Pharmacokinetics,pharmacodynamics and glucose counterregulation following subcutaneous injection of the monomeric insulin analogue [Lys(B28),Pro(B29)] in IDDM

Summary The aim of these studies was to compare the pharmacokinetics, pharmacodynamics, counterregulatory hormone and symptom responses, as well as cognitive function during hypoglycaemia induced by s. c. injection of 0.15 IU/kg of regular human insulin (HI) and the monomeric insulin analogue [Lys(B28),Pro (B29)] (MI) in insulin-dependent-diabetic (IDDM) subjects. In these studies glucose was infused whenever needed to prevent decreases in plasma glucose below 3 mmol/l. After MI, plasma insulin increased earlier to a peak (60 vs 90 min) which was greater than after HI (294±24 vs 255±24 pmol/l), and plasma glucose decreased earlier to a 3 mmol/l plateau (60 vs 120 min) (p<0.05). The amount of glucose infused to prevent plasma glucose falling below 3 mmol/l was three times greater after MI than HI (293±26 vs 90±25 mol · kg^–1 · 60–375 min^–1, p<0.05). After MI, hepatic glucose production was more suppressed (0.7±1 vs 5.9±0.54 mol · kg^–1 · min^–1) and glucose utilization was less suppressed than after HI (11.6±0.65 vs 9.1±0.11mol · kg^–1 · min^–1) (p<0.05). Similarly, plasma NEFA, glycerol, and -OH-butyrate were more suppressed after MI than HI (p<0.05), whereas plasma lactate increased only after MI, but not after HI. Responses of counterregulatory hormones, symptoms and deterioration in cognitive function during plasma glucose plateau of 3 mmol/l were superimposable after MI and HI (p=NS). Post-hypoglycaemia hyperglycaemia was greater after MI than HI (at 480 min 12.1±1 vs 11±1 mmol/l) because of greater hepatic glucose production during insulin waning which occurred at least 135 min earlier with MI as compared to HI (p<0.05). It is concluded that counterregulatory hormones, symptoms and deterioration in cognitive function during hypoglycaemia respond similarly after MI and HI. The biological effect of MI appears greater than that of HI for at least 4 h after the s.c. injection and appears as a good candidate for achieving optimal post-prandial glucose control in IDDM.Abbreviations HI Human insulin - MI monomeric insulin - NEFA non-esterified fatty acid - HGO hepatic glucose production rate - -OH-butyrate -hydroxy-butyrate - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus     

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 regulation of glucose and lipid metabolism in massive obesity

Summary Eight obese patients and 12 normal individuals underwent a euglycaemic insulin clamp (20 and 40 mU · m^2–1 · min^–1) along with continuous infusion of 3-³H-glucose and 1-¹⁴C-palmitate and indirect calorimetry. Basal plasma glucose concentration (4.7±0.3 vs 4.4±0.2 mmol/l) was similar in the two groups, whereas hepatic glucose production was slightly higher in obese individuals (1.11±0.06 vs 0.84±0.05 mmol/min) in spite of higher plasma insulin levels (17±2 vs 6±1 mU/l; p<0.01). Insulin inhibition of hepatic glucose production was impaired in obese subjects. Glucose disposal by lean body mass was markedly reduced both at baseline (11.7±1.1 vs 15.6±0.6 mol · kg^–1 · min^–1; p<0.05) and during clamp (15.0±1.1 vs 34.4±2.8 and 26.7±3.9 vs 62.2±2.8 mol · kg^–1 · min^–1; p<0.01) Oxidative (12.2±1.1 vs 17.8±1 and 16.1±1.1 vs 51.1±1.7 mol · kg^–1 · min^–1; p<0.05–0.002) and non-oxidative glucose metabolism (3.9±1.1 vs 15.0±2.8 and 12.8±3.3 vs 38.3±2.2 mol · kg^–1 · min^–1; p<0.01–0.001) were impaired. Basal plasma concentrations of non-esterified fatty acids (635±75 vs 510±71 mol/l) and blood glycerol (129±17 vs 56±5 mol/l; p<0.01) were increased in obese patients. Following hyperinsulinaemia, plasma non-esterified fatty acids (244±79 vs 69±16 and 140±2 vs 36±10 mol/l; p<0.01) and blood glycerol levels (79±20 vs 34±6 and 73±22 vs 29±5 mol/l; p<0.01) remained higher in obese subjects. Baseline non-esterified fatty acid production rate per kg of fat body mass was significantly larger in normal weight subjects (37.7±6.7 vs 14.0±1.8 mol/l; p<0.01) and insulin inhibition was reduced in obese patients (–41±9 vs –74±3 and –53±11 vs –82±3%; p<0.05). Basal plasma non-esterified fatty acid utilization by lean body mass was similar in the two groups (9.8±0.9 vs 8.8±2.0 mol · kg^–1 · min^–1), whereas during clamp it remained higher in obese patients (6.0±1.2 vs 2.8±2.5 and 4.9±1.3 vs 1.5±0.6 mol · kg^–1 · min^–1; p<0.1–0.05). Lipid oxidation was higher in obese individuals in spite of hyperinsulinaemia (3.7±0.3 vs 2.4±0.4 and 2.3±0.4 vs 0.9±0.3 mol · kg^–1 · min^–1; p<0.05– 0.02). An inverse correlation was found between lipid oxidation and glucose oxidation (r=0.82 and 0.93; p<0.001) and glucose utilization (r=0.54 and 0.83; p<0.05–0.001) both in obese and control subjects. A correlation between lipid oxidation and non-oxidative glucose metabolism was present only in normal weight individuals (r=0.75; p<0.01). We conclude that in obesity all tissues (muscles, liver, and adipose tissue) are resistant to insulin action. Insulin resistance involves glucose as well as lipid metabolism.     

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.     

ACE-inhibition increases hepatic and extrahepatic sensitivity to insulin in patients with Type 2 (non-insulin-dependent) diabetes mellitus and arterial hypertension

Summary To assess the effects of ACE-inhibition on insulin action in Type 2 (non-insulin-dependent) diabetes mellitus associated with essential hypertension, 12 patients with Type 2 diabetes (on diet and oral hypoglycaemic agents) and arterial hypertension were examined on two occasions, in a single blind, cross-over study, after two days of treatment with either captopril or a placebo. The study consisted of a euglycaemic-hyperinsulinaemic clamp (two sequential steps of insulin infusion at the rates of 0.25 mU·kg^–1·min^–1 and 1 mU·kg^–1·min^–1, 2 h each step), combined with an infusion of 3-³H-glucose to measure the rate of hepatic glucose production and that of peripheral glucose utilization. The results show that blood pressure was lower after captopril (sitting, systolic 148±5 mmHg, diastolic 89±2 mm Hg) compared to placebo (155±6 and 94±2 mm Hg) (p<0.05). Captopril treatment resulted in a more suppressed hepatic glucose production (2.7±0.4 vs 4.94±0.55 mol·kg^–1·min^–1), and a lower plasma non-esterified fatty acid concentration (0.143±0.05 vs 0.200±0.05 mmol/l) (captopril vs placebo, p<0.05) at the end of the first step of insulin infusion (estimated portal plasma insulin concentration 305±28 pmol/l); and in a greater glucose utilization (36.5±5.1 vs 28±3.6mol·kg^–1·min^–1, p<0.001) at the end of the second step of insulin infusion (arterial plasma insulin concentration of 604±33 pmol/l). We conclude that captopril improved insulin sensitivity in Type 2 diabetes associated with hypertension at the level of the liver and extrahepatic tissues, primarily muscle and adipose tissue. Thus, in contrast to other antihypertensive drugs such as diuretics and beta-blockers which may have a detrimental effect on insulin action, ACE-inhibitors appear to improve insulin action in Type 2 diabetes and essential hypertension, at least on a short-term basis.     

Contribution of glucose/glucose 6-phosphate cycle activity to insulin resistance in Type 2 (non-insulin-dependent) diabetes mellitus

Summary It has been suggested that increased glucose/glucose 6-phosphate substrate cycling impairs net hepatic glucose uptake in Type 2 (non-insulin-dependent) diabetes mellitus and contributes to hyperglycaemia. To investigate glucose/glucose 6-phosphate cycle activity and insulin action in Type 2 diabetes we studied eight patients and eight healthy control subjects, using the euglycaemic glucose clamp and isotope dilution techniques with purified [2-³H]- and [6-³H] glucose tracers, in the post-absorptive state and eight patients and five healthy control subjects during consecutive insulin infusions at rates of 0.4 and 2.0 mU·kg^–1·min^–1. [2-³H]glucose and [6-³H]glucose radioactivity in plasma samples were determined using selective enzymatic detritiation, allowing calculation of glucose turnover rates for each isotope, the difference being glucose/glucose 6-phosphate cycling. Endogenous glucose production ([6-³H]glucose) was greater in diabetic than control subjects in the post-absorptive state (15.6±1.5 vs 11.3±0.4 mol·kg^–1·min^–1, p<0.05) and during the 0.4 mU insulin infusion (10.1±1.3 vs 5.2±0.3 mol·kg^–1·min^–1, p<0.01) indicating hepatic insulin resistance. Glucose/glucose 6-phosphate cycling was significantly greater in diabetic than in control subjects in the post-absorptive state (2.6±0.4 vs 1.6±0.2 mol·kg^–1·min^–1, p<0.05) but not during the 0.4 mU insulin infusion (2.0±0.4 vs 2.0±0.3 mol·kg^–1·min^–1). During the 2.0 mU insulin infusion endogenous glucose production was suppressed to a similar degree in both groups (2.6±0.5 vs 3.4±0.7 mol · kg^–1·min^–1) but glucose disappearance was lower in the diabetic subjects (30.8±2.0 vs 52.4±4.6 mol·kg^–1·min^–1, p<0.01). During the 2.0 mU insulin infusion glucose/glucose 6-phosphate cycling was greater in the diabetic subjects (3.8±0.7 vs 0.8±0.6 mol·kg^–1·min^–1, p<0.05). In conclusion, both hepatic and peripheral insulin action are impaired in Type 2 diabetes. Increased glucose/glucose 6-phosphate cycling is seen in the post-absorptive state and also during marked hyperinsulinaemia, when insulin resistance is predominantly due to reduced peripheral tissue glucose uptake.     

Increased glucose carbon recycling in severely insulin deficient Type 1 (insulin-dependent) diabetic subjects

Summary Six Type 1 (insulin-dependent) diabetic subjects were studied in order to determine the contribution of recycling of glucose carbon to the overproduction of glucose which is characteristic of the fasting hyperglycaemia produced by insulin withdrawal. The subjects were studied on two occasions, once after an overnight insulin infusion and once following 24 h of insulin withdrawal. The difference in turnover rates of 1-¹⁴C-glucose and 3-³H-glucose was used as a measure of glucose recycling. Insulin withdrawal caused a marked metabolic derangement with a rise in non-esterified fatty acids from 0.69±0.23 to 1.11±0.21 mmol/l (mean±SEM, p<0.05), total ketones from 0.27±0.06 to 2.06±0.51 mmol/l (p<0.01), cortisol from 341±43 to 479±31 nmol/l (p<0.05) and growth hormone from 1.1±0.3 to 19+5-mu/l (p<0.05). Glucose turnover rose from 13.8±2.3 mol·kg^–1·min^–1 at a glucose of 6.9±0.7 mmol/l in the insulin infused study to 25.8±4.4 mol·kg^–1·min^–1 (p<0.05) at a glucose of 16.4±0.7 mmol/l in the insulin withdrawn study. Recycling also rose from 3.0±0.4 mol· kg^–1·min^–1 to 9.4±2.2 mol·kg^–1·min^–1 (p<0.05) when insulin withdrawn, accounting for 23±3% and 36±3% of glucose turnover, respectively. We conclude that in the severely insulin deficient Type 1 diabetic subject recycling of glucose carbon is a major contributor to the excess glucose production.     

Lowering of triglycerides by gemfibrozil affects neither the glucoregulatory nor antilipolytic effect of insulin in Type 2 (non-insulin-dependent) diabetic patients

Summary Hypertriglyceridaemia and insulin resistance are closely associated but it is unknown whether hypertriglyceridaemia per se contributes to insulin resistance. In the present study we examined whether gemfibrozil, by lowering triglyceride levels, improves the glucoregulatory and antilipolytic action of insulin in Type 2 (non-insulin-dependent) diabetes mellitus. Twenty patients were randomly allocated to receive either placebo or gemfibrozil 1200 mg daily for 12 weeks in a double-blind study. Very low density lipoprotein triglyceride levels decreased in the gemfibrozil group by 42±12% (p<0.01). Gemfibrozil had no effect on the diurnal concentration of non-esterified fatty acids (NEFA). At the randomization HbA_1c levels were comparable (7.6±0.3 vs 7.8±0.2%, NS) and increased slightly both in the gemfibrozil (8.2±0.4%, p<0.05) and placebo groups (8.0±0.3%, NS). Pre- and post-treatment diurnal glucose and insulin concentrations remained unchanged. Basal pre- and post-treatment hepatic glucose production rates were comparable in both groups and similarly suppressed by insulin. Rate of whole body glucose disposal during a low-dose insulin infusion (serum insulin 90 pmol/l) (pre- vs post-gemfibrozil 11.9±1.1 vs 11.1±0.7, pre- vs post-placebo 9.9±1.1 vs 10.8±0.8 mol·kg^–1·min^–1, NS for both) and a high-dose insulin infusion (serum insulin 500 pmol/l) (16.2+-1.7 vs 17.7±2.7, 17.1±4.2 vs 17.4±2.9 mol·kg^–1·min^–1, respectively, NS for both) remained unchanged. Basal pre- and post-treatment NEFA turnover rates were comparable in both groups and similarly suppressed by insulin. Also rates of total lipid oxidation, plasma NEFA oxidation and non-oxidative NEFA metabolism remained unchanged in both groups. We conclude that gemfibrozil effectively lowers serum triglycerides but has no effect on insulin sensitivity of glucose and NEFA metabolism. The data suggest that hypertriglyceridaemia is a consequence rather than a cause of insulin resistance in Type 2 diabetic patients.     

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.     

Peripheral and hepatic insulin sensitivity in subjects with impaired glucose tolerance

Summary Recent evidence suggests that the post-prandial hyperglycaemia in impaired glucose tolerance is primarily due to impaired suppression of basal hepatic glucose output. This in turn appears to be secondary to decreased first phase insulin secretion, although decreased hepatic insulin sensitivity, which is a feature of non-insulin-dependent diabetes mellitus, might also play a role. Eight mildly overweight subjects with impaired glucose tolerance and eight closely matched control subjects with normal glucose tolerance underwent an intravenous glucose tolerance test to assess first phase insulin secretion. Insulin sensitivity was examined by a 150-min hyperinsulinaemic-euglycaemic clamp. Somatostatin was infused from 150 min to suppress endogenous insulin secretion, and glucagon and insulin were replaced by constant infusion. Glucose with added dideuterated glucose (labelled infusion technique) was infused to maintain euglycaemia. First phase insulin secretion ( 0–10 min insulin area ÷ 0–10 min glucose area) was significantly decreased in the subjects with impaired glucose tolerance (median [range]: 1.2 [0.2–19.4] vs 9.1 [2.6–14.5] mU·mmol^–1; p<0.01). During the clamp, circulating insulin (93±8 [mean±SEM] and 81±10 mU·l^–1) and glucagon (54±4 and 44±6 ng·l^–1) levels were comparable. Total glucose disposal was decreased in subjects with impaired glucose tolerance (2.78±0.27 vs 4.47±0.53 mg·kg^–1·min^–1; p<0.02), and was primarily due to decreased non-oxidative glucose disposal. However, hepatic glucose output rates were comparable during the clamp (0.38±0.10 and 0.30±0.18 mg·kg^–1·min^–1). Therefore, the main defects in subjects with impaired glucose tolerance are decreased first phase insulin secretion and peripheral non-oxidative glucose disposal, but hepatic glucose output shows normal responsiveness to insulin.Abbreviations FPIS First phase insulin secretion - PG plasma glucose - NIDDM non-insulin-dependent diabetes mellitus - IGT impaired glucose tolerance - HGO hepatic glucose output - IVGTT intravenous glucose tolerance test - OGTT oral glucose tolerance test     

Increased hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 in NIDDM

Summary We measured the hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 (VLDL apoB) using a stable isotope gas-chromatography mass-spectrometry method in six patients with non-insulin-dependent diabetes mellitus (NIDDM) (four males, two females, age 57.5±2.2 years (mean±SEM), weight 88.2±5.5 kg, glycated haemoglobin (HbA₁) 8.5±0.5%, plasma total cholesterol concentration 5.7±0.5 mmol/l, triglyceride 3.8±0.9 mmol/l, high-density lipoprotein (HDL) cholesterol 1.0±0.1 mmol/l) and six non-diabetic subjects matched for age, sex and weight (four males, two females, age 55.7±2.8 years, weight 85.8±5.6 kg, HbA₁ 6.5±0.1%, plasma total cholesterol concentration 5.7±0.5 mmol/l, triglyceride 1.2±0.1 mmol/l, HDL cholesterol 1.4±0.1 mmol/l). HbA₁, plasma triglyceride and mevalpnic acid (an index of cholesterol synthesis in vivo) concentrations were significantly higher in the diabetic patients than in the non-diabetic subjects (p=0.006, p=0.02 and p=0.004, respectively). VLDL apoB absolute secretion rate was significantly higher in the diabetic patients compared with the non-diabetic subjects (2297±491 vs 921±115 mg/day, p<0.05), but there was no significant difference in the fractional catabolic rate of VLDL apoB. There was a positive correlation between VLDL apoB secretion rate and (i) fasting C-peptide (r=0.84, p=0.04) and (ii) mevalonic acid concentration (r=0.83, p<0.05) in the diabetic patients but not in the non-diabetic subjects. There was also a significant positive association between plasma mevalonic acid and plasma C-peptide (r=0.82, p<0.05) concentrations in the diabetic patients. We conclude that in NIDDM, there is increased hepatic secretion of VLDL apoB which may partly explain the dyslipoproteinaemia seen in this condition. We suggest that increased secretion of this apolipoprotein may be a consequence of resistance to the inhibitory effect of insulin on VLDL apoB secretion. Insulin resistance may also be the mechanism by which cholesterol synthesis, a regulator of apoB secretion, is increased in NIDDM.Abbreviations ApoB Apolipoprotein B-100 - VLDL very-low-density lipoprotein - GCMS gas-chromatography mass-spectrometry - MVA mevalonic acid - Hep G2 hepatoma G2 - -KIC -ketoisocaproic acid - TC total cholesterol - TG triglyceride - NEFA non-esterified fatty acids - FSR fractional secretion rate - ASR absolute secretion rate - m/z mass to charge ratio - CV coefficient of variation     

Liver and muscle insulin sensitivity,glycogen concentration and glycogen synthase activity in a rat model of non-insulin-dependent diabetes

Summary Mild diabetes was induced in adult rats with streptozotocin (45 mg/kg body weight), and insulin sensitivity, glycogen deposition and glycogen synthase activity assessed in liver and muscle 5 weeks later. Diabetic rats had significantly elevated fasting blood glucose concentrations (5.6±0.1 versus 3.6±0.1 mmol/l, p<0.001), and blood glucose concentrations 2 h after a 1 g/kg glucose load (12.0±0.6 versus 3.7±0.2 mmol/l, p<0.001). After a 20-h fast hepatic glucose output was significantly elevated (58±3 versus 47±3 mol·min^–1·kg^–1, p<0.05), and failed to suppress at high insulin concentrations during a euglycaemic clamp (hepatic glucose output 21±4 versus 2±4 mol·min^–1·kg^–1, p<0.02). Liver glycogen was lower in the diabetic rats by the end of the clamp (16±3 versus 38±6 mol/g wet wt, p<0.05). At the end of the clamp total glucose turnover was lower in the diabetic rats (107±4 versus 161±17 mol·min^–1· kg^–1, p<0.01), as was skeletal muscle glycogen synthase activity (0.46±0.04 versus 0.67±0.05 U/g wet wt, p<0.01) and glycogen concentration (22±2 versus 33±3 mol/g wet wt, p<0.05). Blood lactate and pyruvate responses suggested that glycolytic pathways were similarly affected. Thus, insulin insensitivity develops in both liver and skeletal muscle after 5 weeks of mild streptozotocin-induced diabetes.     

Lipoprotein secretion by isolated perfused livers from streptozotocin-diabetic rats

Summary In liver perfusion from sucrose-fed, streptozotocin-diabetic rats there was in comparison with normal animals, a decrease in very low density lipoprotein concentration in the perfusion medium (38.6 ±6.3 versus 64.4±8.4 g · g liver ^-1 3h^-1, p <0.05) and an increase in high density lipoprotein concentration (33.5±6.5 versus 14.0+1.9 g · g liver^-1 3h^-1, p<0.005), which was paralleled by enhanced secretion of apoprotein A-I. The triglyceride: protein ratio was lower in very low density lipoprotein from diabetic animals (8.8 versus 13.4). Analysis of the apoprotein composition showed that diabetic very low density lipoprotein lacked arginine-rich protein (apo-E) and apo-C peptides; diabetic high density lipoprotein also lacked arginine-rich protein but contained more A-IV and apo-C-peptides. This may indicate net transfer of C peptides to high density lipoprotein from the degradation of very low density lipoprotein particles. The ratio of ³H-leucine: ¹⁴C-glucosamine incorporation was decreased in all diabetic lipoprotein classes suggesting increased glycosylation of apoproteins. These changes in particle composition may influence lipoprotein metabolism in diabetes through their effects on lipoprotein lipase and lecithin cholesterol acyl transferase activity, plasma half-life and tissue binding.     

Intracerebroventricular administration of melanotan II increases insulin sensitivity of glucose disposal in mice

Aims/hypothesis The present study was conducted to evaluate the effects of central administration of melanotan II (MTII), a melanocortin-3/4 receptor agonist, on hepatic and whole-body insulin sensitivity, independent of food intake and body weight.Methods Over a period of 24 h, 225 ng of MTII was injected in three aliquots into the left lateral ventricle of male C57Bl/6 mice. The animals had no access to food. The control group received three injections of distilled water. Whole-body and hepatic insulin sensitivity were measured by hyperinsulinaemic–euglycaemic clamp in combination with [³H]glucose infusion. Glut4 mRNA expression was measured in skeletal muscle.Results Plasma glucose and insulin concentrations under basal and hyperinsulinaemic conditions were similar in MTII- and placebo-treated mice. Endogenous glucose production (EGP) and glucose disposal in the basal state were significantly higher in MTII-treated mice than in the control group (71±22 vs 43±12 mol·min^–1·kg^–1, p<0.01). During hyperinsulinaemia, glucose disposal was significantly higher in MTII-treated mice (151±20 vs 108±20 mol·min^–1·kg^–1, p<0.01). In contrast, the inhibitory effect of insulin on EGP was not affected by MTII (relative decrease in EGP: 45±27 vs 50±20%). Glut4 mRNA expression in skeletal muscle was significantly increased in MTII-treated mice (307±94 vs 100±56%, p<0.01).Conclusions/interpretation Intracerebroventricular administration of MTII acutely increases insulin-mediated glucose disposal but does not affect the capacity of insulin to suppress EGP in C57Bl/6 mice. These data indicate that central stimulation of melanocortin-3/4 receptors modulates insulin sensitivity in a tissue-specific manner, independent of its well-known impact on feeding and body weight.     

Factors associated with basal metabolic rate in patients with Type 2 (non-insulin-dependent) diabetes mellitus

Summary To examine determinants of basal metabolic rate we studied 66 Type 2 (non-insulin-dependent) diabetic and 24 healthy age- and weight-matched control subjects with indirect calorimetry and infusion of [³H-3-] glucose. Eight Type 2 diabetic patients were re-studied after a period of insulin therapy. Basal metabolic rate was higher in Type 2 diabetic patients than in control subjects (102.8 ± 1.9 J · kg LBM^–1-min^–1 vs 90.7 ± 2.8 J · kg LBM^–1;min^–1; p<0.01) and decreased significantly with insulin therapy (p <0.01). The basal rate of hepatic glucose production was higher in Type 2 diabetic patients than in control subjects (1044.0 ± 29.9 vs 789.3 ± 41.7 mol/min; p <0.001) and decreased after insulin therapy (p <0.01). Hepatic glucose production correlated positively with basal metabolic rate both in Type 2 diabetic patients (r = 0.49; p <0.001) and in control subjects (r = 0.50; p<0.05). Lipid oxidation was increased in Type 2 diabetic patients compared with control subjects (1.68 ± 0.05 vs 1.37 ± 0.08 mol · kg LBM^–1 · min^–1'; p <0.01) and decreased significantly after insulin therapy (p <0.05). The rate of lipid oxidation correlated positively with basal metabolic rate both in Type 2 diabetic patients (r = 0.36; p <0.01) and in control subjects (r = 0.51; p <0.01). These data demonstrate that basal metabolic rate, rates of hepatic glucose production and lipid oxidation are interrelated in Type 2 diabetic patients. A reduction of the hepatic glucose production, however, is associated with a reduction in lipid oxidation, which in turn, may result in a reduction in basal metabolic rate.     

Both acute and chronic near-normoglycaemia are required to improve insulin resistance in Type 1 (insulin-dependent) diabetes mellitus

Summary To determine the impact of both short- and longterm near-normoglycaemia on insulin resistance in Type 1 (insulin-dependent) diabetes hepatic glucose production (mg · kg^–1 · min^–1) and peripheral glucose utilisation (M-value, mg · kg^–1 · min^–1) were estimated during an euglycaemic hyperinsulinaemic clamp (10 mU · kg · min) in patients with either good (HbA_1c<5.8%, groups A and B) or poor (HbA_1c>7.5%, groups C and D) long-term metabolic control (time > 12 months) and in healthy subjects (HbA_1c: 5.08±0.20%; n=8). To this end blood glucose was stabilized at 6.7 mmol/l by overnight (t=12 h) i.v. regular insulin in groups (n=8 each) A (HbA_1c: 5.49±0.46%) and C (HbA_1c: 8.83±1.20%),while groups B (HbA_1c:5.55±0.19%) andD (HbA_1c: 8.51±1.09%) were kept overnight on long-acting insulin without feed-back control of blood glucose before euglycaemic clamping. Thereby, pre-equilibration of blood glucose at 6.7 mmol/l was shown to normalize basal hepatic glucose production (A: 2.27±0.48; C 2.50±0.57 mg · kg^–1 · min^–1) despite different HbA_1c values, whereas basal hepatic glucose production stayed elevated in groups B (3.09±0.38 mg · kg^–1 · min^–1) and D (3.21±0.58 mg · kg^–1 · min^–1) with poor actual glycaemia (B: 10.9±4.6; D: 12.1±4.6 mmol/l). To restitute peripheral glucose utilisation close to normal (healthy subjects: 13.99±2.13; A: 12.12±2.67; B: 8.72±3.0; C: 10.27±1.69; D: 7.10±2.31 mg · kg^–1 · min^–1; healthy subjects vs A: NS; healthy subjects vs B, C, D: p<0.05) both long-term (HbA_1c<5.8%) and acute nearnormoglycaemia by 12-h i. v. insulin pre-treatment were required (group A). We conclude that good long-term glucose control per se is unable to normalize hepatic and peripheral glucose metabolism in Type 1 diabetic patients unless actual near-normoglycaemia is provided consistently, e.g. by i.v. overnight infusion of regular insulin.     

In vivo insulin action and muscle glycogen synthase activity in Type 2 (non-insulin-dependent) diabetes mellitus: effects of diet treatment

Summary Insulin resistant glucose metabolism is a key element in the pathogenesis of Type 2 (non-insulin-dependent) diabetes mellitus. Insulin resistance may be of both primary (genetic) and secondary (metabolic) origin. Before and after diet-induced improvement of glycaemic control seven obese patients with newly-diagnosed Type 2 diabetes were studied with the euglycaemic clamp technique in combination with indirect calorimetry and forearm glucose balance. Muscle biopsies were obtained in the basal state and again after 3 h of hyperinsulinaemia (200 mU/l) for studies of insulin receptor and glycogen synthase activities. Similar studies were performed in seven matched control subjects. Insulin-stimulated glucose utilization improved from 110±11 to 183±23 mg·m^–2·min^–1 (p<0.03); control subjects: 219+23 mg·m^–2·min^–1 (p=NS, vs post-diet Type 2 diabetes). Nonoxidative glucose disposal increased from 74±17 to 138+19 mg·m^–2·min^–1 (p<0.03), control subjects: 159±22 mg· m^–2·m^–1 (p=NS, vs post-diet Type 2 diabetic patients). Forearm blood glucose uptake during hyperinsulinaemia increased from 1.58±0.54 to 3.35±0.23 mol·l^–1·min^–1 (p<0.05), control subjects: 2.99±0.86 mol·l^–1·min^–1 (p=NS, vs post-diet Type 2 diabetes). After diet therapy the increase in insulin sensitivity correlated with reductions in fasting plasma glucose levels (r=0.97, p<0.001), reductions in serum fructosamine (r=0.77, p<0.05), and weight loss (r=0.78, p<0.05). Values of muscle glycogen synthase sensitivity to glucose 6-phosphate (A_0.5 for glucose 6-phosphate) were similar in the basal state. However, insulin stimulation of glycogen synthase was more pronounced after diet treatment (A_0.5: 0.43±0.06 (before) vs 0.30±0.04 mmol/l (after); p<0.03; control subjects: 0.22±0.03 mmol/l). Muscle insulin receptor binding and kinase activity were similar before and after diet treatment and comparable to values in the control group. The data suggest that impaired insulin stimulation of in vivo glucose turn-over and muscle glycogen synthase activity tend to be restored during successful diet treatment of patients with Type 2 diabetes.     

Modulation of hepatic glucose production by non-esterified fatty acids in Type 2 (non-insulin-dependent) diabetes mellitus

Summary To study the effect of changes in plasma non-esterified fatty acid concentration on suppression of hepatic glucose production by insulin eight Type 2 (non-insulin-dependent) diabetic patients participated in three euglycaemic, hyperinsulinaemic (108pmol · m^2–1 · min^–1) clamp studies combined with indirect calorimetry and infusion of [3-³H]-glucose and [1-¹⁴C]palmitate; (1) a control experiment with infusion of NaCl 154 mmol/l, (2) heparin was infused together with insulin, and (3) an antilipolytic agent, Acipimox, was administered at the beginning of the experiment. Six healthy volunteers participated in the control experiment. Plasma non-esterified fatty acid concentrations during the insulin clamp were in diabetic patients: (1) 151±36 mol/1, (2) 949±178 mol/l, and (3) 65±9 mol/l; in healthy control subjects 93±13 mol/l. Non-esterified fatty acid transport rate, oxidation and non-oxidative metabolism were significantly higher during the heparin than during the Acipimox experiment (p<0.001). Suppression of hepatic glucose production by insulin was impaired in the diabetic compared to control subjects (255±42 vs 51±29 mol/min, p<0.01). Infusion of heparin did not affect the suppression of hepatic glucose production by insulin (231±49 mol/min), whereas Acipimox significantly enhanced the suppression (21±53 mol/min, p<0.001 vs 154 mmol/l NaCl experiment). We conclude that insulin-mediated suppression of hepatic glucose production is not affected by increased non-esterified fatty acid availability. In contrast, decreased non-esterified fatty acid availability enhances the suppression of hepatic glucose production by insulin.     

Effects of islet amyloid polypeptide on hepatic insulin resistance and glucose production in the isolated perfused rat liver

Summary The impact of (pancreatic) islet amyloid polypeptide on glucose metabolism and insulin sensitivity was examined in isolated rat livers perfused in a non-recirculating system. Continuous infusion of 10^–7mol/l islet amyloid polypeptide affected neither basal nor glucagon (10^–9 mol/l)-stimulated glucose output by livers from fed rats, but it did increase the hepatic cyclic AMP release within 44 min (7.91±12.07 vs control: 0.07±0.03 pmol·100 g body weight^–1). The effect of the peptide on the ability of insulin to inhibit glucagon-induced hepatic glycogenolysis was measured in three experimental groups (n = 6). As expected glucagon (7×10^–11 mol/l) increased integral hepatic glucose release within 84 min (763.4±161.7 vs –25.7±73.2 mol · 100 g body weight^–1 in the control group, p<0.001), while insulin (100 mU/l) decreased the glucagon-stimulated glucose production (395.2±180.0 mol·100 g body weight^–1, p<0.01). Simultaneous infusion of 10^–7 mol/l islet amyloid polypeptide however, was not able to reverse insulin-dependent inhibition of glucagon-stimulated hepatic glucose output (370.0±102.5 mol·100 g body weight^–1, NS) or to enhance lactate-induced gluconeogenesis of livers from 24 h fasted rats (n = 8). The glucose production stimulated by 10^–9 mol/l glucagon was slightly greater in islet amyloid polypeptide-pre-treated livers than in a control group without addition of islet amyloid polypeptide (5 min: 3.60±3.36 vs 1.67±1.28 mol·min^–1·100 g body weight^–1). These results suggest that islet amyloid polypeptide neither directly affects hepatic glycogenolysis nor causes insulin resistance to hormone-sensitive glucose production, but may increase the size of the hepatic glycogen pool by enhancing gluconeogenesis.