Relationship of skeletal muscle glucose 6-phosphate to glucose disposal rate and glycogen synthase activity in insulin-resistant and non-insulin-dependent diabetic rhesus monkeys

Summary Reduced insulin action on skeletal muscle glycogen synthase activity and reduced whole-body insulin-mediated glucose disposal rates in insulin-resistant subjects may be associated with an alteration in muscle glucose transport (or phosphorylation) or with a defect distal to glucose 6-phosphate. To examine this issue we determined the glucose 6-phosphate concentration and glycogen synthase activity in muscle samples obtained under basal and euglycaemic hyperinsulinaemic clamp conditions in 27 rhesus monkeys (Macaca mulatta). They ranged from metabolically normal (n =11) to insulin-resistant (n =8) to overtly diabetic (non-insulin-dependent) (n =8). The glucose 6-phosphate measured under insulin-stimulated conditions was inversely correlated to insulin-stimulated glycogen synthase independent activity (r = –0.54, p<0.005), the change in glycogen synthase independent activity (insulin-stimulated minus basal) (r = –0.58, p<0.002) and to whole-body insulin-mediated glucose disposal rate (r = –0.60, p<0.002). The insulin-resistant and diabetic monkeys had significantly higher insulin-stimulated glucose 6-phosphate concentrations (0.57±0.11 and 0.62±0.11 nmol/mg dry weight, respectively) compared to the normal monkeys (0.29±0.05 nmol/mg dry weight) (p's <0.05). We conclude that under euglycaemic/hyperinsulinaemic conditions, a defect distal to glucose 6-phosphate is a major contributor to reduced whole-body insulin-mediated glucose disposal rates and to reduced insulin action on glycogen synthase in insulin-resistant and diabetic monkeys. [Diabetologia (1994) 37: 127–133] Received: 28 May 1993 and in revised form: 13 August 1993     

Morphometric documentation of abnormal intramyocellular fat storage and reduced glycogen in obese patients with Type II diabetes

Aims/hypothesis. Insulin resistance of skeletal muscle has been associated with increased lipid availability. This study aimed to estimate volume fractions of intramyocellular triglyceride droplets and glycogen granules in skeletal muscle using electron microscopy and furthermore, relate these findings to insulin sensitivity and the level of circulating lipids. Methods. We compared 11 obese patients with Type II (non-insulin-dependent) diabetes mellitus and 11 obese normoglycaemic subjects matched for age and sex. Glucose metabolism was determined using the euglycaemic hyperinsulinaemic clamp technique (40 mU · m^–2· min^–1) coupled with indirect calorimetry and tritiated glucose. On the second day, using an automatic procedure, a fasting muscle biopsy was carried out and processed for electron microscopy. Volume fractions of intramyocellular structures were estimated by pointcounting on photographic pictures in a blinded manner. Results. Insulin-stimulated total glucose disposal rate was lower in the Type II diabetic subjects compared with the obese normoglycaemic subjects (4.96 ± 049 vs 10.35 ± 0.89 mg · min^–1· kg ffm^–1, p < 0.001) as was glucose storage (2.03 ± 0.50 vs 6.59 ± 0.83, p < 0.001). The electron microscopy study revealed that the diabetic subjects had higher intramyocellular amounts of triglyceride (1.43 ± 0.21 vs 0.39 ± 0.07 %, p < 0.001) and lower amounts of glycogen (3.53 ± 0.33 vs 6.94 ± 0.54 %, p < 0.001). Mitochondrial volume was identical indicating equal aerobic capacity. The fractional intramyocellular lipid volume was found to be positively associated with fasting NEFA (r = 0.63, p = < 0.05 and r = 0.79, p = < 0.05) and triglyceride (r = 0.74, p = 0.01 and r = 0.62, p < 0.05) in the obese diabetic and normoglycaemic cohorts respectively. Intramyocellular lipid content was negatively correlated to insulin sensitivity (r = –0.71, p < 0.02) in the obese diabetic group whereas no significant association was found in the obese normoglycaemic group. Conclusion/interpretation. This study shows that fat accumulates intramyocellulary while glycogen stores are simultaneously reduced in obese subjects with Type II (non-insulin-dependent) diabetes mellitus. Quantitatively, a major component of the excessive lipid accumulation could be secondary in origin, related to the diabetic state in itself, although a contribution from the altered insulin action cascade of obesity and diabetes cannot be excluded. In both groups significant positive relations were found between circulating and intramyocellular lipid. [Diabetologia (2001) 44: 824–833] Received: 6 December 2000 and in revised form: 16 March 2001     

Reduced plasma adiponectin concentrations may contribute to impaired insulin activation of glycogen synthase in skeletal muscle of patients with type 2 diabetes

Aims/hypothesis Circulating levels of adiponectin are negatively associated with multiple indices of insulin resistance, and the concentration is reduced in humans with insulin resistance and type 2 diabetes. However, the mechanisms by which adiponectin improves insulin sensitivity remain unclear.Subjects and methods Combining euglycaemic–hyperinsulinaemic clamp studies with indirect calorimetry and skeletal muscle biopsies, we examined the relationship between plasma adiponectin and parameters of whole-body glucose and lipid metabolism, and muscle glycogen synthase (GS) activity in 51 Caucasians (ten lean, 21 obese and 20 with type 2 diabetes).Results Plasma adiponectin was significantly reduced in type 2 diabetic compared with obese and lean subjects. In lean and obese subjects, insulin significantly reduced plasma adiponectin, but this response was blunted in patients with type 2 diabetes. Plasma adiponectin was positively associated with insulin-stimulated glucose disposal (r=0.48), glucose oxidation (r=0.54), respiratory quotient (r=0.58) and non-oxidative glucose metabolism (r=0.38), and negatively associated with lipid oxidation during insulin stimulation (r=−0.60) after adjustment for body fat (all p<0.01). Most notably, we found a positive association between plasma adiponectin and insulin stimulation of GS activity in skeletal muscle (r=0.44, p<0.01).Conclusions/interpretation Our results indicate that plasma adiponectin may enhance insulin sensitivity by improving the capacity to switch from lipid to glucose oxidation and to store glucose as glycogen in response to insulin, and that low adiponectin may contribute to impaired insulin activation of GS in skeletal muscle of patients with type 2 diabetes.     

Intramuscular triglyceride content is increased in IDDM

Summary Increased lipid oxidation is related to insulin resistance. Some of the enhanced lipid utilization may be derived from intramuscular sources. We studied muscle triglyceride (mTG) concentration and its relationship to insulin sensitivity in 10 healthy men (age 29 ± 2 years, BMI 23.3 ± 0.6 kg/m²) and 17 men with insulin-dependent diabetes mellitus (IDDM) (age 30 ± 2 years, BMI 22.8 ± 0.5 kg/m², diabetes duration 14 ± 2 years, HbA_{1 c} 7.7 ± 0.3 %, insulin dose 48 ± 3 U/day). Insulin sensitivity was measured with a 4 h euglycaemic (5 mmol/l) hyperinsulinaemic (1.5 mU or 9 pmol · kg^–1· min^–1) clamp accompanied by indirect calorimetry before and at the end of the insulin infusion. A percutaneous biopsy was performed from m. vastus lateralis for the determination of mTG. At baseline the IDDM patients had higher glucose (10.2 ± 0.9 vs 5.6 ± 0.1 mmol/l, p < 0.001), insulin (40.3 ± 3.2 vs 23.2 ± 4.2 pmol/l, p < 0.01), HDL cholesterol (1.28 ± 0.06 vs 1.04 ± 0.03 mmol/l, p < 0.01) and mTG (32.9 ± 4.6 vs 13.6 ± 2.7 mmol/kg dry weight, p < 0.01) concentrations than the healthy men, respectively. The IDDM patients had lower insulin stimulated whole body total (–25 %, p < 0.001), oxidative (–18 %, p < 0.01) and non-oxidative glucose disposal rates (–43 %, p < 0.001), whereas lipid oxidation rate was higher in the basal state ( + 44 %, p < 0.01) and during hyperinsulinaemia ( + 283 %, p < 0.05). mTG concentrations did not change significantly during the clamp or correlate with insulin stimulated glucose disposal. In healthy men mTG correlated positively with lipid oxidation rate at the end of hyperinsulinaemia (r = 0.75, p < 0.05). In conclusion: 1) IDDM is associated with increased intramuscular TG content. 2) mTG content does not correlate with insulin sensitivity in healthy subjects or patients with IDDM. [Diabetologia (1998) 41: 111–115] Received: 12 June 1997 and in revised form: 8 September 1997     

Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study

Summary Recent muscle biopsy studies have shown a relation between intramuscular lipid content and insulin resistance. The aim of this study was to test this relation in humans by using a novel proton nuclear magnetic resonance (¹H NMR) spectroscopy technique, which enables non-invasive and rapid ( ∼ 45 min) determination of intramyocellular lipid (IMCL) content. Normal weight non-diabetic adults (n = 23, age 29 ± 2 years, BMI = 24.1 ± 0.5 kg/m²) were studied using cross-sectional analysis. Insulin sensitivity was assessed by a 2-h hyperinsulinaemic ( ∼ 450 pmol/l)-euglycaemic ( ∼ 5 mmol/l) clamp test. Intramyocellular lipid concentrations were determined by using localized ¹H NMR spectroscopy of soleus muscle. Simple linear regression analysis showed an inverse correlation (r = –0.692, p = 0.0017) between intramyocellular lipid content and M-value (100–120 min of clamp) as well as between fasting plasma non-esterified fatty acid concentration and M-value (r = –0.54, p= 0.0267). Intramyocellular lipid content was not related to BMI, age and fasting plasma concentrations of triglycerides, non-esterified fatty acids, glucose or insulin. These results show that intramyocellular lipid concentration, as assessed non invasively by localized ¹H NMR spectroscopy, is a good indicator of whole body insulin sensitivity in non-diabetic, non-obese humans. [Diabetologia (1999) 42: 113–116] Received: 3 September 1998 and in revised form: 11 November 1998     

Effect of rosiglitazone on glucose and non-esterified fatty acid metabolism in Type II diabetic patients

Aims/hypothesis: We aimed to examine the mechanisms by which rosiglitazone improves glycaemic control in Type II (non-insulin-dependent) diabetic patients. Methods: Altogether 29 diet-treated diabetic patients were assigned at random to rosiglitazone, 8 mg/day (n = 15), or placebo (n = 14) for 12 weeks. Patients received 75 g OGTT and two-step euglycaemic insulin (40 and 160 mU/m²min) clamp with 3-³H-glucose, ¹⁴C-palmitate and indirect calorimetry. Results: After 12 weeks, rosiglitazone reduced fasting plasma glucose (195 ± 11 to 150 ± 7 mg/dl, p < 0.01), mean plasma glucose (PG) during OGTT (293 ± 12 to 236 ± 9 mg/dl, p < 0.01), and HbA_{1 c} (8.7 ± 0.4 to 7.4 ± 0.3 %, p < 0.01) without changes in plasma insulin concentration. Basal endogenous glucose production (EGP) declined (3.3 ± 0.1 to 2.9 ± 0.1 mg/kg FFM · min, p < 0.05) and whole body glucose metabolic clearance rate increased after rosiglitazone (first clamp step: 2.8 ± 0.2 to 3.5 ± 0.2 ml/kg FFM · min, p < 0.01; second clamp step: 6.7 ± 0.6 to 9.2 ± 0.8, p < 0.05) despite increased body weight (86 ± 4 to 90 ± 4 kg, p < 0.01) and fat mass (33 ± 3 to 37 ± 3 kg, p < 0.01). Fasting plasma non-esterified fatty acid (NEFA) (735 ± 52 to 579 ± 49 μEq/l, p < 0.01), mean plasma NEFA during OGTT (561 ± 33 to 424 ± 35, p < 0.01), and basal NEFA turnover (18.3 ± 1.5 to 15.5 ± 1.2 μEq/kg FM · min, p < 0.05) decreased after rosiglitazone. Changes in EPG and mean plasma glucose (PG) during OGTT correlated with changes in basal EGP (r = 0.54; r = 0.58), first EGP (r = 0.36; r = 0.41), first MCR (r = –0.66; r = –0.68), second MCR (r = –0.49; r = –0.54), fasting plasma NEFA (r = 0.53; r = 0.49), and NEFA during OGTT (r = 0.66; r = 0.66). Conclusion/interpretation: Rosiglitazone increases hepatic and peripheral (muscle) tissue insulin sensitivity and reduces NEFA turnover despite increased total body fat mass. These results suggest that the beneficial effects of rosiglitazone on glycaemic control are mediated, in part, by the drug's effect on NEFA metabolism. [Diabetologia (2001) 44: 2210–2219] Received: 20 May 2001 and in revised form: 9 August 2001     

Intracellular Glucose and Fat Metabolism in Identical Twins Discordant for Non-insulin-dependent Diabetes Mellitus (NIDDM): Acquired Versus Genetic Metabolic Defects?

Intracellular glucose and lipid metabolism was studied in 12 identical twin pairs discordant for non-insulin-dependent (Type 2) diabetes mellitus (NIDDM) and 13 control subjects without family history of diabetes during low (baseline) and high plasma insulin concentrations, using the hyperinsulinaemic clamp technique combined with indirect calorimetry, tritiated water glycolytic flux rates and biopsy skeletal muscle glycogen synthase activity determinations. Baseline and insulin stimulated rates of lipid oxidation were elevated—and glucose oxidation decreased—in the NIDDM twins compared with the non-diabetic co-twins and controls (all p < 0.05). Baseline and insulin stimulated rates of glucose and lipid oxidation were similar in non-diabetic twins and controls. Exogenous glycolytic flux was decreased in NIDDM twins compared with both their non-diabetic co-twins and controls during clamp insulin measurements (p < 0.02), but similar in all study groups during baseline measurements. Insulin stimulated glucose disposal, exogenous glucose storage (glucose disposal–exogenous glycolytic flux) and skeletal muscle glycogen synthase activity were all significantly decreased in NIDDM twins compared with both their non-diabetic co-twins and controls. Furthermore, glucose disposal and glucose storage were decreased in the non-diabetic twins (n = 12) compared with controls (p < 0.05 both). However, insulin stimulated fractional skeletal muscle glycogen synthase activity was not significantly decreased in non-diabetic twins compared with controls. In conclusion: (1) the glucose fatty acid cycle plays a major role in the secondary—but not the primary—abnormalities of glucose metabolism in NIDDM; (2) insulin resistance in non-diabetic identical co-twins of NIDDM patients is restricted exclusively to the pathway of exogenous glucose storage; (3) however, the decreased glucose storage is not explained solely by an impairment of insulin stimulated skeletal muscle glycogen synthase activity; and finally (4) the impairment of skeletal muscle glycogen synthase activity in NIDDM has an apparent non-genetic component and can be escaped (or postponed) in individuals (twins) with a 100 % genetic predisposition to NIDDM.     

Muscle enzyme activity and insulin sensitivity in Type 1 (insulin-dependent) diabetes mellitus

Summary The mechanisms of insulin insensitivity in diabetes are poorly understood. We have therefore assessed the relationship between glucose disposal during a euglycaemic clamp, muscle glycogen formation, and the activities of insulin regulated enzymes within skeletal muscle in five Type 1(insulin-dependent) diabetic patients, both on conventional injection therapy (HbA₁ 11.0±1.0 (SD) %) and after 6 weeks continuous subcutaneous insulin infusion (HbA₁ 7.6±1.4%,p < 0.01). On both regimens, overnight euglycaemia before the clamp was maintained with an intravenous insulin infusion. The increase in clamp glucose requirements (insulin 0.1 U kg^–1·h^–1) between injection therapy and continuous subcutaneous insulin infusion was significant (6.2±0.9 (SE) to 7.0 ± 0.9 mg·kg^–1·min^–1,p<0.05), but small compared to differences between subjects. Glucose requirement remained lower than in control subjects (10.4 ± 0.7 mg·kg^–1·min^–1,p < 0.05). The increase in muscle glycogen with the clamp was slightly higher on continuous subcutaneous insulin infusion (9.5 ± 2.5 mg/g protein) than on injection therapy (8.5 ± 2.4 mg/g,p < 0.05), but less than in control subjects (17.9 ± 2.1 mg/g,p < 0.05). The expressed activity of glycogen synthase and pyruvate dehydrogenase increased significantly between fasting and the end of the clamps in the patients (p < 0.001 and < 0.005), but was not significantly different between the two treatment regimens. Expressed glycogen synthase activity at the end of the clamp was lower on both treatments than in control subjects (p < 0.05). Both enzyme activities were, however, highly correlated with glucose requirement between patients, (r=0.89–0.94,p<0.05-0.02), and glycogen synthase was similarly correlated in the control subjects (r = 0.84,p < 0.05). Patients had significantly different enzyme activities, glucose requirement, and glycogen stored by analysis of variance (p < 0.05-0.01). Correlation of each enzyme activity between subjects on the two treatment regimens was also high (r=0.94–0.98,p < 0.02–0.01). At the end of the clamp the enzyme activities were themselves closely related (injectionsr = 0.99,p < 0.001; infusionr = 0.88,p < 0.05), and glycogen synthase activity predicted muscle glycogen deposition (r=0.94–0.97,p < 0.02–0.01). We suggest that: (1) preceding metabolic control has a relatively small influence on whole body insulin sensitivity measured immediately after careful overnight control; (2) insulin sensitivity derived from glucose clamp data is strongly related to skeletal muscle glycogen deposition and skeletal muscle enzyme activities.     

Post-marathon Paradox in IDDM: Unchanged Insulin Sensitivity in Spite of Glycogen Depletion

Acute physical exercise usually enhances insulin sensitivity. We examined the effect of a competitive 42 km marathon run on glucose uptake and lipid oxidation in 7 runners with insulin-dependent diabetes mellitus (IDDM), aged 36 ± 3 yr, BMI 23.9 ± 0.5 kg m⁻², VO_{2 max} 46 ± 1 ml kg⁻¹ min⁻¹, HbA_1c 7.7 ± 0.3 %, duration of diabetes 16 ± 5 yr, runtime 3 h 47 ± 8 min. On the marathon day, they reduced pre-race insulin doses by 26 ± 8 %, and ingested 130 ± 33 g carbohydrate before, 91 ± 26 g during, and 115 ± 20 g after the race. During the run, blood glucose concentration fell from 14.4 ± 2.0 to 7.4 ± 3.0 mmol l⁻¹ (p < 0.05) and serum insulin from 51 ± 8 to 33 ± 8 pmol l⁻¹ (p < 0.05). Serum NEFA increased by 4-fold (p < 0.05), but fell to the normal level by next morning. Muscle glycogen content was 56 % lower (p < 0.05) and glycogen synthase fractional activity 40 % greater (p < 0.05) in the morning after the marathon as compared to the resting control day. In spite of glycogen depletion, whole body glucose disposal (euglycaemic insulin clamp) was unchanged, while glucose oxidation (indirect calorimetry) was decreased by 49 % (p < 0.05) and lipid oxidation increased by 41 % (p < 0.01). There was an inverse correlation between the rates of lipid oxidation and glucose uptake after the marathon (r = −0.75; p < 0.05). In conclusion: after successfully managed marathon running in patients with IDDM, insulin sensitivity was not increased in spite of low glycogen content and enhanced glycogen synthase activity after marathon, probably because of increased lipid oxidation. © 1997 by John Wiley & Sons, Ltd.     

Effects of non-esterified fatty acid availability on insulin stimulated glucose utilisation and tissue pyruvate dehydrogenase activity in the rat

Summary Fatty acids in cardiac muscle compete with glucose for oxidation, thereby inhibiting glucose utilisation. It is not clear whether a similar mechanism is important in resting skeletal muscle. We used the hyperinsulinaemic euglycaemic clamp technique in conscious rats fasted for 20 h to examine the effects of increased plasma non-esterified fatty acid levels (1 mmol/l) on glucose metabolism. Insulin was infused at 75 mU/h (plasma insulin, 2.27±0.21 g/l) or 300mU/h (16.41±0.47 g/l). An increase in non-esterified fatty acid levels decreased clamp glucose requirement and 3–³H-glucose turnover by 35% (p<0.001) when the higher insulin dose was used but there was no change at the lower dose. At both insulin infusion rates, clamp blood lactate and pyruvate responses suggested inhibition of muscle glycolysis by elevated plasma non-esterified fatty acid concentrations. Quadriceps muscle glycogen deposition during the clamps was enhanced by increased non-esterified fatty acid availability at the lower insulin dose (p<0.001) but not at the higher insulin concentration. Activation of pyruvate dehyrogenase during the clamps was partially inhibited by increased plasma non-esterified fatty acid in the heart, adipose tissue and quadriceps muscle. This was evident at both insulin levels in heart but only at the higher insulin concentration in muscle (p<0.002). The findings are consistent with an inhibition of glycolysis in skeletal muscle of mixed fibre type as a result of increased fatty acid availability. At low rates of glucose flux glycogen synthesis may compensate for decreased glycolysis so that glucose turnover is not decreased. The role of pyruvate dehydrogenase in the glucose-fatty acid cycle in muscle may depend on the prevailing plasma insulin concentration and the degree of activation of this enzyme.     

A Mediterranean and a high-carbohydrate diet improve glucose metabolism in healthy young persons

Aims/hypothesis: Insulin resistance usually precedes the diagnosis of Type II (non-insulin-dependent) diabetes mellitus. However, in most patients, the clinical expression of the disease could be prevented by dietary and lifestyle changes. We investigated the effects of a diet enriched in monounsaturated fatty acids (Mediterranean diet) and a low fat, high-carbohydrate diet on in vivo and in vitro glucose metabolism in 59 young subjects (30 men and 29 women). Methods: We carried out an intervention dietary study with a saturated fat phase and two randomized-crossover dietary periods: a high-carbohydrate diet and a Mediterranean diet for 28 days each. We analysed the plasma lipoproteins fractions, free fatty acids, insulin sensitivity and glucose uptake in isolated monocytes at the end of the three dietary periods. Results: In comparison to the saturated fat diet, the CHO and Mediterranean diets induced a decrease of LDL-cholesterol (p < 0.001) and HDL-cholesterol (p < 0.001). Steady-state plasma glucose decreased (p = 0.023) and basal and insulin-stimulated 2-deoxiglucose uptake in peripheral monocytes increased in both diets (CHO and Mediterranean), (p = 0.007) indicating an improvement in insulin sensitivity. Fasting free fatty acids plasma values were correlated positively with steady state plasma glucose (r = 0.45; p < 0.0001). In addition, there was an inverse correlation between the mean glucose of the steady state plasma glucose period and logarithmic values of basal (r = –0.34; p = 0.003) and insulin stimulated glucose uptake in monocytes (r = –0.32; p = 0.006). Conclusion/interpretation: Isocaloric substitution of carbohydrates and monounsaturated fatty acids for saturated fatty acids improved insulin sensitivity in vivo and in vitro, with an increase in glucose disposal. Both diets are an adequate alternatives for improving glucose metabolism in healthy young men and women. [Diabetologia (2001) 44: 2038–2043] Received: 19 February 2001 and in revised form: 9 July 2001     

Insulin secretion and insulin sensitivity in diabetic subgroups: studies in the prediabetic and diabetic state

Aims/hypothesis. To evaluate insulin sensitivity and insulin secretion in prediabetic and diabetic subjects with mutations in MODY1 (HNF-4α) and MODY3 (HNF-1α) genes, in subjects with GAD antibodies, latent autoimmune diabetes in adults and in subjects with the common form of Type II (non-insulin-dependent) diabetes mellitus. Methods. Insulin secretion was measured as the incremental 30-min insulin (I30) and insulin glucose ratio (I:G30) during OGTT whereas insulin sensitivity was measured as the insulin sensitivity index during OGTT in 131 carriers of MODY mutations [NGT = 38, IFG/IGT = 21, diabetes mellitus (DM) = 72], in 293 subjects with GADA (NGT = 47, IFG/IGT = 29, DM = 217) and in 2961 subjects with a family history of the common form of Type II diabetes but without MODY mutations or GADA (NGT = 1360, IFG/IGT = 857, DM = 744). A subgroup of the subjects underwent a euglycaemic clamp (n = 210) and intravenous glucose tolerance test (n = 337) for the estimation of insulin sensitivity and first-phase insulin secretion. Results. Non-diabetic subjects with MODY mutations had pronounced impaired insulin secretion (I30, I:G30) compared with the two other groups (p = 0.005). Normal or non-diabetic glucose tolerance was maintained by enhanced insulin sensitivity compared with the other two groups (p < 0.05 and p < 0.005). In contrast to patients with Type II diabetes and with adult latent autoimmune diabetes, MODY patients showed only a modest deterioration in insulin sensitivity at onset of diabetes. The 2-h glucose values inversely correlated with insulin sensitivity in subjects with GADA (r = –0.447, p < 0.001) and subjects from Type II diabetic families (r = –0.426, p < 0.001), whereas no such relation was observed in subjects with MODY mutations (r = 0.151, p = NS). There were no statistically significant differences in insulin secretion or insulin sensitivity between subjects with GADA or subjects with a family history of Type II diabetes, either at the NGT or the IFG/IGT stage. Conclusion/interpretation. Glucose-tolerant carriers of MODY mutations are characterised by a severe impairment in insulin secretion. Enhanced insulin sensitivity is the most likely explanation for the normal glucose tolerance. Whereas subjects with positive GADA or Type II diabetes have impaired insulin sensitivity with increasing glucose concentrations, MODY mutation carriers seem to be protected from the effect of glucose toxicity. [Diabetologia (2000) 43: 1476–1483] Received: 23 March 2000 and in revised form: 29 August 2000     

The relation of proinsulin and insulin to insulin sensitivity and acute insulin response in subjects with newly diagnosed Type II diabetes: the Insulin Resistance Atherosclerosis Study

Abstract Aims/hypothesis. Proinsulin concentrations are increased relative to insulin concentrations in subjects with Type II (non-insulin-dependent) diabetes mellitus. This could be secondary to hyperglycaemia or insulin resistance or due to a defect in insulin secretion. Methods. We investigated the association between fasting insulin, intact proinsulin and the intact proinsulin: insulin ratio with insulin sensitivity, estimated by a frequently sampled intravenous glucose tolerance test and the minimal model and with acute insulin response (AIR) in 182 newly diagnosed Type II diabetic subjects aged 40 to 69 years. None of the subjects was receiving hypoglycaemic medication. Results. Insulin sensitivity correlated inversely with fasting insulin (r _s = –0.42) and intact proinsulin (r _s = –0.32) (p < 0.001). The intact proinsulin:insulin ratio was not correlated with insulin sensitivity. AIR correlated positively with intact proinsulin (r _s = 0.23) and inversely with the intact proinsulin:insulin ratio (r _s = –0.29, p < 0.001). Fasting glucose correlated positively with intact proinsulin (r _s = 0.34) and the intact proinsulin:insulin ratio (r _s = 0.24, p < 0.001). The intact proinsulin:insulin ratio increased by decreasing AIR (quartiles of AIR from high to low: 7.8, 8.2, 9.7 and 12.1 %, p < 0.001). This association was independent of age, sex, ethnicity, body mass index, fasting glucose, and insulin sensitivity. Conclusion/interpretation. Insulin resistance (low insulin sensitivity) was not related to the intact proinsulin:insulin ratio in subjects with Type II diabetes. In contrast, both low AIR and high fasting glucose concentrations were associated with a disproportionate increase in proinsulin concentration. These results suggest that increased intact proinsulin:insulin ratio is a marker of a defect in insulin secretion in Type II diabetic subjects. [Diabetologia (1999) 42: 1060–1066] Received: 25 February 1999 and in revised form: 12 April 1999     

Vitamin D, glucose tolerance and insulinaemia in elderly men

Summary Vitamin D status was assessed in 142 elderly Dutchmen participating in a prospective population-based study of environmental factors in the aetiology of non-insulin-dependent diabetes mellitus. Of the men aged 70–88 years examined between March and May 1990, 39 % were vitamin D depleted. After adjustment for confounding by age, BMI, physical activity, month of sampling, cigarette smoking and alcohol intake the 1-h glucose and area under the glucose curve during a standard 75-g oral glucose tolerance test (OGTT) were inversely associated with the serum concentration of 25-OH vitamin D (r = −0.23, p < 0.01; r = −0.26, p < 0.01, respectively). After excluding newly diagnosed diabetic patients total insulin concentrations during OGTT were also inversely associated with the concentration of 25-OH vitamin D (r = −0.18 to −0.23, p < 0.05). Hypovitaminosis D may be a significant risk factor for glucose intolerance. [Diabetologia (1997) 40: 344–347] Received: 3 September 1996 and in revised form: 2 January 1997     

Insulin resistance in non-diabetic patients with non-alcoholic fatty liver disease: sites and mechanisms

Aims/Hypothesis Non-alcoholic fatty liver disease (NAFLD) has been associated with the metabolic syndrome. However, it is not clear whether insulin resistance is an independent feature of NAFLD, and it remains to be determined which of the in vivo actions of insulin are impaired in this condition.Methods We performed a two-step (1.5 and 6 pmol min^–1 kg^–1) euglycaemic insulin clamp coupled with tracer infusion ([6,6-²H₂]glucose and [²H₅]glycerol) and indirect calorimetry in 12 non-obese, normolipidaemic, normotensive, non-diabetic patients with biopsy-proven NAFLD and six control subjects.Results In NAFLD patients, endogenous glucose production (basal and during the clamp) was normal; however, peripheral glucose disposal was markedly decreased (by 30% and 45% at the low and high insulin doses, respectively, p<0.0001) at higher plasma insulin levels (p=0.05), due to impaired glucose oxidation (p=0.003) and glycogen synthesis (p<0.001). Compared with control subjects, glycerol appearance and lipid oxidation were significantly increased in NAFLD patients in the basal state, and were suppressed by insulin to a lesser extent (p<0.05–0.001). The lag phase of the in vitro copper-catalysed peroxidation of LDL particles was significantly shorter in the patients than in the control subjects (48±12 vs 63±13 min, p<0.04). Lipid oxidation was significantly related to endogenous glucose production, glucose disposal, the degree of hepatic steatosis, and LDL oxidisability.Conclusions/interpretation Insulin resistance appears to be an intrinsic defect in NAFLD, with the metabolic pattern observed indicating that adipose tissue is an important site.     

Involvement of non-esterified fatty acid oxidation in glucocorticoid-induced peripheral insulin resistance in vivo in rats

Summary The mechanism by which glucocorticoids induce insulin resistance was studied in normal rats administered for 2 days with corticosterone then tested by euglycaemic hyperinsulinaemic clamps. Corticosterone administration induced a slight hyperglycaemia, hyperinsulinaemia and increased non-esterified fatty acid levels. It impaired insulin-stimulated total glucose utilization (corticosterone 15.7±0.7; controls 24.6±0.8 mg·kg⁻¹·min⁻¹), as well as residual hepatic glucose production (corticosterone 4.9±1.0; controls 2.0±0.7 mg·kg⁻¹·min⁻¹). During the clamps, insulin did not decrease the elevated non-esterified fatty acid levels in corticosterone-administered rats (corticosterone 1.38±0.15, controls 0.22±0.04 mmol/l). Corticosterone administration decreased the in vivo insulin-stimulated glucose utilization index by individual muscles by 62±6%, and the de novo glycogen synthesis by 78±2% (n=8–9 muscles). GLUT4 protein and mRNA levels were either unchanged or slightly increased by corticosterone administration. Inhibition of lipid oxidation by etomoxir prevented corticosterone-induced muscle but not hepatic insulin resistance. In conclusion, glucocorticoid-induced muscle insulin resistance is due to excessive nonesterified fatty acid oxidation, possibly via increased glucose fatty-acid cycle ultimately inhibiting glucose transport, or via decreased glycogen synthesis, or by a direct effect on glucose transporter translocation or activity or both.     

Changes in hepatic glycogen cycling during a glucose load in healthy humans

Aims/hypothesis Glycogen cycling, i.e. simultaneous glycogen synthesis and glycogenolysis, affects estimates of glucose fluxes using tracer techniques and may contribute to hyperglycaemia in diabetic conditions. This study presents a new method for quantifying hepatic glycogen cycling in the fed state. Glycogen is synthesised from glucose by the direct and indirect (gluconeogenic) pathways. Since glycogen is also synthesised from glycogen, i.e. glycogen→glucose 1-phosphate→glycogen, that synthesised through the direct and indirect pathways does not account for 100% of glycogen synthesis. The percentage contribution of glycogen cycling to glycogen synthesis then equals the difference between the sum of the percentage contributions of the direct and indirect pathways and 100. Materials and methods The indirect and direct pathways were measured independently in nine healthy volunteers who had fasted overnight. They ingested ²H₂O (5 ml/kg body water) and were infused with [5-³H]glucose and acetaminophen (paracetamol; 1 g) during hyperglycaemic clamps (7.8 mmol/l) lasting 8 h. The percentage contribution of the indirect pathway was calculated from the ratio of ²H enrichments at carbon 5 to that at carbon 2, and the contribution of the direct pathway was determined from the ³H-specific activity, relative to plasma glucose, of the urinary glucuronide excreted between 2 and 4, 4 and 6, and 6 and 8 h. Results Glucose infusion rates increased (p<0.01) to ∼50 μmol kg⁻¹ min⁻¹. Plasma insulin and the insulin : glucagon ratio rose ∼3.6- and ∼8.3-fold (p<0.001), respectively. From the difference between 100% and the sum of the direct (2–4 h, 54±6%; 4–6 h, 59±5%; 6–8 h, 63±4%) and indirect (32±3, 38±4, 36±3%) pathways, glycogen cycling was seen to be decreased (p<0.05) from 14±4% (2–4 h) to 4±3% (4–6 h) and 1±3% (6–8 h). Conclusions/interpretation This method allows measurement of hepatic glycogen cycling in the fed state and demonstrates that glycogen cycling occurs most in the early hours after glucose loading subsequent to a fast.     

Insulin resistance and coronary artery disease

Summary The purpose of the present study was to quantitate insulin-mediated glucose disposal in normal glucose tolerant patients with angiographically documented coronary artery disease (CAD) and to define the pathways responsible for the insulin resistance. We studied 13 healthy, normal weight, normotensive subjects with angiographically documented CAD and 10 age-, weight-matched control subjects with an oral glucose tolerance test and a 2-h euglycaemic insulin (40 mU · m⁻²· min⁻¹) clamp with tritiated glucose and indirect calorimetry. Lean body mass was measured with tritiated water. All CAD and control subjects had a normal oral glucose tolerance test. Fasting plasma insulin concentration (66 ± 6 vs 42 ± 6 pmol/l, p < 0.05) and area under the plasma insulin curve following glucose ingestion (498 ± 54 vs 348 ± 42 pmol · l⁻¹· min⁻¹, p < 0.001) were increased in CAD vs control subjects. Insulin-mediated whole body glucose disposal (27.8 ± 3.9 vs 38.3 ± 4.4 μmol · kg fat free mass (FFM)⁻¹· min⁻¹, p < 0.01) was significantly decreased in CAD subjects and this was entirely due to diminished non-oxidative glucose disposal (8.9 ± 2.8 vs 20.0 ± 3.3 μmol · kg FFM⁻¹· min⁻¹, p < 0.001). The magnitude of insulin resistance was positively correlated with the severity of CAD (r = 0.480, p < 0.05). In the CAD subjects basal and insulin-mediated rates of glucose and lipid oxidation were normal and insulin caused a normal suppression of hepatic glucose production. In conclusion, subjects with angiographically documented CAD are characterized by moderate-severe insulin resistance and hyperinsulinaemia and should be included in the metabolic and cardiovascular cluster of disorders that comprise the insulin resistance syndrome or ’syndrome X'. [Diabetologia (1996) 39: 1345–1350] Received: 6 February 1996 and in revised form: 29 May 1996     

Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene

Aims/hypothesis Recently we reported the coexistence of postprandial hypoglycaemia and moderate insulin resistance in heterozygous carriers of the Arg1174Gln mutation in the insulin receptor gene (INSR). Controlled studies of in vivo insulin signalling in humans with mutant INSR are unavailable, and therefore the cellular mechanisms underlying insulin resistance in Arg1174Gln carriers remain to be clarified.Subjects, materials and methods We studied glucose metabolism and insulin signalling in skeletal muscle from six Arg1174Gln carriers and matched control subjects during a euglycaemic–hyperinsulinaemic clamp.Results Impaired clearance of exogenous insulin caused four-fold higher clamp insulin levels in Arg1174Gln carriers compared with control subjects (p<0.05). In Arg1174Gln carriers insulin increased glucose disposal and non-oxidative glucose metabolism (p<0.05), but to a lower extent than in controls (p<0.05). Insulin increased Akt phosphorylation at Ser473 and Thr308, inhibited glycogen synthase kinase-3α activity, reduced phosphorylation of glycogen synthase at sites 3a+3b, and increased glycogen synthase activity in Arg1174Gln carriers (all p<0.05). In the insulin-stimulated state, Akt phosphorylation at Thr308 and glycogen synthase activity were reduced in Arg1174Gln carriers compared with controls (p<0.05), whereas glycogen synthase kinase-3α activity and phosphorylation of glycogen synthase at sites 3a+3b were similar in the two groups.Conclusions/interpretation In vivo insulin signalling in skeletal muscle of patients harbouring the Arg1174Gln mutation is surprisingly intact, with modest impairments in insulin-stimulated activity of Akt and glycogen synthase explaining the moderate degree of insulin resistance. Our data suggest that impaired insulin clearance in part rescues in vivo insulin signalling in muscle in these carriers of a mutant INSR, probably by increasing insulin action on the non-mutated insulin receptors.