Albumin excretion rate levels in non-diabetic offspring of NIDDM patients with and without nephropathy

Summary Familial clustering of diabetic nephropathy points to genetic susceptibility. The observation that in non-diabetic subjects microalbuminuria occurs more frequently in the presence of a parental history of diabetes supports this hypothesis. However, the role of inherited factors is poorly understood in non-insulin dependent diabetes mellitus (NIDDM). This study investigated the albumin excretion rate in non-diabetic offspring of NIDDM patients with increased albumin excretion rate (>20 g/min) or normal albumin excretion rate (<20 g/min). We recruited 20 offspring of NIDDM patients with increased albumin excretion rate (A-off) and 20 offspring of NIDDM patients with normal albumin excretion rate (N-off), matched for age, sex, body mass index, blood pressure and estimated protein intake. All offspring were normotensive, had normal creatinine clearance, normal glucose tolerance and sterile urine collection. Albumin excretion rate was measured on three sterile overnight urine collections and median values were used for calculations. Albumin excretion rate was significantly higher in A-off than in N-off (7.7±1.2 vs 3.4±0.6 g/min p<0.01) and significantly related to parents' albumin excretion rate (p<0.01, r=0.53). These results suggest that an increased glomerular permeability is present in non-diabetic offspring of NIDDM patients with increased albumin excretion rate.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - AER albumin excretion rate - OGTT oral glucose tolerance test - A-off offspring of NIDDM patients with increased albumin excretion rate - N-off offspring of NIDDM patients without increased albumin excretion rate     

Albumin excretion and vascular deaths in NIDDM

Summary Non-insulin-dependent diabetes mellitus (NIDDM) is associated with premature mortality, generally thought to be exaggerated in patients with microalbuminuria. This prospective 8-year follow-up study aimed to determine outcome, mortality and cause of death in NIDDM patients with abnormal urinary albumin excretion compared to those with normal albumin excretion. We recruited 153 NIDDM patients with abnormal urinary albumin excretion and 153 control subjects with albumin excretion within the normal non-diabetic range, matched for age, sex and duration of diabetes, from three University hospital diabetic clinics in Newcastle upon Tyne. The outcome measures were status at follow-up, mortality and cause of death. Subjects with abnormal albumin excretion had a significantly higher 8-year mortality than matched control subjects (Odds Ratio 1.47, p=0.02; 108 vs 66 per 1000 person years follow-up, p<0.001). This difference was seen at all levels of abnormal albumin excretion, from just outside the normal range (10.6–29.9 g/min: 104 vs 61 per 1000 person years follow-up, p<0.001) to more conventional definitions of microalbuminuria (30 g/min: 111 vs 71 per 1000 person years follow-up, p<0.01). Those with abnormal albumin excretion had an excess of vascular deaths compared to matched control subjects (Odds Ratio 1.70, p = 0.009), again at different levels of albumin excretion (10.6–29.9 g/min p<0.01, 30–150 g/min p<0.05). On multivariate analysis, age, initial ischaemic heart disease and initial albumin excretion rates were independent predictors of death from all causes. Even a minor elevation of albumin excretion above the normal non-diabetic range is associated with excess mortality from vascular causes in NIDDM.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - AER albumin excretion rate - SBP systolic blood pressure - DBP diastolic blood pressure - CI confidence interval - MAP mean arterial pressure     

Sulphonylurea stimulates glucose uptake in rats through an ATP-sensitive K+ channel dependent mechanism

Summary We studied the effect of gliclazide, a second-generation sulphonylurea, on rat skeletal muscle glucose uptake using perfused hindquarter muscle preparations. Gliclazide at concentrations of 10 to 1000 g/ml increased (p<0.05) the basal glucose uptake. The effect of gliclazide on glucose uptake was immediate and dose-dependent, reaching a plateau at a concentration of 300 g/ml; the half-maximal effect was obtained between 25 and 50 g/ml. The glucose uptake stimulated by gliclazide (300–1000 g/ ml) did not differ from that achieved by 10^–9 mol/l insulin, and was lower (p<0.05) than that obtained with 10^–7 mol/l insulin. The combination of gliclazide (300 g/ml) and 10^–9 mol/l insulin produced an increase in glucose uptake (7.7±0.6 mol · g^–1 · h^–1, n=8, mean±SEM) which was higher (p<0.05) than that achieved with 10^–9 mol/l insulin (5.6±0.7 mol · g^–1 · h^–1, n=11) and not different from that obtained with 10^–7 mol/l insulin (9.8±1.0 mol · g^–1 · h^–1, n=11). Diazoxide (100 mol/l), an ATP-sensitive K⁺ channel opener, reversed the stimulatory effect of gliclazide (100 g/ml) on muscle glucose uptake from 3.1±0.4 to 0.5±0.2 mol · g^–1 · h^–1, (n=7, p<0.001). The addition of diazoxide prior to gliclazide into the perfusion medium blocked the gliclazide-induced glucose uptake by the hindquarter muscle preparations. In conclusion, gliclazide alone has an immediate stimulatory effect on glucose uptake by skeletal muscle and together with insulin has an additive effect on muscle glucose uptake. The effect of gliclazide on muscle glucose uptake seems to be due to the inhibition of ATP-sensitive K⁺ channels.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - GLUT glucose transporter     

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     

Effect of 14 days' subcutaneous administration of the human amylin analogue,pramlintide (AC137), on an intravenous insulin challenge and response to a standard liquid meal in patients with IDDM

Summary Individuals with insulin-dependent diabetes mellitus (IDDM or type 1 diabetes) are deficient in both insulin and amylin, peptides secreted by the beta cell. We have investigated the effects of amylin replacement therapy employing the human amylin analogue, pramlintide (25, 28, 29-pro-human amylin, previously referred to as AC137), upon the responses to a standardized insulin infusion (40 mU · kg^–1 · h^–1) for 100 min and a liquid Sustacal meal (360 kcal) in 84 healthy IDDM patients. Following baseline evaluations, patients were randomly assigned to receive subcutaneous injections of placebo, 30, 100 or 300 g pramlintide 30 min before meals for 14 days. There was no meaningful difference between adverse events reported by the 30-g pramlintide and the placebo groups, but ten subjects withdrew due to nausea, eight of these in the 300-g dose group. Peak plasma pramlintide concentrations for the 30-g group were 21±3 and 29±5 pmol/l on Days 1 and 14, respectively. These values are similar to postprandial plasma amylin concentrations in normal volunteers. The plasma glucose, free insulin, glucagon, epinephrine and norepinephrine concentrations during the insulin infusion test before and after therapy were identical in each of the groups. Prior to pramlintide therapy, Sustacal ingestion produced a 4.0–4.8 mmol/l rise in plasma glucose concentrations in each of the groups. Pramlintide therapy reduced postprandial hyperglycaemia as reflected by the 3-h incremental AUC_glucose (AUC_glucose above or below fasting glucose concentration) Day 1 vs Day 14: 30 g, 322±92 vs –38±161 mmol/l · min, p=0.010; 100 g, 317±92 vs –39±76 mmol/l · min, p=0.001; and 300 g, 268±96 vs –245±189 mmol/l · min, p=0.077. Thus, pramlintide therapy with these regimens did not appear to impair either in vivo insulin action or the counter-regulatory response to hypoglycaemia but did show a clear effect of blunting postprandial hyperglycaemia following a standardized meal.Abbreviations IDDM Insulin-dependent diabetes mellitus - AUC_glucose area under the plasma glucose concentration curve - ANOVA analysis of variance     

Comparison of the effects of human recombinant insulin-like growth factor I and insulin on plasma amino acid concentrations and leucine kinetics in humans

Summary We examined the effects of recombinant human insulin-like growth factor I (IGF-I) and insulin on the plasma amino acid (AA) profile and leucine kinetics in eight normal subjects. IGF-I was infused at 52 pmol·kg^–1·min^–1, in combination with prime-continuous [1-¹⁴C] leucine infusion, to obtain steady-state plasma concentrations of total (54±3 nmol/l) and free (7.3±1 nmol/l) IGF-I (study 1). In response to IGF-I, plasma AA levels declined by 37±3% (1975±198 to 1368±120 mol/l) and total branched chain amino acids (BCAA) declined by 34±3% (390±21 to 256±13 mol/l). This hypoaminoacidaemic effect was associated with a decline in endogenous leucine flux of 17±2% (1.88±0.05 to 1.57±0.04 mol·kg^–1·min^–1) and leucine oxidation of 17±1% (0.31±0.02 vs 0.26±0.02 mol·kg^–1·min^–1) (both p<0.01 vs basal). The same subjects underwent a second study (study 2) in which insulin was infused at 6.22 pmol·kg^–1·min^–1 to obtain a steady-state plasma insulin concentration of 530±25 pmol/l while maintaining euglycaemia. The infusion rate was designed to match the declines in plasma BCAA levels and leucine turnover observed during IGF-I infusion. The rates of glucose infusion necessary to maintain euglycaemia during IGF-I and insulin infusion were 4.9±1.0 and 7.8±0.6 mg·kg^–1 ·min^–1, respectively. During insulin infusion total BCAA declined by 39% from 369±23 to 226±20 mol/l, leucine flux declined by 16±2% from 1.90±0.05 to 1.61±0.03 mol·kg^–1·min^–1, and leucine oxidation declined by 19±2% from 0.32±0.02 to 0.26±0.02 mol·kg^–1·min^–1. On a molar basis IGF-I was 7.3% as potent as insulin in inhibiting proteolysis. These results demonstrate that in humans: (i) the hypoaminoacidaemic response to IGF-I can be entirely ascribed to the inhibition of proteolysis; (ii) qualitatively, the effects of IGF-I and insulin on plasma AA profile and protein metabolism are similar; (iii) quantitatively, IGF-I is 14-fold less potent than insulin in suppressing protein degradation.Abbreviations IGF-I Insulin-like growth factor I - AA aminoacid - BCAA branched chain amino acids - KIC alphaketoisocaproate - ELF endogenous leucine flux - NOLD non-oxidative leucine disposal     

The relationship of urinary albumin excretion rate to ambulatory blood pressure and erythrocyte sodium-lithium countertransport in NIDDM

Summary Increased erythrocyte sodium-lithium countertransport rate is found in non-diabetic subjects with essential hypertension, and in insulin-dependent diabetic subjects with nephropathy. However, relationships between these variables in non-insulin-dependent diabetic subjects are ill-defined. In order to characterise the relationships between blood pressure, urinary albumin excretion, and erythrocyte sodium-lithium countertransport, 66 subjects with non-insulin-dependent diabetes were studied. Urinary albumin excretion rate correlated with mean 24-h ambulatory systolic blood pressure (r=0.57; p<0.001), but not with sodium-lithium countertransport (r=0.06; p=0.31). No significant relationship was observed between 24-h systolic blood pressure and erythrocyte sodium-lithium countertransport (r = 0.16; p=0.17). The principal differences between microalbuminuric and normoalbuminuric subjects (albumin excretion rate >15 g·min^–1 [n=20], and <15 g·min^–1, [n=46]) were: higher 24-h systolic blood pressure (145.9 [16.8] mm Hg vs 131.9 [16.8] mm Hg; p=0.006), nocturnal heart rate (72.4 [8.9] vs 67.4 [8.9] beats·min^–1; p=0.042), and HbA₁ (11.3 [1.5]% vs 10.1 [2.0]%; p=0.028), and a longer median duration of diabetes (10.0 vs 5.0 years; p = 0.02). In contrast, there was no significant difference in sodium-lithium countertransport rate between microalbuminuric (0.41 [0.18] mmol·l^–1·h^–1) and normoalbuminuric subjects (0.39 [0.15] mmol·l^–1· h^–1; p=0.687). In multiple regression analysis controlling for race, age, body mass index and HbA₁, the significant determinants of albumin excretion rate were 24-h systolic blood pressure (B [regression coefficient]=0.029, SE[B] [standard error of B]=0.009, t=2.95, p=0.005), duration of diabetes (B=0.430, SE[B]=0.169, t=2.54, p=0.016) and male gender (B=–1.170, SE[B]=0.457, t=–2.56, p=0.015). In conclusion, albumin excretion rates in non-insulin-dependent diabetic subjects are linked to hypertension and glycaemic exposure, but show no relationship to erythrocyte sodium-lithium countertransport.Abbreviations IDDM Insulin-dependent diabetes mellitus - NIDDM non-insulin dependent diabetes mellitus - SLC sodium lithium countertransport - AER albumin excretion rate - B regression - SE coefficient; standard error of B     

Effects of ingested fructose and infused glucagon on endogenous glucose production in obese NIDDM patients,obese non-diabetic subjects,and healthy subjects

Summary Increased endogenous glucose production (EGP) and gluconeogenesis contribute to the pathogenesis of hyperglycaemia in non-insulin-dependent diabetes mellitus (NIDDM). In healthy subjects, however, EGP remains constant during administration of gluconeogenic precursors. This study was performed in order to determine whether administration of fructose increases EGP in obese NIDDM patients and obese non-diabetic subjects. Eight young healthy lean subjects, eight middle-aged obese NIDDM patients and seven middle-aged obese non-diabetic subjects were studied during hourly ingestion of ¹³C fructose (0.3 g · kg fat free mass^–1 · h^–1) for 3 h. Fructose failed to increase EGP (measured with 6,6 ²H glucose) in NIDDM (17.7±1.9 mol · kg fat free mass^–1 · min^–1 basal vs 15.9±0.9 after fructose), in obese non-diabetic subjects (12.1±0.5 basal vs 13.1±0.5 after fructose) and in lean healthy subjects (13.3±0.5 basal vs 13.8±0.6 after fructose) although ¹³C glucose synthesis contributed 73.2% of EGP in lean subjects, 62.6% in obese non-diabetic subjects, and 52.8% in obese NIDDM patients. Since glucagon may play an important role in the development of hyperglycaemia in NIDDM, healthy subjects were also studied during ¹³C fructose ingestion + hyperglucagonaemia (232±9 ng/l) and during hyperglucagonaemia alone. EGP increased by 19.8% with ingestion of fructose + glucagon (p<0.05) but remained unchanged during administration of fructose or glucagon alone. The plasma ¹³C glucose enrichment was identical after fructose ingestion both with and without glucagon, indicating that the contribution of fructose gluconeogenesis to the glucose 6-phosphate pool was identical in these two conditions. We concluded that during fructose administration: 1) gluconeogenesis is increased, but EGP remains constant in NIDDM, obese non-diabetic, and lean individuals; 2) in lean individuals, both an increased glucagonaemia and an enhanced supply of gluconeogenic precursors are required to increase EGP; this increase in EGP occurs without changes in the relative proportion of glucose 6-phosphate production from fructose and from other sources (i. e. glycogenolysis + gluconeogenesis from non-fructose precursors).Abbreviations EGP Endogenous glucose production - CHO carbohydrate - APE atom percent excess - GRd glucose rate of disappearance - FFM fat-free mass     

Insulin sensitivity,insulin secretion and glucose effectiveness in diabetic and non-diabetic cirrhotic patients

Summary In cirrhotic patients with normal fasting glucose levels both insulin insensitivity and a blunted early insulin response to oral glucose are important determinants of the degree of intolerance to oral glucose. It is not known whether the ability of hyperglycaemia per se to enhance glucose disposal (glucose effectiveness) is also impaired. It is also unclear whether overt diabetes is due to (1) more marked insulin insensitivity; (2) impaired insulin secretion; (3) reduced glucose effectiveness; or (4) a combination of these mechanisms. We used the minimal model to analyse the results of a 3-h intravenous glucose tolerance test to assess glucose effectiveness, insulin sensitivity and insulin responses in 12 non-diabetic cirrhotic patients, 8 diabetic cirrhotic patients and 10 normal control subjects. Fasting blood glucose levels were 4.8±0.2, 7.5±0.6 and 4.7±0.1 mmol/l, respectively. Fasting insulin and C-peptide levels were higher in both cirrhotic patient groups compared with control subjects. The glucose clearance between 6 and 19 min after i.v. glucose was lower in both cirrhotic groups (non-diabetic, 1.56±0.14, diabetic, 0.76±0.06, control subjects, 2.49±0.16 min^–1%, both p<0.001 vs control subjects). Serum insulin peaked at 3 and 23 min in the non-diabetic cirrhotic patients and control subjects; both peaks were higher in the non-diabetic cirrhotic patients and showed a delayed return to basal levels. In the diabetic cirrhotic patients, the first phase insulin and C-peptide response to i.v. glucose was absent; their early (22–27 min) incremental insulin response to i. v. tolbutamide was however similar to that of control subjects but 43% lower than in the non-diabetic cirrhotic patients (p<0.05). Insulin sensitivity was markedly reduced in both cirrhotic groups (non-diabetic, 1.11±0.24×10^–4, diabetic, 0.33±0.53×10^–4, control subjects, 4.37±0.53×10^–4 min^–1 per mU·l^–1, both p<0.001 vs controls). Glucose effectiveness was normal in the non-diabetic cirrhotic patients but 29% lower in the diabetic group. It would appear that overt diabetes develops in those cirrhotic patients who in addition to insulin insensitivity have a marked impairment of insulin secretion. An associated reduction in glucose effectiveness may be a contributory factor.     

A quantitative ultrastructural study of juxtaglomerular arterioles in IDDM patients with micro- and normoalbuminuria

Summary Hyalinization of juxtaglomerular arterioles is prominent in advanced diabetic nephropathy and may have important functional consequences. We studied the early stages of diabetic renal disease using kidney biopsy material from insulin-dependent diabetic patients, 8 with normal albumin excretion rate (<15 /min) and 16 with microalbuminuria (15–200 g/min). Ten living non-diabetic kidney donors served as a control group. Median duration of diabetes was 9.5 years (range 5–31) in patients with normoalbuminuria, and 12 years (7–22) in patients with microalbuminuria (p=0.27). The tissue was sectioned systematically, 1-m thick sections for light microscopy at 10-m intervals, and thin sections for electron microscopy taken at 60-m intervals. The arterioles were identified as afferent or efferent, and total profiles were photographed (magnification 7500×), providing a systematic independent sample for measurements using standard stereological methods. Patients with microalbuminuria had significantly increased arteriole parameters compared with the control group: for afferent and efferent arterioles the volume fraction of matrix/media, means and (coefficient of variation, CV), was 0.47 (0.16) vs 0.33 (0.19) (p=0.0009), and 0.62 (0.14) vs 0.45 (0.23) (p=0.0004) and matrix-T, expressing amount of matrix per unit arteriolar surface, 2.38 (0.38) m vs 1.44 (0.30) m (p=0.004), and 1.62 (0.28) m vs 1.03 (0.34) (p=0.0009). Patients with normoalbuminuria showed no significant differences from the control group, and had lower values than microalbuminuric patients for all parameters except the afferent matrix-T. In the normoalbuminuric group a correlation was found between parameters for afferent arterioles and those for glomerular structure. In conclusion there is arteriolar accumulation of extracellular material in the early phase of diabetic nephropathy, concomitant with early glomerulopathy.Abbreviations IDDM Insulin-dependent diabetes mellitus - GFR glomerular filtration rate - AER albumin excretion rate - matrix-T matrix thickness - ND non-diabetic subjects - D_NA IDDM patients with normal albumin excretion rate - D_MI IDDM patients with microalbuminuria - RPF renal plasma flow - CV coefficient of variation     

Effect of insulin on GLUT-4 mRNA and protein concentrations in skeletal muscle of patients with NIDDM and their first-degree relatives

Summary We examined whether insulin resistance, i. e. impaired insulin stimulated glucose uptake in NIDDM patients and their first-degree relatives is associated with alterations in the effect of insulin on the expression of the GLUT-4 gene in skeletal muscle in vivo. Levels of GLUT-4 mRNA and protein were measured in muscle biopsies taken before and after a euglycaemic insulin clamp from 14 NIDDM patients, 13 of their first-degree relatives and 17 control subjects. Insulin stimulated glucose uptake was decreased in the diabetic subjects (19.8±3.0 mol · kg LBM^–1 · min^–1, both p<0.001) compared with control subjects (44.1±2.5 mol · kg LBM^–1 · min^–1) and relatives (39.9±3.3 mol · kg LBM^–1 · min^–1). Basal GLUT-4 mRNA levels were significantly higher in diabetic subjects and relatives compared to control subjects (99±8 and 108±9 pg/g RNA vs 68±5 pg/g RNA; both p<0.01). Insulin increased GLUT-4 mRNA levels in all control subjects (from 68±5 to 92±6 pg/ug RNA; p<0.0001), but not in the diabetic patients (from 99±8 to 90±8 pg/g RNA, NS), or their relatives (from 94±9 to 101±11 pg/g RNA, NS). In the relatives, individual basal GLUT-4 mRNA concentrations varied between 55 and 137 pg/g RNA. Insulin-resistant (n=6, mean glucose uptake rate=30.6±3.4 mol · kg LBM^–1 · min^–1) but not insulin-sensitive relatives (n=7, mean glucose uptake rate=47.4±3.2 mol · kg LBM^–1 · min^–1) had higher basal GLUT-4 mRNA concentrations compared to control subjects (108±9 vs 68±5 pg/ug RNA, p<0.01). GLUT-4 protein content in muscle did not differ between the groups in the basal state and remained unchanged in all groups after insulin infusion. Neither insulin-stimulated GLUT-4 mRNA nor protein concentrations correlated with insulin-stimulated glucose uptake in any of the groups studied. We conclude, that impaired glucose uptake in NIDDM is not related to insulin-stimulated GLUT-4 mRNA or protein concentrations. Acute stimulation of GLUT-4 mRNA by insulin is altered in skeletal muscle of NIDDM patients and their first-degree relatives. This might be a consequence of chronic hyperinsulinaemia elevating basal GLUT-4 mRNA concentrations rather than the cause of insulin resistance.Abbreviations NIDDM Non-insulin dependent diabetes mellitus - GLUT-4 glucose transporter 4 - LBM lean body mass - IDDM insulin-dependent diabetes mellitus     

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     

Excessive glucose production,rather than insulin resistance,accounts for hyperglycaemia in recent-onset streptozotocin-diabetic rats

Summary Glucose production and utilization and activities of key enzymes involved in liver and muscle glucose metabolism were studied in post-absorptive streptozotocin-diabetic rats after 12 h of severe hyperglycaemia (17.5±0.5 mmol/l) and insulinopenia (5±1 U/ml). Basal glucose production was increased: 36.6±3.0 mg·kg·min^–1, vs 24.4±2.5 in controls (p<0.05); liver glycogen concentration was decreased by 40% (p<0.05); liver phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities were increased by 375 and 156%, respectively (p<0.001 and <0.01). During a euglycaemic clamp at a plasma insulin level of 200 U/ml, glucose production was totally suppressed in controls, but persisted at 20% of basal in diabetic rats. In these rats, glucose production was suppressed at a plasma insulin level of 2500 U/ml. Basal whole body glucose utilization rate, 2-deoxy-1-[³H]-d-glucose ([³H]-2dG) uptake by muscles and muscle glycogen concentrations were similar in both groups, as well as total and active forms of pyruvate dehydrogenase and glycogen synthase activities. During the euglycaemic clamp, the total body glucose utilization rates and [³H]-2dG uptake by muscles were similar in control and diabetic rats at a plasma insulin level of 200 U/ ml, but lower in diabetic rats at a plasma insulin level of 2500 U/ml. We conclude 1) in recent-onset severely insulinopenic rats, an excessive glucose production via gluconeogenesis prevailed, mainly accounting for the concomitant hyperglycaemia. This excess glucose output cannot be attributed to liver insulin resistance: the gluconeogenic pathway is physiologically less sensitive than glycogenolysis to the inhibition by insulin. 2) Glucose utilization was apparently normal under hyperglycaemic conditions and at a lower insulin plateau of the euglycaemic clamp but suboptimal in the presence of maximal insulin concentrations, suggesting an early appearance of peripheral insulin resistance.Abbreviations STZ streptozotocin - IDDM insulin-dependent diabetes mellitus - PEPCK phosphoenolpyruvate carboxykinase - PDH pyruvate dehydrogenase - G6Pase glucose 6-phosphatase - NEFA non-esterified fatty acids - DCA sodium dichloroacetate     

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     

Reduced glycogen synthase activity in skeletal muscle from obese patients with and without Type 2 (non-insulin-dependent) diabetes mellitus

Summary In order to evaluate the importance of a defect in insulin mediated non-oxidative glucose metabolism and glycogen synthase activity in skeletal muscles in obese subjects with and without Type 2 (non-insulin-dependent) diabetes mellitus we studied: 10 lean and 10 obese control subjects and 12 obese diabetic patients using the euglycaemic hyperinsulinaemic clamp technique (basal, 20 mU · (m²)^–1 · min^–1, 80mU·(m²)^–1·min^–1) in combination with indirect calorimetry. Muscle biopsies were taken from m. vastus lateralis at each insulin level. We found that non-oxidative glucose metabolism could be stimulated by insulin in all three groups (p<0.01). The values obtained at the highest insulin levels (around 140 U/ml) were lower in both obese groups compared to the lean control subjects (118±21, 185±31, 249±14 mg·(m²)^–1·min^–1 (p< 0.01)). Insulin stimulation of the glycogen synthase activity at a glucose-6-phosphate concentration of 0.1 mmol/l was absent in both obese groups, while activities increased significantly in the lean control subjects (19.6±4.2% to 45.6±6.8%, p< 0.01). Glycogen synthase activities at the highest insulin concentrations only differed significantly between lean control subjects and obese diabetic patients (45±7% and 31±5%, p< 0.05). We conclude that insulin resistance in peripheral tissues in obese subjects with and without Type 2 diabetes may be partly explained by a reduced insulin mediated non-oxidative glucose metabolism and that this abnormality might be due to an absent insulin stimulation of glycogen synthase in skeletal muscles. This enzyme defect is correlated to obesity itself.     

Lack of relationship between muscle sympathetic nerve activity and skeletal muscle vasodilation in response to insulin infusion

Summary Increases in plasma insulin concentration result in vasodilation in skeletal muscle but also in an increase in muscle sympathetic nerve activity (MSNA) which is thought to cause vasoconstriction. The increase in MSNA could therefore be a response to vasodilation (baroreflex), or MSNA could cause vasodilation via putative sympathetic vasodilatory fibres. To examine the relationship between vasodilation, MSNA and insulin action we studied nine non-diabetic Pima Indian men (age 29±7 years, weight 91±19 kg, 29±6% body fat, mean ± SD) during sequential euglycaemic clamps at low and high insulin doses (80 and 600 mU · m^–2 min^–1). Leg blood flow was measured by thermodilution, leg glucose uptake by the balance technique, arterial pressure by invasive monitoring and MSNA by microneurography of the peroneal nerve. Whole body glucose uptake (M) ranged from 6.7 to 48.3 during low dose and from 9.4 to 67.7 mol · kg fat free mass^–1 · min^–1 during high dose insulin infusion. At both insulin doses, incremental leg blood flow correlated with M (r=0.63 and 0.71, respectively). No correlation was found between incremental MSNA and leg blood flow, M or leg glucose uptake. Blood pressure was unchanged throughout the study. MSNA increased after 15–40 min of insulin infusion in all the subjects, whereas leg blood flow started to increase only after 45 min in the most insulin sensitive but not in the most insulin resistant subjects. Thus, insulin stimulates MSNA more rapidly than vasodilation. In conclusion, insulin-mediated MSNA: 1) is neither a response to nor a cause of the vasodilation observed in insulin sensitive men, 2) has no net pressor effect even in the most insulin resistant men in whom insulin-mediated vasodilation was impaired. We conclude that the effect of insulin to stimulate MSNA is dissociated from its acute haemodynamic action.Abbreviations MSNA Muscle sympathetic nerve activity - LBF leg blood flow - M whole body glucose uptake - LGU leg glucose uptake - NIDDM non-insulin-dependent diabetes mellitus - EDNO endothelium derived nitric oxide, FFM, fat free mass - MAP mean arterial blood pressure     

Effect of metformin on insulin-stimulated glucose transport in isolated skeletal muscle obtained from patients with NIDDM

Summary Metformin has been demonstrated to lower blood glucose in vivo by a mechanism which increases peripheral glucose uptake. Furthermore, the therapeutic concentration of metformin has been estimated to be in the order of 0.01 mmol/l. We investigated the effect of metformin on insulin-stimulated 3-0-methylglucose transport in isolated skeletal muscle obtained from seven patients with non-insulin-dependent diabetes mellitus (NIDDM) and from eight healthy subjects. Whole body insulin-mediated glucose utilization was decreased by 45% (p<0.05) in the diabetic subjects when studied at 8 mmol/l glucose, compared to the healthy subjects studied at 5 mmol/l glucose. Metformin, at concentrations of 0.1 and 0.01 mmol/l, had no effect on basal or insulin-stimulated (100 U/ml) glucose transport in muscle strips from either of the groups. However, the two control subjects and three patients with NIDDM which displayed a low rate of insulin-mediated glucose utilization (<20 mol·kg^–1·min^–1), as well as in vitro insulin resistance, demonstrated increased insulin-stimulated glucose transport in the presence of metformin at 0.1 mmol/l (p<0.05). In conclusion, the concentration of metformin resulting in a potentiating effect on insulin-stimulated glucose transport in insulin-resistant human skeletal muscle is 10-fold higher than the therapeutic concentrations administered to patients with NIDDM. Thus, it is conceivable that the hypoglycaemic effect of metformin in vivo may be due to an accumulation of the drug in the extracellular space of skeletal muscle, or to an effect of the drug distal to the glucose transport step.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - KHB Krebs-Henseleit's bicarbonate buffer - HEPES N-2, hydroxyethyl-piperazine-N'-2-ethanesulfonic acid - BSA bovine serum albumin - RIA radioimmunoassay - HbA_1c glycated haemoglobin A_1c - BMI body mass index (kg/m²) - GLUT 1 HepG2/erythrocyte - GLUT 4 insulin-regulatable glucose transporter     

Simple empirical assessment of Beta-cell function by a constant infusion of glucose test in normal and Type 2 (non-insulin-dependent) diabetic subjects

Summary The plasma insulin or C-peptide response to a 90-min constant glucose infusion 5 mg · kg ideal body weight^–1·min^–1 provides Beta-cell assessment comparable to more intensive methods. In 14 diet-treated Type 2 (non-insulin-dependent) diabetic subjects and 12 non-diabetic subjects, plasma insulin and C-peptide concentrations gave near linear plots against simultaneous glucose values. The glucose-insulin and glucose-C-peptide vectors (G-I and G-C vectors), could be extrapolated to predict insulin and C-peptide levels during a 12 mmol/l hyperglycaemic clamp. Predicted concentrations correlated with clamp concentrations, r = 0.94 and r = 0.98 respectively, p<0.001, validating the vectors as empirical glucose dose-response curves. The vector slopes correlated highly with % Beta, a mathematical model-derived measure of Beta-cell function using constant infusion of glucose model assessment, Spearman r = 0.95 and 0.93 for insulin and C-peptide, respectively. G-I vector slopes in 21 diet-treated Type 2 diabetic subjects with fasting glucose (mean +1 SD) 7.5±2,3 mmol/1, were lower than in 28 non-diabetic subjects, (geometric mean, 1 SD range, 8.4 pmol/mmol (3.3–21.0) and 25.1 pmol/mmol (14.3–44.1), p<0.001, respectively), indicating an impaired Beta-cell response. The G-I vector slopes correlated with obesity in both groups (r = 0.54 p<0.02 and 0.72, p<0.001 respectively), and, in 15 non-diabetic subjects, correlated inversely with insulin sensitivity as measured by a euglycaemic clamp (r = –0.66, p<0.01).Thus,Beta-cell function needs to be interpreted in relation to obesity/insulin resistance and, taking obesity into account, only 4 of 21 diabetic patients had Betacell function (G-I vector slope) in the non-diabetic range. The fasting plasma glucose in the diabetic subjects correlated inversely with the obesity-corrected G-I and G-C vector slopes (partial r = –0.57, p <0.01 and –0.86, p<0.001, respectively). The insulin or C-peptide response to the glucose infusion provides a direct empirical measure of the Beta-cell function, which can be interpreted in relation to obesity or to insulin resistance to assess underlying pancreatic responsiveness.     

Nitric oxide in ischaemic acute renal failure of streptozotocin diabetic rats

Summary Changes in nitric oxide (NO) levels were determined in ischaemic acute renal failure in streptozotocin-induced diabetes mellitus rats. Two weeks after streptozotocin administration and immediately after right nephrectomy, the left renal artery was occluded for 60 min. Similar procedures were carried out in non-diabetic rats. The nitrite (NO₂) + nitrate (NO₃) levels were measured in plasma and urine. The effects of chronic oral supplementation with l-arginine and an NO synthase inhibitor (N-omega-nitro-l-arginine) were also studied in both diabetic and non-diabetic rats before and after renal artery clamping. The rats with diabetic acute renal failure had a much lower creatinine clearance (90±22 l · min^–1 · 100 g body weight^–1, p<0.005), and higher fractional excretion of sodium (FENa)% (10.90±4.2, p<0.001) and protein excretion (2078±69 g/ml creatinine clearance, p<0.001) compared with the respective values in the non-diabetic groups (163±30; 1.46±86; 453.3±31). The plasma and urine NO₂ + NO₃ levels were significantly higher in the untreated diabetic rats compared with the untreated normal rats before ischaemia (p<0.001). The ischaemic acute renal failure in non-diabetic rats increased the plasma and urinary NO₂ + NO₃ excretion after ischaemia. The urinary excretion of these metabolites decreased significantly and their plasma levels remained unchanged in the ischaemic diabetic rats. The l-arginine administration resulted in a small but significantly higher creatinine clearance after clamping in the non-diabetic rats. The NO synthase inhibitor caused deterioration in renal function in all ischaemic and non-ischaemic groups. In summary, the greater vulnerability to ischaemia of the diabetic kidney seems to be associated with both impaired response to and impaired production of NO.Abbreviations IARF Ischaemic acute renal failure - STZ streptozotocin - NOSi nitric oxide synthase inhibitor - ARF acute renal failure - Ccr creatinine clearance - FENa% fractional excretion of sodium     

Insulin-like growth factor-I in man enhances lipid mobilization and oxidation induced by a growth hormone pulse

Summary Growth hormone (GH) secretion is suppressed during insulin-like growth factor-I (IGF-I) administration. The aim of the study was to examine whether IGF-I alters the metabolic response to a GH pulse. Seven healthy male subjects (age 27±4 years, BMI 21.8±1.7 kg/m²) were treated with NaCl 0.9% (saline) or IGF-I (8 g · kg^–1 · h^–1) for 5 days by continuous subcutaneous infusion in a randomized, crossover fashion while receiving an isocaloric diet (30 kcal · kg^–1 · day^–1). On the third treatment day an intravenous bolus of 0.5 U GH was administered. Forearm muscle metabolism was examined by measuring arterialized and deep venous blood samples, forearm blood flow by occlusion plethysmography and substrate oxidation by indirect calorimetry. IGF-I treatment significantly reduced insulin concentrations by 80% (p<0.02) and C-peptide levels by 78% (p<0.02), as assessed by area under the curve. Non-esterified fatty acid (NEFA), glycerol and 3-OH-butyrate levels were elevated and alanine concentration decreased. Forearm blood flow rose from 2.10±0.43 (saline) to 2.79±0.37 ml · 100ml^–1 · min^–1 (IGF-I) (p<0.02). GH-pulse: 10 h after i.v. GH injection serum GH peaked at 40.9±7.4 ng/ml. GH did not influence circulating levels of total IGFI, C-peptide, insulin or glucose, but caused a further increase in NEFA, glycerol and 3-OH-butyrate levels, indicating enhanced lipolysis and ketogenesis. This effect of GH was much more pronounced during IGF-I: NEFA rose from 702±267 (saline) and 885±236 (IGF-I) to 963±215 (saline) (p<0.05) and 1815±586 mol/l (IGF-I) (p<0.02), respectively; after 5 h, 3-OH-butyrate rose from 242±234 (saline) and 340±280 (IGF-I) to 678±638 (saline) (p<0.02) and 1115±578 mol/l (IGF-I) (p<0.02) respectively. After injection of GH, forearm uptake of 3-OH-butyrate was markedly elevated only in the subjects treated with IGF-I: from 44±195 to 300±370 after 20 min (p<0.03) and to 287±91 nmol · 100 ml^–1 · min^–1after 120 min (p<0.02). In conclusion, the lipolytic and ketogenic response to GH was grossly enhanced during IGF-I treatment, and utilization of ketone bodies by skeletal muscle was increased.Abbreviations AUC Area under the curve - C-peptide connecting peptide - EE energy expenditure - FFM fat-free mass - GH growth hormone - IGF-I insulin-like growth factor-I - NEFA non-esterified fatty acid - Ra rate of glucose appearance - Rd rate of glucose disposal - FGU forearm glucose uptake - CV coefficient of variation