucose fluxes. It should be noted that other previous studies have found either a normal [ 6, 16, 38 ] or a decreased [ 7, 39, 40 ] basal rate of whole-body glucose oxidation in fasting and hyperglycaemic Type 2 diabetic patients. These differences may be explained by differences between study subjects in BMI, or in different degrees of elevated fasting plasma insulin, glucose or NEFA concentrations. These are all factors known to influence the rate of whole-body glucose oxidation in human subjects. However, it may be important to emphasize that, in the basal postabsorptive state, only a small proportion of the rate of total wholebody glucose disposal and glucose oxidation is accounted for by skeletal muscle ( < 20 %) [ 41 ]. This means that the direct measurements by Kelley and Mandarino of muscle glucose oxidation, using the limb balance technique, cannot be directly compared with the measurements of whole-body glucose oxidation in the basal postprandial state. In addition, this implies that the different findings of basal rates of whole-body glucose oxidation in Type 2 diabetic patients may be due to the influence of the abovementioned factors on the rate of glucose oxidation in tissues other than skeletal muscle. In contrast to the condition during low fasting insulin concentrations, skeletal muscle accounts for the majority of whole-body glucose disposal during high physiological insulin concentrations ( > 80 % ) [ 41 ]. Therefore, the rates of whole-body glucose oxidation and non-oxidative glucose disposal during insulin infusion in the present study are likely to represent skeletal muscle glucose metabolism. The normal muscle glycogen synthase activation during the isoglycaemic insulin infusion in the present study does not mean that the activation by insulin of muscle glycogen synthase is normal in patients with Type 2 diabetes. In contrast, these data demonstrate that higher plasma glucose and intracellular glucose concentrations, together with a higher muscle G6P content, are required to compensate for the previously demonstrated defective glycogen synthase insulin activation in these patients during euglycaemia [ 4, 8, 13 ]. It was previously shown that hyperglycaemia per se does not stimulate the fractional activity of glycogen synthase in muscle biopsies from normal subjects during somatostatin infusion [ 42 ] or in Type 1 (insulin-dependent) diabetic subjects [ 43 ]. However, we demonstrate in the present study that hyperglycaemia normalizes the defective insulin activation of glycogen synthase in skeletal muscle in Type 2 diabetic patients during euglycaemia. This is in accordance with the data from Kelley and ManA. Vaaget al.:Hyperglycaemiaand muscleglucosemetabolismin Type2 diabetes darino for Type 2 diabetes [ 8, 37 ], and is also consistent with the finding that hyperglycaemia normalizes the defective insulin sensitivity of muscle glycogen synthase fractional activity in insulin resistant Type 1 diabetic patients [44]. Finally, the influence of glucose on glycogen synthase insulin activation is in accordance with data obtained in the isolated mouse soleus muscle demonstrating that, whereas glucose alone had no effect on muscle glycogen synthase activity, incubation with insulin plus glucose increased glycogen synthase activity more than incubation with insulin alone [ 45 ]. Thus, it appears that whereas glucose per se does not stimulate the fractional glycogen synthase activity in skeletal muscle, glucose has a permissive effect on the insulin activation of the enzyme. Insulin sensitivity of muscle glycogen synthase is decreased in obese compared to lean normal subjects [ 4 ]. In the present study, the Type 2 diabetic patients were significantly more obese than the control subjects. Thus, hyperglycaemia in the present study normalized both the obesity-related and the diabetes-related defects in muscle glycogen synthase activation in the Type 2 diabetic patients. The mechanism responsible for the compensatory effect of hyperglycaemia on fractional glycogen synthase activity may be through an enhancing effect of either the increased glucose or G6P concentration on the activity of glycogen synthase phosphatase. Glycogen synthase phosphatase is responsible for the dephosphorylation, and thus the activation, of glycogen synthase. The individual roles of glucose vs G6P in compensating glycogen synthase activity may be complicated. Studies on the intact mouse diaphragm have indicated that especially G6R and only to a small extent glucose per se, may stimulate the activity of glycogen synthase phosphatase [ 46 ]. The high correlation between intracellular free glucose concentrations and glycogen synthase insulin sensitivity in the present study suggests a primary role of glucose per se in increasing muscle glycogen synthase insulin sensitivity in patients with Type 2 diabetes. The inverse correlation between G6P concentrations and glycogen synthase insulin sensitivity, however, may suggest that low glycogen synthase activities causes an accumulation of G6R On the other hand, an increasing G6P concentration may stimulate glycogen synthase allosterically [ 47, 48 ], which in turn may decrease the G6P concentration due to a higher turn-over rate of the substrate. Nevertheless, the muscle G6P concentration in vivo is determined not only by the rate of glycogen synthesis, but also by the rate of glucose uptake and phosphorylation (hexokinase), glycolysis and glycogenolysis. Therefore, we cannot exclude the increased G6P concentration in the Type 2 diabetic patients as being responsible for the compensated (normalized) fractional glycogen synthase activation. The correlations between increments in fractional glycogen synthase activation and whole-body rates of nonoxidative glucose uptake confirm an important role of muscle glycogen synthase activity in determining in vivo insulin action. The present study suggests this to be the case not only during euglycaemia, but also during hyperglycaemia in patients with Type 2 diabetes. The fractional glycogen synthase activity is supposed to merely reflect the covalent activation (degree of dephosphorylation) of the enzyme. However, it is unclear whether the increased muscle G6P concentration in the hyperglycaemic diabetic patients stimulates the rate of glycogen synthesis further through an additional in vivo allosteric activation of glycogen synthase, or alternatively, whether the increased muscle G6P content is reflected solely by the compensated (normalized) fractional glycogen synthase activity. The completely normalized insulin stimulated increase in muscle glycogen content and in in vivo non-oxidative glucose metabolism indicate that the increased muscle G6P content does not cause an additional increase in the rate of muscle glycogen synthesis in the diabetic patients. On the other hand, it is important to realize that measurements of insulin-stimulated non-oxidative glucose metabolism and increments in muscle glycogen content represent the net product of the rate of glycogen synthesis and the rate of glycogenolysis. There are some data to indicate that a major part of the G6P content in rat muscle biopsies, which have not been freezeclamped, represents G6P coming from glycogen due to glycogenolysis [ 49 ]. If this is the case in the present study, it may in fact suggest the presence of an increased rate of glycogenolysis in the diabetic patients, and it may furthermore provide an additional explanation for the inverse correlation betwen the muscle G6P content and glycogen synthase activity in these subjects. Therefore, we cannot exclude the theoretical possibility that the compensated glucose metabolism in the hyperglycaemic Type2 diabetic patients includes an increased turnover rate of glycogen in skeletal muscle, i.e. an increased rate of glycogen synthesis together with an increased rate of glycogenolysis. The higher free glucose and G6P concentrations in the Type 2 diabetic patients compared with the control subjects during similar glucose utilization rates and insulin concentrations suggests that glycogen synthase activity may be rate limiting for the insulin stimulated muscle glucose metabolism in patients with Type 2 diabetes. A defective glycogen synthase activity may cause an accumulation of muscle G6P and free glucose, in turn causing a decrease in glucose transport over the cell membrane due to the smaller glucose gradient. Thereafter, the increasing glucose and G6P concentrations outside and inside the cell may gradually improve glycogen synthase insulin sensitivity, and an increasing muscle G6P concentration may furthermore increase the allosteric activation of glycogen synthase. When the glucose concentration reaches a level where the glycogen synthase activity is sufficient to store glucose at a rate equal to the rate of glucose appearance, a newly compensated state may be achieved. Thus, the physiological implication of our data may be that hyperglycaemia in Type 2 diabetic patients may serve to compensate for insulin resistance and a defective endogenous insulin secretion [ 50 ] in order to maintain a normal intracellular glucose metabolism in skeletal muscles. In conclusion, the present study demonstrates that both basal and insulin-stimulated rates of glucose oxidation and non-oxidative glucose disposal are fully compensated (normalized) in obese patients with Type 2 diabetes d u r i n g f a s t i n g h y p e r g l y c a e m i a a n d i s o g l y c a e m i c i n s u l i n i n f u s i o n . M o s t i m p o r t a n t l y , w e h a v e d e m o n s t r a t e d in p a t i e n t s w i t h T y p e 2 d i a b e t e s t h a t t h e m u s c l e g l y c o g e n synt h a s e a c t i v i t y is s t i m u l a t e d a d e q u a t e l y w h e n d e t e r m i n e d d u r i n g a p h y s i o l o g i c a l i s o g l y c a e m i c i n s u l i n i n f u s i o n , a n d t h u s d u r i n g a n o r m a l i z e d g l u c o s e t u r n o v e r r a t e . F u r t h e r m o r e , t h e p a t i e n t s w i t h T y p e 2 d i a b e t e s h a d a n i n c r e a s e d i n t r a c e l l u l a r c o n c e n t r a t i o n o f f r e e g l u c o s e a n d G 6 P d u r ing t h e p h y s i o l o g i c a l i n s u l i n i n f u s i o n , s u g g e s t i n g t h a t t h e r a t e l i m i t i n g s t e p in m u s c l e g l u c o s e m e t a b o l i s m in p a t i e n t s w i t h T y p e 2 d i a b e t e s is l o c a t e d a f t e r G 6 E A h i g h c o r r e l a t i o n b e t w e e n m u s c l e f r e e g l u c o s e c o n c e n t r a t i o n a n d insulin s e n s i t i v i t y o f m u s c l e g l y c o g e n s y n t h a s e i n d i c a t e s an i m p o r t a n t c o m p e n s a t o r y r o l e o f g l u c o s e in t h e i n s u l i n a c t i v a t i o n o f m u s c l e g l y c o g e n s y n t h a s e in p a t i e n t s w i t h T y p e 2 d i a b e t e s . Acknowledgements. The authors would like to acknowledge the expert technical assistance of Ms. A. 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A. Vaag, P. Damsbo, O. Hother-Nielsen, H. Beck-Nielsen. Hyperglycaemia compensates for the defects in insulin-mediated glucose metabolism and in the activation of glycogen synthase in the skeletal muscle of patients with Type 2 (non-insulin-dependent) diabetes mellitus, Diabetologia, 1992, 80-88, DOI: 10.1007/BF00400856