Presence of very low density lipoprotein compositional abnormalities in Type 1 (insulin-dependent) diabetic patients; effects of blood glucose optimisation

Summary Plasma lipoprotein compositional abnormalities were investigated in eight normolipidaemic (plasma cholesterol <5.70 mmol/l; triglyceride <2.03 mmol/l) young male Type 1 (insulin-dependent) diabetic patients (before and after a short period of optimised blood glucose control) and in nine healthy control subjects, matched for sex, age and body mass index. Free and esterified cholesterol, triglyceride, phospholipids were assayed in all lipoprotein classes (VLDL, IDL, LDL) and in HDL subclasses (HDL2 and HDL3); apoB was measured only in very low density lipoproteins (VLDL). All VLDL constituents were increased in the diabetic group, the differences being more striking for apoB (6.0±1.1 mg/dl vs 2.0±0.1 mg/dl, p<0.02), free cholesterol (0.27±0.04 mmol/l vs 0.13±0.02 mmol/l, p<0.02) and esterified cholesterol (0.32±0.08 mmol/l vs 0.13±0.01 mmol/l, p<0.05). Also HDL subfractions showed differences between the two groups: all HDL2 constituents were increased, while in HDL3 only triglyceride was significantly increased (0.11±0.01 mmol/l vs 0.08±0.004 mmol/l, p<0.02). After two weeks of optimised blood glucose control all VLDL constituents were reduced and particularly: esterified cholesterol (–39%, p<0.02), free cholesterol (–37%, p<0.05), apoB (– 35%, p<0.05). Expressing each VLDL constituent as percent of the total lipoprotein mass, it was evident that the diabetic VLDL was rich in cholesterol both esterified (8.4±1.0% vs 5.4±0.5%, p<0.02) and free (8.5±0.7% vs 5.5±0.3%, p<0.001), apo B (5.1±0.6% vs 2.6±0.3%, p<0.001) and depleted in triglyceride (57.0±1.7% vs 64.1±1.7%, p<0.001). Two weeks of optimised blood glucose control were not able to correct the abnormal composition of VLDL. In conclusion, Type 1 (insulin-dependent) diabetic patients, although normolipidaemic, show an abnormal VLDL composition suggesting an increased prevalence of smaller and, possibly, more atherogenic VLDL particles. This abnormality is not corrected by a short period of blood glucose optimisation.     

Normal metabolism of apolipoprotein B100-containing lipoproteins despite qualitative abnormalities in type 1 diabetic men

Aims/hypothesis Type 1 diabetic subjects are at increased risk of cardiovascular disease and exhibit multiple qualitative abnormalities of apolipoprotein (apo) B100-containing lipoproteins. This stable isotope kinetic experiment was designed to study whether these abnormalities are associated with changes in the synthesis and fractional catabolic rates of VLDL-, IDL- and LDL-apoB100.Methods Using a bolus followed by a 16-h constant infusion of ¹³C-leucine, we performed a kinetic study in eight men with type 1 diabetes treated with a continuous subcutaneous insulin infusion administered by an external pump and in seven healthy men, in the fed state.Results The mean HbA₁c level in the type 1 diabetic patients was 8.00±1.48%. Plasma triglyceride, and total, LDL and HDL cholesterol levels were similar in patients and control subjects. VLDL were less triglyceride rich in type 1 diabetic patients than in control subjects (VLDL triglyceride : apoB 6.91±0.81 vs 8.29±1.24 mmol/g, p=0.05). Conversely, the IDL and LDL of the type 1 diabetic patients contained relatively higher levels of triglycerides (IDL triglycerides : apoB 2.16±0.36 vs 1.57±0.30 mmol/g, p<0.01; LDL triglycerides : apoB 0.27±0.06 vs 0.16±0.04 mmol/g, p<0.05). The apoB100 pool size, production and fractional catabolic rates in the two groups of subjects were similar for all lipoprotein fractions.Conclusions/interpretation Despite qualitative abnormalities, especially abnormalities of triglyceride content, the metabolism of apoB100-containing lipoproteins is not altered in type 1 diabetic men with fair glycaemic control with continuous subcutaneous insulin infusion. The high risk of atherosclerosis in these patients cannot be explained by kinetic abnormalities of apoB100-containing lipoproteins.     

Changes of lipolytic enzymes cluster with insulin resistance syndrome

Summary The activities of hepatic and lipoprotein lipase and the levels of lipo- and apoproteins were compared in two groups of normoglycaemic men representing the highest (n=18) and lowest (n=15) fasting insulin quintiles of first degree male relatives of non-insulin-dependent diabetic patients. The high insulin group representing insulin-resistant individuals had significantly lower post-heparin plasma lipoprotein lipase activity than the low insulin group (14.2±4.0 vs 20±5.8 mol NEFA·ml^–1·h^–1, p<0.001); hepatic lipase activity did not differ between the two groups (24.2±11 vs 18.0±5.3 mol NEFA·ml^–1·h^–1, NS). The lipoprotein lipase/hepatic lipase ratio in the high insulin group was decreased by 66% as compared to the low insulin group (0.75±0.57 vs 1.25±0.65, p<0.01). In the high insulin group both total and VLDL triglycerides were higher than in the low insulin group (1.61±0.57 vs 0.86±0.26 mmol/l, p< 0.001 and 1.00±0.47 vs 0.36±0.16 mmol/l, p<0.001, respectively) whereas HDL cholesterol and HDL₂ cholesterol were lower (1.20±0.30 vs 1.43±0.22 mmol/l, p<0.05 and 0.49±0.21 vs 0.71±0.17 mmol/l, p<0.05, respectively). Total cholesterol, LDL cholesterol or HDL₃ cholesterol did not differ between the two groups. The mean particle size of LDL was smaller in the high insulin group than in the low insulin group (258±7 vs 265±6 å, p<0.05). We propose that the changes of lipoprotein lipase and lipoprotein lipase/hepatic lipase ratio cluster with insulin resistance and provide a possible mechanism to explain the lowering of HDL cholesterol and elevation of triglyceride concentrations observed in insulin-resistant subjects.Abbreviations LPL Lipoprotein lipase - HL hepatic lipase - VLDL very low density lipoprotein - IDL intermediate density lipoprotein - LDL low density lipoprotein - HDL high density lipoprotein - chol cholesterol - TG triglycerides - NEFA non-esterified fatty acids     

Insulin resistance and abnormal albumin excretion in non-diabetic first-degree relatives of patients with NIDDM

Summary Microalbuminuria has recently been associated with insulin resistance in both insulin-dependent and non-insulin-dependent (NIDDM) diabetes mellitus. To establish whether microalbuminuria in non-diabetic subjects as well is associated with insulin resistance and associated abnormalities in glucose and lipid metabolism, oral glucose tolerance tests were performed with measurement of urinary albumin excretion rate, lipids and lipoproteins in 582 male non-diabetic first-degree relatives of patients with NIDDM. In addition, insulin sensitivity was assessed in 20 of these subjects with the euglycaemic hyperinsulinaemic clamp technique. Abnormal albumin excretion rate (AER), defined as AER 15–200 g/min, was associated with higher systolic blood pressure (p<0.05), higher fasting glucose values (p<0.05), lower HDL-cholesterol (p<0.05) and lower apolipoprotein A-I (p<0.05) concentrations than observed in subjects with normal AER. The rate of glucose metabolism was lower in subjects with abnormal compared to subjects with normal albumin excretion rate (38.0±2.8 vs 47.3±2.4 mol·kg lean body mass^–1. min^–1; p=0.028). This difference was almost completely accounted for by a reduction in non-oxidative glucose metabolism (17.7±1.9 vs 27.4±2.7 mol·kg lean body mass^–1. min^–1; p = 0.010), which correlated inversely with the AER (r=–0.543; p=0.013). These results suggest that in non-diabetic individuals genetically predisposed to NIDDM, abnormal AER is associated with insulin resistance and abnormalities in glucose and lipid metabolism.Abbreviations LBM lean body mass - IDDM Insulin-dependent diabetes mellitus - HDL high-density lipoprotein - NIDDM non-insulin-dependent diabetes mellitus - VLDL very low density lipoprotein - AER albumin excretion rate - OGTT oral glucose tolerance test     

Regulation of low-density lipoprotein particle size distribution in NIDDM and coronary disease: importance of serum triglycerides

Summary An increase of low-density lipoprotein triglycerides (LDL-Tg) was found to be an independent coronary artery disease (CAD) risk factor for non-insulin-dependent diabetic (NIDDM) patients in a recent prospective study. We examined the composition and size of LDL particles in 50 NIDDM men with angiographically verified CAD (NIDDM+ CAD+) and in 50 NIDDM men without CAD (NIDDM+ CAD–) as compared to 50 non-diabetic men with CAD (NIDDM– CAD+) and 31 non-diabetic men without CAD (NIDDM– CAD–). The groups had similar ranges of age and BMI LDL particle size was determined by gradient gel electrophoresis, and LDL was isolated by sequential ultracentrifugation for compositional analyses. Serum Tg was increased in NIDDM patients as compared to non-diabetic subjects (p<0.05), and in patients with CAD as compared to subjects without the disease (p<0.05). LDL cholesterol was lower in NIDDM patients than in non-diabetic subjects (p<0.001). Mean diameter of LDL particles was less than 255 å, but closely comparable in all groups. The presence of NIDDM was associated with increases of Tg and protein but lowering of free cholesterol in LDL (p<0.005 for all). In multivariate regression analyses neither NIDDM nor CAD were associated with LDL particle size, but serum Tg was the major determinant of LDL size in both NIDDM and non-diabetic subjects (p<0.001). When the patients were divided into quartiles according to fasting serum Tg levels, the LDL particle size and free cholesterol content decreased, but Tg and protein contents of LDL particles increased from the lowest to the highest Tg quartile (analysis of variance p<0.001 for all). When the subjects were categorized into two groups according to the median of VLDL-Tg (1.10 mmol/l) LDL size was associated with VLDL-Tg in the high but not in the low VLDL-Tg group. We conclude that in NIDDM patients with or without CAD serum Tg is the major determinant of the properties of LDL particles. The clinical implication is that in NIDDM serum Tg should be as low as possible to prevent atherogenic changes in LDL.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - CAD coronary artery disease - HDL high-density lipoprotein - LDL low-density lipoprotein - VLDL very-low-density lipoprotein - apoB apolipoprotein B - HL hepatic lipase - LPL lipoprotein lipase - CETP cholesteryl ester transfer protein - PL phospholipids - ANOVA analysis of variance - Tg triglycerides - FC free cholesterol     

Asymptomatic hyperglycaemia is associated with increased intimal plus medial thickness of the carotid artery

Summary Atherosclerotic changes have not been demonstrated directly in asymptomatic hyperglycaemic non-diabetic subjects, although high mortality due to coronary heart disease has been reported. We measured arterial wall thickness non-invasively, in order to directly demonstrate atherosclerosis of the carotid arteries of hyperglycaemic non-diabetic subjects and to evaluate its risk factors.The thicknesses of the intimal plus medial complex (IMT) of the carotid arteries of 112 asymptomatic hyperglycaemic non-diabetic subjects (aged 22–81, 95 males and 17 females) were compared with those of 55 healthy male subjects and 211 non-insulin-dependent NIDDM male diabetic patients. The subjects were subgrouped into impaired glucose-tolerant (IGT) subjects who had a 2-h glycaemic level of more than 7.8 mmol/l, and non-IGT subjects whose 2-h glycaemic levels were within 6.7–7.7 mmol/l.Non-IGT and IGT subjects showed significantly greater IMTs than age-matched healthy males and showed no significant differences compared to age-matched NIDDM patients. Multivariate analysis demonstrated that the risk factors for IMT of non-IGT and IGT subjects were age and systolic blood pressure. According to data on the accumulation of atherogenic risks (hypertension, dyslipidaemia, and smoking), IMT increased linearly in non-IGT and IGT subjects. However, non-IGT and IGT subjects without hyperlipidaemia, hypertension, or smoking risk still had significantly greater IMT than age-matched normal males (1.019±0.063 vs 0.770±0.111 mm, p<0.05). Prevalence of ECG-indicated coronary heart disease was significantly higher in hyperglycaemic non-diabetic subjects and NIDDM with increased carotid arterial wall thickness (IMT 1.1 mm) than in those without increased thickness (IMT<1.1 mm). Asymptomatic hyperglycaemic non-diabetic subjects have increased thickness of their carotid arteries compared to age-matched male NIDDM patients. As one of several independent risk factors, mild hyperglycaemia advances atherosclerosis, which leads to coronary heart disease.Abbreviations IMT Intimal plus medial complex - NIDDM non-insulin-dependent diabetes mellitus - IGT impaired glucose tolerance - CHD coronary heart disease - T-Chol serum total cholesterol - HDL-C high-density lipoprotein cholesterol - TG serum triglycerides     

Long-term intensive insulin therapy in IDDM: effects on HbA1c,risk for severe and mild hypoglycaemia,status of counterregulation and awareness of hypoglycaemia

Summary The present studies were designed to assess the percentage of HbA_1c, frequency, and awareness of hypoglycaemia (H) during long-term intensive therapy (IT) of insulin-dependent diabetes mellitus (IDDM). From 1981 to 1994, 112 IDDM patients were on IT. HbA_1c was 7.17±0.16% (non-diabetic subjects 3.8–5.5%), the frequency of severe H 0.01±0.009 episodes/patient-year, frequency of mild symptomatic H 35.6±2.9 episodes/patient-year. IDDM patients with HbA_1c 5.5% (Group I, n=10), between 6.1–7.0% (Group II, n=12), and 7.6% (Group III, n=11) were studied to assess responses of counterregulatory hormones, symptoms and cognitive function during experimental, stepped H. Compared to 18 non-diabetic subjects, Group I exhibited high thresholds (plasma glucose had to decrease more than normal to evoke responses), and impaired responses of adrenaline, unawareness of H and delayed onset of cognitive dysfunction at the lowest glycaemic plateau (2.3 mmol/l). Group II had normal thresholds and responses, whereas Group III had low thresholds. Frequency of mild H was higher in Group I (54.5±1.9 episodes/patient-year) than in Group II and III (33.7±3.5 and 20.4±2.5 episodes/ patient-year, respectively, p<0.001) and correlated with percentage of HbA_1c (r=–0.82). In conclusion: IT can maintain near-normal HbA_1c and is compatible with low frequency of severe H. However, if HbA_1c is less than 6.0%, mild, symptomatic H is excessively frequent and causes impaired counterregulation and H unawareness. Efforts should be made not only to maintain HbA_1c 7.0%, but also to prevent, recognize and reverse iatrogenic H unawarenes during long-term IT of IDDM by maintaining HbA_1c>6.0%.Abbreviations IDDM Insulin-dependent diabetes mellitus - DCCT Diabetes Control and Complications Trial     

Metabolic effects of darglitazone,an insulin sensitizer,in NIDDM subjects

Summary Insulin resistance is a significant pathogenetic factor in the development of non-insulin-dependent diabetes mellitus (NIDDM). A new class of drugs, the thiazolidinediones, have been shown to lower blood glucose levels without stimulating insulin secretion. We report the metabolic effect of the thiazolidinedione, darglitazone, in obese NIDDM subjects. Nineteen subjects were enrolled in a doubleblind placebo-controlled study in which 25 mg of darglitazone was given once a day for 14 days. Nine subjects received the active drug and ten subjects received placebo. Darglitazone-treated subjects showed; 1) a decrease in 24-h plasma glucose area under the curve from 292.8±31.2 to 235.2±21.6 mmol · h^–1 · l^–1 p=0.002; 2) a decrease in 24-h serum insulin area under the curve from 1027.2±254.4 to 765.6±170.4 U · h^–1 · l^–1 p=0.045; 3) a decrease in 24-h non-esterified fatty acid area under the curve from 1900±236 to 947±63 g · h^–1 · l^–1 p=0.002; 4) a decrease in mean 24-h serum triglyceride by 25.9±6.2% as compared to – 3.9±4.8% for the placebo-treated group, p=0.012. Placebo-treated subjects showed no change in their metabolic parameters after treatment. Thus, darglitazone is effective in increasing insulin effectiveness in obese NIDDM subjects. The potential for this and similar drugs to treat or prevent NIDDM as well as the insulin-resistance syndrome needs to be explored.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - NEFA non-esterified fatty acids - IVGTT intravenous glucose tolerance test - AUC area under the curve     

Glucose intolerance and impairment of insulin secretion in relation to vitamin D deficiency in East London Asians

Summary Vitamin D deficiency reduces insulin secretion and still occurs in East London Asians in whom the prevalence of diabetes mellitus is at least four times that of Caucasians. Vitamin D status was assessed in 44 of 65 non-diabetic subjects at risk of diabetes (spot blood glucose level >6.0 mmol/l <2 h post cibum, or >4.6 mmol/l >2 h post cibum on two separate occasions) and in 15 of 60 age and sex-matched low-risk control subjects who attended for oral glucose tolerance test (OGTT) after screening of 877 omnivorous subjects not known to have diabetes. It was found that 95% of at-risk and 80% of low-risk subjects were vitamin D deficient (serum 25-hydroxy-vitamin D <11 ng/ml). Diabetes was present in 16, impaired glucose tolerance in 12 and normoglycaemia in 19 at-risk subjects, impaired glucose tolerance in 2, and normoglycaemia in 13 low-risk subjects. Correlations of 30-min OGTT blood glucose, specific insulin and C-peptide levels with 25-hydroxy-vitamin D concentrations in 44 at-risk subjects were –0.31 (p=0.04), 0.59 (p=0.0001) and 0.44 (p=0.006). In 15 not-at-risk subjects 30-min OGTT specific insulin and C-peptide levels correlated with 25-hydroxy-vitamin D, r=0.39 (p=0.04) and 0.16 (p=0.43), respectively. Serum alkaline phosphatase concentration was higher in at-risk than not-at-risk subjects (59.6 vs 46.5 IU/l, p=0.012); corrected calcium concentrations were comparable (2.38 vs 2.39 mmol/l, p=0.7). Following treatment with 100,000 IU vitamin D by i.m. injection, specific insulin, C-peptide [30 min on OGTT] and 25-hydroxy-vitamin D concentrations had risen 8–12 weeks later [means±SD] from 57±62 to 96.2±82.4 mU/l [p=0.0017], 1.0±0.4 to 1.7±0.8 pmol/ml [p=0.0001] and 3.6±1.8 to 13.5±7.4 ng/ml [p=0.0001], (but not to low-risk group values of 179±89 mU/l, 2.7±1.14 pmol/ml and 8.16±6.4 ng/ml), respectively. Both total serum alkaline phosphatase and corrected calcium concentrations rose following vitamin D treatment in the at-risk subjects by 11.1±8.22 (from 44 to 55 IU/l) and 0.15±0.18, (2.43 to 2.57 mmol/l), respectively (p=0.004). Abnormal glucose tolerance was unchanged by vitamin D treatment. The value of early and sustained repletion with vitamin D in diabetes prophylaxis should be examined in communities where vitamin D depletion is common.Abbreviations OGTT Oral glucose tolerance test - IGT impaired glucose tolerance - p.c. post cibum - CV coefficient of variation - NEFA non-esterified fatty acids     

Troglitazone,an insulin action enhancer,improves metabolic control in NIDDM patients

Summary The effects of troglitazone, a novel thiazolidinedione, in non-insulin-dependent diabetic (NIDDM) patients were studied in a double-blind, parallel-group, placebo-controlled, dose-ranging trial. A total of 330 patients (63% male), mean age 57 years (range 39–72), with two fasting capillary blood glucose values 7 and 15 mmol/l (within 2.5 mmol/l of each other) were randomised to treatment with placebo or troglitazone at doses of 200, 400, 600 or 800 mg once daily, or 200 or 400 mg twice daily, for 12 weeks. Prior to the study, treatment had been with diet alone (38% patients) or with oral hypoglycaemic agents which were stopped 3–4 weeks before study treatment started. During treatment, HbA_1c tended to rise in patients taking placebo (7.2–8.0%), but remained unchanged with all doses of troglitazone. After 12 weeks of treatment, HbA_1c was significantly lower in the troglitazone-treated (mean 7.0–7.4%) compared to the placebo-treated (8.0%) patients (p=0.055 to <0.001), as was fasting serum glucose concentration (troglitazone, 9.3–11.0 mmol/l vs placebo, 12.9 mmol/l, p<0.001). All doses of troglitazone were equally effective. Troglitazone also lowered fasting plasma insulin concentration, by 12–26% compared to placebo (p=0.074 to <0.001). Insulin sensitivity assessed by homeostasis model assessment (HOMA) was greater after 12 weeks of treatment in troglitazone-treated patients (troglitazone, 34.3–42.8% vs placebo, 29.9%, p<0.05). In addition, serum triglyceride and non-esterified fatty acid concentrations were significantly lower and HDL cholesterol higher at troglitazone doses of 600 and 800 mg/day. LDL cholesterol increased at 400 and 600 mg doses only (from 4.3 and 3.9 mmol/l at baseline to 4.8 and 4.5 mmol/l, respectively at 12 weeks, p<0.05), but not at doses of 800 mg once daily or 400 mg twice daily. LDL/HDL ratio did not change during treatment. All doses were well tolerated; incidence of adverse events in troglitazone-treated patients was no higher than in those treated with placebo. However, a tendency to reduced neutrophil counts was observed in patients taking the highest doses of troglitazone. We conclude that troglitazone is effective and well-tolerated and shows potential as a new therapeutic agent for the treatment of NIDDM.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - HDL high density lipoprotein - LDL low density lipoprotein - NEFA non-esterified fatty acids - HOMA homeostasis model assessment     

Very low density lipoprotein subfraction abnormalities in IDDM patients: any effect of blood glucose control?

Summary Normolipidaemic insulin-dependent diabetic (IDDM) patients are characterized by an increase in the smaller VLDL particles, considered to be the most atherogenic. Since blood glucose control is one of the main regulators of lipid metabolism in diabetic patients, it could influence the shift in the distribution of VLDL subfractions towards smaller particles. To evaluate this possibility, VLDL subfractions, post-heparin lipoprotein lipase and hepatic lipase activities have been evaluated in male IDDM patients with either unsatisfactory blood glucose control (group 1, HbA_1c>8%, n=18) or good blood glucose control (group 2, HbA_1c<8%, n=16) and in 16 normoglycaemic individuals. The three groups were comparable for sex, age, body mass index, and plasma lipid levels. Three VLDL subfractions (large, Svedberg flotation unit (Sf) 175–400; intermediate, Sf 100–175; small, Sf 20–100) were separated by density gradient ultracentrifugation and analysed for cholesterol, triglyceride, and phospholipid levels. When compared to control subjects both groups of IDDM patients showed a clear shift in VLDL subfraction distribution with a significant increase in the proportion of small VLDL (group 1; 49±2%; p<0.005; group 2: 51±3%, p<0.01; control subjects 40±2%) (mean ± SEM) in relation to total VLDL. By contrast, the absolute lipid concentration of small VLDL was higher only in group 1, compared to control subjects (35±4 vs 27±3 mg/dl, p=0.05). Post-heparin hepatic lipase activity was significantly reduced in both IDDM groups (group 1: 254±19 mU/ ml, p<0.05; group 2: 202±19 mU/ml, p<0.005; control subjects 317±31 mU/ml). In conclusion, normolipidaemic IDDM patients show an increase in the smallest VLDL, whatever their degree of blood glucose control. However, this abnormality may be clinically relevant only in patients with unsatisfactory blood glucose control, since absolute lipid concentration of these potentially atherogenic particles is only increased in this group.Abbreviations IDDM Insulin-dependent diabetes mellitus - VLDL very low density lipoprotein - LPL lipoprotein lipase - HL hepatic lipase     

Effects of glycaemia on glucose transport in isolated skeletal muscle from patients with NIDDM: in vitro reversal of muscular insulin resistance

Summary We investigated the influence of altered glucose levels on insulin-stimulated 3-0-methylglucose transport in isolated skeletal muscle obtained from NIDDM patients (n=13) and non-diabetic subjects (n=23). Whole body insulin sensitivity was 71% lower in the NIDDM patients compared to the non-diabetic subjects (p <0.05), whereas, insulin-mediated peripheral glucose utilization in the NIDDM patients under hyperglycaemic conditions was comparable to that of the non-diabetic subjects at euglycaemia. Following a 30-min in vitro exposure to 4 mmol/l glucose, insulin-stimulated 3-0-methylglucose transport (600 pmol/l insulin) was 40% lower in isolated skeletal muscle strips from the NIDDM patients when compared to muscle strips from the non-diabetic subjects. The impaired capacity for insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle was normalized following prolonged (2 h) exposure to 4 mmol/l, but not to 8 mmol/l glucose. Insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle exposed to 8 mmol/l glucose was similar to that of the non-diabetic muscle exposed to 5 mmol/l glucose, but was decreased by 43% (p <0.01) when compared to non-diabetic muscle exposed to 8 mmol/l glucose. Despite the impaired insulin-stimulated 3-0-methylglucose transport capacity demonstrated by skeletal muscle from the NIDDM patients, skeletal muscle glycogen content was similar to that of the non-diabetic subjects. Kinetic studies revel a K_m for 3-0-methylglucose transport of 9.7 and 8.8 mmol/l glucose for basal and insulin-stimulated conditions, respectively. In conclusion, the impaired capacity for insulinstimulated glucose transport in skeletal muscle from patients with NIDDM appears to protect the cell from excessive glucose uptake under hyperglycaemic conditions. Furthermore, the in vitro normalization of the decreased insulin-stimulated glucose transport in NIDDM skeletal muscle following exposure to 4 mmol/l glucose suggests that glycaemia per se has a profound effect on the regulation of muscular glucose transport.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - KHB Krebs-Henseleit bicarbonate buffer - BSA bovine serum albumin - ANOVA analysis of variance - GLUT 4 insulin regulated glucose transporter     

Partial recovery of insulin secretion and action after combined insulin-sulfonylurea treatment in Type 2 (non-insulin-dependent) diabetic patients with secondary failure to oral agents

Summary Metabolic control, insulin secretion and insulin action were evaluated in seven Type 2 (non-insulin-dependent) diabetic patients with secondary failure to oral antidiabetic agents before and after two months of combined therapy with supper-time insulin (Ultratard: 0.4 U/kg body weight/day) plus premeal glibenclamide (15 mg/day). Metabolic control was assessed by 24 h plasma glucose, NEFA, and substrate (lactate, alanine, glycerol, ketone bodies) profile. Insulin secretion was evaluated by glucagon stimulation of C-peptide secretion, hyperglycaemic clamp (+7 mmol/l) and 24 h free-insulin and C-peptide profiles. The repeat studies, after two months of combined therapy, were performed at least 72 h after supper-time insulin withdrawal. Combining insulin and sulfonylurea agents resulted in a reduction in fasting plasma glucose (12.9±7 vs 10.4±1.2 mmol/l; p<0.05) and hepaic glucose production (13.9±1.1 vs 11.1±1.1 mol·kgc-min^–1; p<0.05). Mean 24 h plasma glucose was also lower (13.7±1.2 vs 11.1±1.4 mmol/l; p<0.05). Decrements in fasting plasma glucose and mean 24 h profile were correlated (r=0.90; p<0.01). HbA_1c also improved (11.8±0.8 vs 8.9±0.5%; p<0.05). Twenty-four hour profile for NEFA, glycerol, and ketone bodies was lower after teatment, while no difference occurred in the blood lactate and alanine profile. Insulin secretion in response to glucagon (C-peptide =+0.53±0.07 vs +0.43±0.07 pmol/ml) and hyperglycaemia (freeinsulin = 13.1±2.0 vs 12.3±2.2 mU/l) did not change. On the contrary, mean 24 h plasma freeinsulin (13.2±2.6 vs 17.5±2.2 mU/l; p<0.01) and C-peptide (0.76±0.10 vs 0.98±0.13 pmol/l; p<0.02) as well as the area under the curve (19.1±4.1 vs 23.6±3.1 U/24 h;p<0.01 and 1.16±0.14 vs 1.38±0.18 mol/24 h; p<0.02 respectively) were significantly increased. The ratio between glucose infusion (M) and plasma insulin concentration (I) during the hyperglycaemic clamp studies (M/I, an index of insulin sensitivity), was not statistically different (1.40±0.25 vs 1.81±0.40 mol·kg^–1· min^–1/mU·l^–1). These data suggest that, in Type 2 diabetic patients with secondary failure to oral antidiabetic agents, the combination of supper-time longacting insulin and premeal sulfonylurea agents can improve metabolic control. This positive effect is possibly mediated through an increased secretion of insulin in response to physiologic stimuli.     

Nerve conduction velocity in man: influence of glucose,somatostatin and electrolytes

Summary Insufficient metabolic control in diabetes mellitus is associated with a reversible reduction in nerve conduction velocity, but the mechanism behind this phenomenon is unknown. To examine the effect of acute hyperglycaemia on nerve conduction eight non-diabetic men (20–49 years of age) with no signs of peripheral neuropathy were studied before and after 3 h of hyperglycaemic clamping (plasma glucose 15 mmol/l), while insulin secretion was suppressed by somatostatin [Study 1]. Nerve conduction velocity, as determined in the proximal part of the median nerve, fell by 2.8±3.0 m/s (2p-value: 0.033). However, during euglycaemic clamping (plasma glucose 5 mmol/l) in five non-diabetic men (19–38 years of age) infused solely with somatostatin [Study 2], a comparable decrement in nerve conduction velocity was found (1.7±1.3 m/s, 2p-value: 0.043). In both studies relative hypoinsulinaemia was present. Serum-sodium decreased significantly (143±1 mmol/l vs 137±1 mmol/l [Study 1] and 143±1 mmol/l vs 142±2 mmol/l [Study 2]), while serum-potassium increased. In conclusion, the slight but significant reduction in nerve conduction velocity observed in both studies appears to be correlated to electrolyte changes. However, an effect of hypersomatostatinaemia or the hormonal changes associated with this cannot be excluded, while short-term hyperglycaemia per se seems to be without effect on nerve conduction velocity.Abbreviations NCV Nerve conduction velocity     

Delayed intracellular dissociation of the insulin-receptor complex impairs receptor recycling and insulin processing in cultured Epstein-Barr virus-transformed lymphocytes from insulin-resistant subjects

Summary Insulin-receptor internalization and processing are defective in insulin-resistant subjects. To assess the reversibility of these defects, we cultured Epstein-Barr virus-transformed-lymphoblasts from six normal, six obese, and six non-insulin-dependent diabetic (NIDDM) subjects in media containing low (5 mmol/l) or high (25 mmol/l) glucose concentrations, and studied the insulin-receptor internalization and processing in vitro. In cells from normal, obese, and NIDDM subjects cultured in low glucose concentrations, exposure to 100 nmol/l insulin for 30 min at 37C reduced cell-surface ¹²⁵I-insulin binding to a similar extent (82±2, 77±5, and 82±5 % of initial values, respectively). The same results were obtained with cells cultured in high glucose concentrations. In cells cultured under both glucose conditions, and exposed to 100 nmol/l insulin for 30 min at 37C, a complete recovery of the initial ¹²⁵I-insulin binding was observed in normal but not in obese and NIDDM subjects. Release of intracellular insulin and its degradation in vitro was determined by incubating cells with 600 pmol/l of ¹²⁵I-insulin for 60 min at 37C, acid washing cells, and re-incubating in insulin-free buffer at 37C. The radioactivity released by cells was characterized by trichloroacetic acid precipitability, Sephadex G-50 column Chromatograph, and rebinding to fresh cells. Rates of release of internalized radioactivity were reduced in obese and NIDDM subjects (t_1/2=61±9 min, p<0.02; 58±10 min, p<0.05; and 38±4 min in obese, NIDDM, and normal subjects, respectively). The percentage of intact insulin released from cells was significantly higher in obese and NIDDM subjects than in the normal subjects. The t_1/2 of intracellular dissociation of insulin-receptor complexes measured by a polyethylene glycol assay was lower in normal (6±1 min) than in obese (12±2 min, p<0.03) and NIDDM subjects (14±3 min, p<0.02). The results suggest that in insulin-resistant subjects a primary defect in intracellular dissociation of insulin is responsible for alterations of receptor recycling and insulin processing.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - EBV Epstein-Barr virus - RPMI - FCS fetal calf serum - PEG polyethylene glycol - ANOVA analysis of variance     

Response of adipose tissue lipoprotein lipase activity and serum lipoproteins to acute hyperinsulinaemia in man

Summary In order to assess the short-term effects of hyperinsulinaemia and hyperglycaemia on adipose tissue lipoprotein lipase activity and on serum lipoproteins, we measured these variables in ten normal subjects during euglycaemic and hyperglycaemic hyperinsulinaemic clamps. The mean steady-state plasma glucose and insulin concentrations, respectively, were 4.7 mmol/l and 101 mU/l during euglycaemic moderate-insulin clamp, 4.9 mmol/l and 565 mU/l during euglycaemic high-insulin clamp, and 8.8 mmol/l and 148 mU/l during hyperglycaemic clamp. Saline infusion was used as control. The adipose tissue lipoprotein lipase activity rose significantly over 5 h during high-insulin clamp (p<0.01) and during hyperglycaemic clamp (p<0.05), but did not change during the moderate-insulin clamp. The magnitude of change of lipoprotein lipase activity from baseline (either rise or fall) was inversely related to the preclamp activity during euglycaemic moderate-insulin clamp (r= -0.67), during hyperglycaemic clamp (r= -0.68) and during infusion of saline (r= -0.75, p<0.05). Total serum triglyceride concentration decreased significantly during all clamp studies compared with the control experiment. This change was mainly accounted for by a decrease of VLDL triglyceride. The LDL cholesterol level fell by an average of 5% (p<0.05) during the high-insulin clamp and by 10% (p<0.05) during the hyperglycaemic clamp. The HDL cholesterol level did not change significantly. It is concluded that adipose tissue lipoprotein lipase activity in man is increased by physiological insulin levels during hyperglycaemia and also by supraphysiological insulin levels during euglycaemia, but is not influenced by physiological hyperinsulinaemia without hyperglycaemia. Low basal lipoprotein lipase activity is more sensitive to insulin-glucose stimulation than primarily high lipoprotein lipase activity. Acute hyperinsulinaemia decreases VLDL triglyceride and LDL cholesterol concentrations.     

Low density lipoprotein cholesterol: An association with the severity of diabetic retinopathy

Summary Diurnal profiles of total and lipoprotein cholesterol and triglycerides were measured in 11 insulin-dependent diabetic subjects without retinopathy, 10 with background and 10 with proliferative retinopathy. The groups were closely matched for age and duration of diabetes. Total cholesterol levels were higher in patients with proliferative (5.6±0.5 mmol/l) than background (5.1±0.7 mmol/l) or no retinopathy (4.6±0.8 mmol/l, trend test; p < 0.003), due to raised levels of low density lipoprotein (LDL) cholesterol (3.8±0.9, 3.2±0.6 and 2.8±0.8 mmol/l respectively; p < 0.02). High density lipoprotein (HDL) levels were similar in patients with and without retinopathy and HDL/ LDL ratios were lower with more severe retinopathy (p < 0.025). Cholesterol levels were similar in diabetic subjects without retinopathy and in 12 normal subjects. Triglyceride levels were not related to retinopathy and no measure of plasma lipids correlated with HbA₁ or 24-h mean plasma glucose. Total and LDL cholesterol were weakly inversely correlated with creatinine clearance but the association with retinopathy was independent of this effect.     

Oxidation of low-density lipoprotein in NIDDM: its relationship to fatty acid composition

Summary The increased risk of atherosclerotic disease in diabetic subjects may be due to enhanced foam cell formation following an increased susceptibility of low density lipoprotein to oxidative modification. This study has compared fatty acid content and lipoprotein oxidisability in 10 non-insulin-dependent diabetic subjects with that in 10 control subjects. Both groups were normocholesterolaemic and the diabetic subjects had higher triglyceride levels (2.2±0.4 vs 1.2±0.2 mmol/l, p<0.05). The fatty acid composition was compared in low density lipoprotein following Folch extraction, separation by thin layer chromatography (for the lipid classes) and analysis by gas liquid chromatography. Low density lipoprotein oxidisability was assessed by conjugated diene and thiobarbituric acid reacting substance formation in the presence of copper ions. The esterified/free cholesterol ratio was higher in the low density lipoprotein from patients compared to control subjects (2.9±0.1 vs 1.9±0.3, p<0.05). Linoleic acid in the cholesteryl ester fraction of the lipoprotein was higher in the patients than in the control subjects (48.2±2.2% vs 42.4±3.4%, p<0.05) as was the total quantity of linoleic acid in the cholesteryl ester fraction (317.8±68.0 vs 213.2±28.0 g/mg protein, p<0.05) and in the low-density lipoprotein as a whole (443.2±70.0 vs 340.2±28.2 g/mg protein, p<0.05). Lipoprotein oxidisability was also increased in the diabetic group with increased formation of thiobarbituric acid reacting substances (35.6±7.2 vs 22.3±3.5 nmol/mg protein, p<0.05, increased total diene formation (502±60 vs 400±30 nmol/mg protein, p<0.05) and increased rate of diene formation (7.2±0.6 vs 5.1±0.9 nmol diene · mg protein^–1 · min^–1, p<0.05). This study indicates that low-density lipoprotein from diabetic subjects is more susceptible to oxidation. This could, in vivo, accelerate foam-cell formation thereby increasing atherosclerotic risk in diabetic subjects.Abbreviations BHT Butylated hydroxytoluene - EDTA ethylenediaminetetraacetic acid - TBARS thiobarbituric reacting substances - HPLC high performance liquid chromatography - MDA malondialdehyde - HbA_1c glycated haemoglobin     

The biochemical basis of increased hepatic glucose production in a mouse model of type 2 (non-insulin-dependent) diabetes mellitus

Summary The mechanism of increased hepatic glucose production in obese non-insulin-dependent diabetic (NIDDM) patients is unknown. The New Zealand Obese (NZO) mouse, a polygenic model of obesity and NIDDM shows increased hepatic glucose production. To determine the mechanism of this phenomenon, we measured gluconeogenesis from U-¹⁴C-glycerol and U-¹⁴C-alanine and relevant gluconeogenic enzymes. Gluconeogenesis from glycerol (0.07±0.01 vs 0.21±0.02 mol · min^–1 · body mass index (BMI)^–1, p<0.005) and alanine (0.57±0.07 vs 0.99±0.07 mol · min^–1 · BMI^–1, p<0.005) was elevated in control mice NZO vs as was glycerol turnover (0.25±0.02 vs 0.63±0.09 mol · min^–1 · BMI^–1, p<0.05). Fructose 1,6-bisphosphatase activity (44.2±1.9 vs 55.7±4.1 nmol · min^–1 · mg protein^–1, p<0.05) and protein levels (6.9±1.1 vs 16.7±2.3 arbitrary units, p<0.01) were increased in NZO mouse livers, as was the activity of pyruvate carboxylase (0.12±0.01 vs 0.17±0.02 nmol · min^–1 · mg protein^–1, p<0.05). To ascertain whether elevated lipid supply is responsible for these biochemical changes in NZO mice, we fed lean control mice a 60% fat diet for 2 weeks. Fat-fed mice were hyperinsulinaemic (76.37±4.06 vs 98.00±7.07 pmol/l, p=0.05) and had elevated plasma non-esterified fatty acid levels (0.44±0.05 vs 0.59±0.03 mmol/l, p=0.05). Fructose 1,6-bisphosphatase activity (43.86±2.54 vs 52.93±3.09 nmol · min^–1 · mg protein^–1, p=0.05) and protein levels (33.03±0.96 vs 40.04±1.26 arbitrary units, p=0.005) and pyruvate carboxylase activity (0.10±0.003 vs 0.14±0.01 nmol · min^–1 · mg protein^–1, p<0.05) were elevated in fat-fed mice. We conclude that in NZO mice increased hepatic glucose production is due to elevated lipolysis resulting from obesity.Abbreviations HGP Hepatic glucose production - NZO New Zealand Obese - FBPase fructose 1,6-bisphosphatase - PC pyruvate carboxylase - PEPCK phosphoenolpyruvate carboxykinase - BMI body mass index - NIDDM non-insulin-dependent diabetes mellitus - NZC lean control mice - NEFA non-esterified fatty acids     

Insulin resistance in Type 2 (non-insulin-dependent) diabetic patients with hypertriglyceridaemia

Summary Hypertriglyceridaemia, which is frequently seen in Type 2 (non-insulin-dependent) diabetes mellitus, is associated with insulin resistance. The connection between hypertriglyceridaemia and insulin resistance is not clear, but could be due to substrate competition between glucose and lipids. To address this question we measured glucose and lipid metabolism in 39 Type 2 diabetic patients with hypertriglyceridaemia, i. e. mean fasting serum triglyceride level equal to or above 2 mmol/l (age 59±1 years, BMI 27.4±0.5 kg/m², HbA_1c8.0±0.2%, serum triglycerides 3.2±0.2 mmol/l) and 41 Type 2 diabetic patients with normotriglyceridaemia, i. e. mean fasting serum triglyceride level below 2 mmol/l (age 58±1 years, BMI 27.0±0.7 kg/m², HbA_1c7.8±0.2 %, serum triglycerides 1.4±0.1 mmol/l). Insulin sensitivity was assessed using a 340 pmol·(m²)^–1· min^–1 euglycaemic insulin clamp. Substrate oxidation rates were measured with indirect calorimetry and hepatic glucose production was estimated using a primed (25 Ci)-constant (0.25 Ci/min) infusion of [3-³H]-glucose. Suppression of lipid oxidation by insulin was impaired in patients with hypertriglyceridaemia vs patients with normal triglyceride levels (3.5±0.2 vs 3.0±0.2mol·kg^–1· min^–1; p<0.05). Stimulation of glucose disposal by insulin was reduced in hypertriglyceridaemic vs normotriglyceridaemic patients (27.0±1.3 vs 31.9±1.6 mol·kg^–1·min^–1; p<0.05) primarily due to impaired glucose storage (9.8±1.0 vs 14.6±1.4mol·kg^–1·min^–1; p<0.01). In contrast, insulinstimulated glucose oxidation was similar in patients with hypertriglyceridaemia and in patients with normal triglyceride concentrations (16.9±0.8 vs 17.2±0.7mol·kg^–1·min^–1). Hepatic glucose production in the basal state and during the clamp did not differ between the two groups. We conclude therefore that oxidative substrate competition between glucose and lipids does not explain insulin resistance associated with hypertriglyceridaemia in Type 2 diabetes. The question remains whether the reduced nonoxidative glucose disposal observed in the patients with hypertriglyceridaemia is genetically determined or a consequence of increased lipid oxidation.