Influence of severe diabetes mellitus early in pregnancy in the rat: effects on insulin sensitivity and insulin secretion in the offspring

Summary We studied the influence of severe diabetes early in pregnancy on insulin sensitivity and insulin secretion in the offspring. Diabetes (blood glucose >20 mmol/l) was induced in female Sprague-Dawley rats before mating. Diabetic dams were insulin treated during the second half of pregnancy (mean blood glucose 10.6 mmol/l). The offspring were reared by foster mothers. Offspring of both sexes were insulin resistant at four and seven months of age as evidenced by normal glucose tolerance after glucose (2 g/kg body weight intraperitoneally) concomitant with higher than normal rises in insulin levels. Regardless of fetal environment the male rats had higher glucose and insulin levels than the female rats. Insulin responses to glucose (27 mmol/l) in vitro in perfused pancreases were not increased by maternal diabetes, male gender or higher age. Conversely responses to 3-isobutyl-1-methylxanthine (1.0 mmol/l) were enhanced by all three conditions. The pancreatic content of insulin was only marginally affected by maternal diabetes. We conclude that severe diabetes during early pregnancy affects glucose homeostasis in the offspring primarily by diminishing insulin sensitivity and that susceptibility to this effect is not sex- or age-dependent.     

Effect of islet activating protein on glucose tolerance,insulin secretion and insulin responsiveness in the NZO mouse

Summary The effect of islet activating protein on glucose tolerance, insulin secretion and insulin responsiveness was studied in the NZO mouse, a model of non-insulin dependent diabetes and obesity. A single IV injection of 5 ng/g body weight islet activating protein markedly lowered plasma glucose and the glucose response to IP glucose administration, measured 5 days later (mean±SEM, plasma glucose levels 0, 10, 30 and 60 min after glucose 6.0±0.9, 14.6±1.3, 14.1±1.3 and 13.2±1.7 mmol/l in islet activating protein-treated NZO mice versus 12.8±1.6, 27.8±3.4, 34.7±4.1, 39.1±3.8 mmol/l in carrier-treated NZO mice). There was no difference in fasting plasma insulin levels between islet activating protein and carrier-treated mice. No response of plasma insulin to glucose occurred in the carrier-treated mice, but a highly significant insulin response to glucose was seen in the islet activating protein-treated mice. The in vitro responsiveness of pancreatic islets of islet activating protein-treated NZO mice to glucose was improved, and the inhibitory effect of adrenaline on insulin secretion was reduced. The in vivo hypoglycaemic response to exogenous insulin was not improved by islet activating protein and a demonstrated defect in the insulin sensitivity and responsiveness of glucose utilization by isolated soleus muscle was not reversed by islet activating protein treatment. It is concluded that islet activating protein is highly effective in improving glucose tolerance and insulin secretion in NZO mice, and that the improvement in glucose tolerance occurs without demonstrable improvement in the responsiveness to exogenous insulin or sensitivity of soleus muscle to insulin.     

Insulin resistance in Type 1 (insulin-dependent) diabetes: dependence on plasma insulin concentration

Summary Sensitivity to insulin in vivo was studied in six Type 1 diabetic patients without residual insulin secretion and without clinical insulin resistance, and in eight non-diabetic subjects, using the euglycaemic insulin clamp technique. Insulin was infused for four periods of 2 h sequentially at 0.5, 1.0, 2.0 and 5.0 mU · kg^-1 · min^-1; for each insulin infusion period the steady-state plasma free insulin levels were comparable in the diabetic and non-diabetic subjects. The mean ±SEM plasma glucose concentration was 4.9±0.03 mmol/l in the diabetic subjects (coefficient of variation of plasma glucose values: 5.7±0.7%) and 4.6±0.01 mmol/l in the control subjects (coefficient of variation: 5.1±0.6%). Insulin-mediated glucose disposal was lower in the diabetic than in the non-diabetic subjects at the two lower insulin infusion rates (mean±SEM = 2.03±0.27 versus 4.8±0.64 mg · kg^-1 · min^-1 at the first insulin infusion rate, p<0.01, and 5.59±0.59 versus 8.36±0.61 mg · kg^-1 · min^-1 at the second insulin infusion rate, p<0.01). However, insulin-induced glucose uptake did not differ significantly between the two groups at the third and fourth rates of insulin infusion. These results show that impaired insulin sensitivity in Type 1 diabetes is dependent on insulin concentration.     

Long-term gliclazide treatment improves the in vitro glucose-induced insulin release in rats with Type 2 (non-insulin-dependent) diabetes induced by neonatal streptozotocin

Summary Neonatal rats treated with streptozotocin on the day of birth (n0-STZ) or on day 5 (n5-STZ) exhibited when fully grown a very mild or frank basal hyperglycaemia respectively and a specific failure of insulin release in response to glucose. To determine whether short (1 day) or long-term (30 days) gliclazide treatment modifies the pancreatic insulin content and the B-cell response to secretagogues, diabetic rats were given oral gliclazide (10 mg/kg per day) and compared to control diabetic and non-diabetic rats. Insulin secretion in the isolated perfused pancreas was studied the day after the last gliclazide administration. In severely hyperglycaemic n5-STZ rats (plasma glucose levels >16 mmol/l) long-term gliclazide treatment did not lower the plasma glucose values, did not affect the pancreatic insulin stores, nor did it significantly modify the insulin release in vitro in response to glucose or arginine. In moderately hyperglycaemic n5-STZ rats (plasma glucose levels <16 mmol/l) the plasma glucose levels declined progressively reaching 8 mmol/l as a mean at the end of the gliclazide therapy. In the n5-STZ rats responsive to gliclazide the pancreatic insulin stores were increased twofold as compared to values in untreated n5-STZ rats, however, this difference did not reached significance and the pancreatic in sulin stores in the responsive gliclazide treated rats remained depleted by 76% compared to normal insulin stores. In the n0-STZ rats (very mild hyperglycaemia) the long-term gliclazide treatment did not significantly modify the plasma glucose levels or the pancreatic insulin stores. After the 30-day gliclazide therapy in both the n5-STZ gliclazide responder group and the n0-STZ group: (1) the in vitro glucose-induced insulin secretion was increased three to fivefold, (2) the response to arginine which was basically increased in the untreated diabetic rats was again amplified two to threefold, (3) the insulin release in response to gliclazide was unchanged. In conclusion, long-term gliclazide therapy augments stimulated insulin secretion in these two rat models of Type 2 (non-insulin-dependent) diabetes and does not induce any refractoriness to acute sulfonylurea stimulation. The improvement of B-cell function observed here was not related to the concomitant variations of hyperglycaemia and/or pancreatic insulin contentThis work was presented in part at the 24th Annual Meeting of the European Association of the Study of Diabetes, Paris, France, 5–8 September 1988     

The effect of treatment of Type 2 (insulin independent) diabetes mellitus on plasma concentrations of pancreatic polypeptide and glucagon

Summary The effect of the control of diabetes with diet and insulin upon plasma levels of human pancreatic polypeptide and glucagon was determined in eight patients with Type 2 (insulin independent) diabetes mellitus. The mean±SEM fasting plasma glucose was 15.9±1.3mmol/l for 5 days of diet treatment and 5.9±0.4 mmol/l for the last 5 days of treatment with diet plus insulin (p < 0.0001); corresponding fasting plasma pancreatic polypeptide levels were 328±97 and 247±71 pg/ml (p < 0.05) and immunoreactive glucagon levels were 95±11 and 62±6 pg/ml (p < 0.005). Cooked ground beef was administered on the first day of diet treatment and on the last day of treatment with diet plus insulin; mean maximal rise of pancreatic polypeptide, and total and incremental plasma pancreatic polypeptide response areas were significantly lower following treatment (p < 0.01), as was total area for immunoreactive glucagon (p < 0.05). Normalisation of fasting plasma glucose by short-term treatment with diet plus insulin is associated with decreases in basal and stimulated secretory activity of the pancreatic polypeptide cells in insulin independent diabetes mellitus.     

Pulsatile intravenous insulin replacement in streptozotocin-diabetic rats is more efficient than continuous delivery: effects on glycaemic control,insulin-mediated glucose metabolism and lipolysis

Summary Short-term exposure of tissues to pulses of insulin generally leads to an enhancement of insulin action. We have investigated the possible beneficial effects of long-term near-physiological continuous vs pulsatile intravenous insulin treatment of insulin-deficient streptozotocin (70 mg/kg) diabetic rats on blood glucose control, in vivo insulin action and in vitro insulin action in isolated adipocytes. First, we determined the 24-h peripheral plasma insulin profiles in normal rats under precisely controlled meal-feeding conditions. Basal plasma insulin levels (40±9 U/ml) oscillate with a periodicity of 11.9±0.9 min (p<0.05), and an amplitude of 60±10%. Subsequently, the 24-h insulin profile was mimicked in diabetic (D) rats by a continuous (c) or pulsatile (p) (6-min double, 6-min off) insulin infusion rate for 2 weeks, using a programmable pump-swivel unit. Control (C) rats received vehicle treatment. In Cc, Dc, Cp and Dp daily urinary glucose loss and average plasma glucose levels were 0±0, 7.5±4.4, 0±0, 0.8±0.4 mmol and 6.7±0.2, 11.5±2.7, 6.6±0.1, 5.9±1.4 mmol/l, respectively. Hypoglycaemia (<3 mmol/l) was observed in 10 and 20% of the blood samples collected from Dc and Dp rats, respectively. After 2 weeks of treatment, in vivo peripheral and hepatic insulin action was measured by the hyperinsulinaemic euglycaemic (6 mmol/l) clampwith[3-³H]-glucoseinfusion. Pre-clampcounter-regulatory hormone levels were similar among rats. Compared to Cc and Cp, Dc showed a reduction in insulin sensitivity and responsiveness for peripheral glucose uptake whereas Dp only showed a reduction in insulin sensitivity. Suppression of hepatic glucose production by insulin was similar among rats. After 2.5 weeks of treatment, epididymal adipocytes were isolated. Specific [¹²⁵I]-insulin binding, basal and insulin-stimulated [U-¹⁴C]-glucose uptake and isoproterenol-stimulated glycerol output were comparable among rat adipocytes. The inhibition of glycerol output by insulin was identical in Cp and Dp (V_max=48.6±6.1 and 42.3±4.6%) but blunted in Dc vs Cc (V_max=8.2±4.6 vs 44.0±7.2%, p<0.01) adipocytes, suggesting a post-binding defect in the antilipolytic action of insulin in Dc rats. In conclusion, long-term near-physiological pulsatile intravenous insulin replacement in insulin-deficient diabetic rats is more efficient than continuous delivery in reducing blood glucose, lowering glucosuria, increasing insulin sensitivity and inhibiting lipolysis.Abbreviations IDDM Insulin-dependent diabetes mellitus - AUC area under the curve - CV coefficient of variation     

The effect of continuous subcutaneous insulin infusion and conventional insulin regimes on 24-hour variations of blood glucose and intermediary metabolites in the third trimester of diabetic pregnancy

Summary Twenty-four hour metabolic profiles were performed in the third trimester of pregnancy in seven diabetic women; first when optimally controlled using conventional insulin regimes and subsequently when controlled with continuous subcutaneous insulin infusion. Seven non-diabetic women were also studied. Mean ±SD 24 h metabolite levels in the diabetics before and during continuous subcutaneous insulin infusion and in the controls were respectively: glucose -5.8±1.2; 5.0±0.9; 4.7±0.8 mmol/l; total ketone bodies -0.2±0.06; 0.15±0.05; 0.11±0.04 mmol/l; lactate -0.90±0.33; 0.90±0.24; 1.05 ±0.18 mmol/l; alanine -0.29±0.06; 0.30±0.06; 0.31±0.03 mmol/l. Total ketone body levels were significantly elevated (p<0.05) on conventional therapy but not on continuous subcutaneous insulin infusion compared with controls. Variations in metabolites over 24 h, as measured by mean standard deviations, were increased for glucose (p<0.001) and for total ketone bodies (p<0.05) on the conventional regimes we employed compared with controls. On continuous subcutaneous insulin infusion variations of blood glucose were not affected whereas variations in total ketone bodies were no different from controls. The best possible maternal metabolic control is necessary for normal foetal development and continuous subcutaneous insulin infusion provides a method of achieving this.     

Acetyl-salicylic acid impairs insulin-mediated glucose utilization and reduces insulin clearance in healthy and non-insulin-dependent diabetic man

Summary The effect of acetyl-salicylic acid (ASA, 3 g per day for 3 days) on glucose utilization and insulin secretion was studied in healthy volunteers and Type 2 diabetic patients using the hyperglycaemic and euglycaemic insulin clamp technique. When in healthy subjects arterial plasma glucose was acutely raised and maintained at +7 mmol/l above fasting level, the plasma insulin response was enhanced by ASA (70±7 vs. 52±7mU/l), whereas the plasma C-peptide response was identical. Despite higher insulin concentrations, glucose utilization was not significantly altered (control, 61±7; ASA, 65±6mol·kg^–1·min^–1) indicating impairment of tissue sensitivity to insulin by ASA. Inhibition of prostaglandin synthesis was not likely to be involved in the effect of ASA, since insulin response and glucose utilization were unchanged following treatment with indomethacin. In the euglycaemic insulin (1 mU·kg^–1·min^–1) clamp studies, glucose utilization was unaltered by ASA despite higher insulin concentrations achieved during constant insulin infusion (103±4vs. 89±4mU/l). In Type 2 diabetic patients, fasting hyperglycaemia (10.6 ±1.1 mmol/l) and hepatic glucose production (15±2 mol·kg^–1·min^–1) fell upon ASA treatment (8.6±0.7 mmol/l; 13±1 mol·kg^–1· min^–1). During the hyperglycaemic clamp study, the plasma response of insulin, but not of C-peptide, was enhanced by ASA, whereas tissue sensitivity to insulin was reduced by 30 percent. It is concluded that in healthy and Type 2 diabetic man, ASA impairs tissue sensitivity to the action of insulin. This effect is counterbalanced by an augmented plasma insulin response to glucose, which results from a reduced insulin clearance rate. In Type 2 diabetic patients, the reduction in hepatic glucose production may be responsible for the amelioration of hyperglycaemia following ASA treatment.     

Attenuation of the pancreatic beta cell response to a meal following hypoglycaemia in man

Summary The plasma concentration of C-peptide, insulin (IRI) and glucose was measured in 9 healthy subjects during insulin-induced hypoglycaemia followed by a meal. Identical observations were made in the same subjects after an equivalent period of fasting without hypoglycaemia (control study). Endogenous secretion of insulin was suppressed following administration of exogenous insulin and this persisted long after the blood glucose concentration had returned to normal. After the meal the mean blood glucose rose to a peak of 8.4±0.3 mmol/l (mean ± SEM) at 60 min and was still raised at 7.5±0.3 mmol/l at 120 min, compared with a peak value of only 5.1±0.2 mmol/l at 30 min after the meal in the control study. Following hypoglycaemia the mean plasma IRI rose from 8.3±1.3 mU/l to a delayed peak of 81.6±12.7 mU/l at 60 min and was 123.5±14 mU/l at 120 min post-prandially, compared with a peak of 72.4±0.5 mU/l at 30 min after the meal in the control study. Acute hypoglycaemia may thus induce an abnormal pattern of insulin secretion in response to a meal, with impaired carbohydrate tolerance in normal subjects.     

Beta-cell hypersensitivity for glucose precedes loss of glucose-induced insulin secretion in 90% pancreatectomized rats

Summary Glucose-induced insulin secretion is impaired in the presence of chronic hyperglycaemia. Insulin secretion was studied in a diabetic rat model prior to the beta cells becoming non-responsive to glucose in order to map out the sequence of changes that accompany chronic hyperglycaemia. In vitro pancreas perfusions were carried out 1 and 2 weeks after a 90% pancreatectomy; controls underwent a sham pancreatectomy. One week post 90% pancreatectomy: (i) non-fasting plasma glucose values were 2–3 mmol/l above normal, (ii) the in vitro insulin response to 16.7 mmol/l glucose was 20 ± 4% of shams, a response that was appropriate for the surgical reduction in beta-cell mass, (iii) the beta-cell sensitivity for glucose was increased as reflected by left-shifted dose-response curves for glucose-induced insulin secretion (half maximal insulin output 5.7 mmol/l glucose vs 16.5 mmol/l glucose in shams) and glucose potentiation of arginine-induced insulin secretion (half maximal insulin output 3.5 mmol/l glucose vs 14.8 mmol/l glucose in shams). This heightened beta-cell sensitivity for glucose was not a result of the hyperglycaemia, because similarly reduced half-maximal insulin responses were found after a 60% pancreatectomy, a surgical procedure in which plasma glucose values remained normal. In summary, a rise in beta-cell sensitivity for glucose precedes the loss of glucose-induced insulin secretion in diabetic rats.     

Hormonal and metabolic effects of chlorpropamide,glibenclamide and placebo in a cross-over study in diabetics not controlled by diet alone

Summary Twenty diabetic patients, whose hyperglycaemia had been shown to fail to respond to at least one month's dietary treatment, completed a crossover study in order to: 1) compare the effectiveness of two sulphonylureas, chlorpropamide and glibenclamide, and 2) study the effects of sulphonylureas on insulin secretion and on biochemical indices of glucose intolerance. Fasting blood glucose fell on active treatment from 10.7±0.6 (mean ± SEM) to 6.6±0.7 mmol/l and rose again to 10.6±0.7 after 4 months placebo. A second period of 4 months sulphonylurea therapy resulted in a comparable fall in blood glucose (to 6.9±0.7 mmol/l) and a similar relapse was seen after the second placebo period (to 10.5±0.9 mmol/l). Glucose tolerance and associated insulin secretion improved markedly on active treatment, with ketone bodies, non-esterified fatty acids, and glycerol falling to within the reference range. Sulphonylurea therapy was associated with a small but significant increase in the fasting insulin level. These effects were nearly all reversed 4 months after withdrawal of the sulphonylureas. No marked changes were found in growth hormone, lactate, pyruvate, lactate/pyruvate ratio or fasting cholesterol, triglycerides and lipoproteins. On a weight basis, glibenclamide was 26 times more potent than chlorpropamide and, in the doses used in this study, their biochemical effects were indistinguishable. The effects of these two sulphonylureas seem most likely to be mediated by a direct stimulation of insulin secretion by the B-cell.     

Insulin action and determinants of glycaemia in a rat model of Type 2 (non-insulin-dependent) diabetes mellitus

Summary We aimed to assess prandial responses, basal glucose turnover and insulin action (euglycaemic clamp) in a very low-dose neonatal streptozotocin model of Type 2 (noninsulin-dependent) diabetes mellitus. Male Wistar rats were injected at 2 days of age with 45 mg/kg streptozotocin or vehicle (control). At 8 weeks, the groups were subdivided and fed either a high-fat or high-starch diet for 3 weeks. Both the fat diet and streptozotocin treatments had independent hyperglycaemic effects (streptozotocin/fat 9.3±0.3 mmol/l; streptozotocin/starch 7.5±0.3 mmol/l; control/fat 7.4±0.1 mmol/l; all p<0.01 vs control/starch 6.4±0.1 mmol/l). The fat diet effect was associated with both a reduction in basal glucose clearance (p<0.001) and in basal hepatic glucose output (p<0.05). Streptozotocin increased basal hepatic glucose output. Significantly higher prandial glycaemia in the streptozotocin/starch group occurred despite similar insulin levels and appeared to be related to an impaired early insulin response. Whole-body and tissue-specific insulin sensitivity were significantly depressed in fat-fed animals compared to starch-fed animals, however there were no significant effects of streptozotocin treatment. We conclude that fasting hyperglycaemia associated with abnormalities in both glucose production and clearance can exist in the presence of a basal hepatic glucose output which is reduced compared to control animals. Furthermore, dietary-fat-induced insulin resistance is not exacerbated by the relative insulin deficiency and/or mild hyper glycaemia observed when dietary fat and neonatal streptozotocin-treatments are combined.     

Relationship between plasma insulin levels and high density lipoprotein cholesterol levels in healthy men

Summary Insulin and high density lipoproteins are considered to play a role in the development of atherosclerosis. In order to study whether there was a relationship between endogenous plasma insulin response and high density lipoproteins, an acute intravenous glucose tolerance test (0.5 g glucose/kg body weight) was performed in 94 healthy men, aged 20–49 years. Cholesterol and triglyceride levels were measured in very low density lipoproteins, low density lipoproteins and high density lipoproteins isolated from fasting serum by preparative ultracentrifugation. The subjects were divided into quartiles according to their fasting and post-glucose load plasma insulin and high density lipoprotein cholesterol levels. The results obtained in the subjects of the upper quartiles were compared with the results obtained in the subjects of the lower quartiles. The mean glucose disappearance rates were within the normal range and did not differ between the upper and lower quartiles. Subjects with high fasting plasma insulin had lower high density lipoprotein cholesterol levels (1.11±0.34 mmol/l, p=0.01) than men with low fasting plasma insulin (1.40±0.37 mmol/l). Higher mean post-glucose plasma insulin was associated with lower high density lipoprotein cholesterol levels (1.18±0.32 mmol/l, p<0.05) and increased high density lipoprotein triglyceride levels (0.14±0.07 mmol/l, p<0.01) when compared with the men with low post-glucose plasma insulin (1.40±0.36 mmol/l and 0.09±0.03 mmol/l respectively). These observations reflect the close relationship between endogenous insulin and lipoprotein metabolism.     

The effect of oral galactose on GIP and insulin secretion in man

Summary The insulinotropic effect of 50 g galactose given orally to 5 normal volunteers on two occasions — once with and once without a period of hyperglycaemia produced by an intravenous glucose infusion — was studied. Oral galactose caused a rise in plasma GIP from fasting levels of 260±50 ng/l (mean ± S. E. M.) to a maximum of 900±65 ng/l 30 min after ingestion, but in the presence of induced hyperglycaemia the GIP response was significantly diminished and delayed (maximum plasma GIP levels 595+110 ng/l at 45 min, p<0.05). The insulin response to galactose was greatly enhanced by IV glucose (mean area under plasma insulin curve with galactose alone 236.5±66.0, with galactose + IV glucose 451.9+81.6, p<0.025). The mean rise in plasma galactose was significantly lower in the presence of IV glucose (mean peak level 1.97±0.28 mmol/l with galactose alone, 0.69±0.16 mmol/l galactose + IV glucose, p <0.025). Oral galactose caused the release of GIP, which is powerfully insulinotropic in the presence of moderate hyperglycaemia. The lower plasma GIP and galactose levels observed following oral galactose in the presence of IV glucose may be accounted for either by postulating that insulin inhibits the absorption of oral galactose, or that insulin exerts a negative feed-back control on GIP release and accelerates galactose disposition in the body.     

Increased glucose disposal after jejuno-ileostomy

Summary Nine patients were studied 1.5–3 years after jejuno-ileostomy for obesity by an intravenous glucose infusion technique designed to imitate blood glucose concentrations after glucose ingestion. Whereas serum insulin and gastrin concentrations were normal, blood glucose concentrations were significantly depressed compared to preoperative levels as well as to levels in matched normal subjects. Thus, in the fasting state mean concentrations (± S.E.M.) of blood glucose, serum insulin and gastrin in the patients were, respectively, 3.3±0.2 mmol/l, 95±22 pmol/l and 38±4 pmol/l. The corresponding concentrations in the matched normals were 4.3±0.2 mmol/l, 70±18 pmol/l and 39±6 pmol/l. The glucose concentrations in the patients were low in all situations, i. e. in the fasting state, after oral glucose ingestion and during the intravenous glucose infusion. The results indicate that jejuno-ileostomy in obesity greatly facilitates peripheral glucose disposal. The mechanism behind this phenomenon is not yet known.     

Effects of the combination of insulin and glibenclamide in Type 2 (non-insulin-dependent) diabetic patients with secondary failure to oral hypoglycaemic agents

Summary The effects of combined insulin and sulfonylurea therapy on glycaemic control and B-cell function was studied in 15 Type 2 (non-insulin-dependent) diabetic patients who had failed on treatment with oral hypoglycaemic agents. The patients were first treated with insulin alone for four months. Five patients were given two daily insulin doses and ten patients one dose. During insulin treatment the fasting plasma glucose fell from 14.5±0.8 to 8.8±0.4 mmol/l and the HbA₁ concentration from 12.6±0.4 to 9.2±0.2%. This improvement of glycaemic control was associated with a suppression of basal (from 0.31±0.04 to 0.10±0.02 nmol/l) and glucagon-stimulated (from 0.50±0.08 to 0.19±0.04 nmol/l) C-peptide concentrations. Four months after starting insulin therapy the patients were randomised to a four-month double-blind cross-over treatment with insulin combined with either 15 mg glibenclamide per day or with placebo. Addition of glibenclamide to insulin resulted in a further reduction of the fasting plasma glucose (7.9±0.5 mmol/l) and HbA₁ (8.3±0.2%) concentration whereas the basal (0.21±0.03 nmol/l) and glucagon-stimulated C-peptide concentrations (0.34±0.06 nmol/l) increased again. Addition of placebo to insulin had no effect. The daily insulin dose could be reduced by 25% after addition of glibenclamide to insulin, while it remained unchanged when insulin was combined with placebo. The fasting free insulin concentration did not differ between the glibenclamide and placebo periods (28±6 vs 30±5 mmol/l). The fasting free insulin concentration correlated, however, positively with the insulin dose (r=0.76, p<0.01) indicating that the insulin dose was the main determinant of the free insulin concentration. In contrast, the basal C-peptide concentration was higher during the insulin plus glibenclamide than during the insulin plus placebo period (0.21±0.03 vs 0.16±0.03 nmol/l; p<0.05). Addition of glibenclamide to insulin therapy increased the treatment cost by 30–50%, was associated with increased frequency of mild hypoglycaemic reactions and with a slight, but significant fall in HDL cholesterol concentration (from 1.40±0.07 to 1.29±0.06; p<0.05) compared with insulin plus placebo. We conclude that in Type 2 diabetic patients, who have failed on treatment with oral hypoglycaemic agents, the combination of insulin and glibenclamide resulted in slightly improved glycaemic control and allowed reduction of the insulin dose. The price for this improvement was higher treatment costs, more (mild) hypoglycaemic reactions and a marginal fall in the HDL cholesterol concentration. Whether the same effect could have been achieved with divided insulin doses in all patients is not known.     

Indirect two-site immunoradiometric assay of rat and mouse proinsulin

Summary An indirect two-site immunoradiometric assay for rat and mouse proinsulin using a rabbit antibody to synthetic rat C-peptide has been developed. The sensitivity of the assay is 0.006 pmol/ml. Proinsulin was 4.95% of the total proinsulin and insulin in extracts of rat pancreas and 5.45% in extracts of isolated rat islets. The mean fasting rat insulin and proinsulin concentrations were 0.13±0.09 pmol/ml (n=5) and 0.008±0.002 pmol/ml (n=5) respectively. The mean fasting mouse proinsulin concentration was 0.019±0.006 pmol/ml (n=8). In rats intravenous glucose produced a biphasic insulin response but proinsulin rose progressively to 0.021±0.011 pmol/ml at 45 min. In mouse oral glucose increased the proinsulin concentration to 0.13 pmol/ ml at 30 min. Proinsulin release from isolated rat islets was studied during intermittent or continuous high glucose (20 mmol/l) stimulation in static incubation. Significant increases in proinsulin release were only observed 90 min after initial exposure to high glucose whether glucose stimulation was continuous or intermittent. Both in vivo and in vitro glucose stimulation led initially to a fall in the proinsulin/ insulin molar ratio but later upon prolonged stimulation this progessively increased to above the basal value.     

Effect of pretreatment with a high fat diet on the gastric inhibitory polypeptide and insulin responses to oral triolein and glucose in rats

Summary Male Wistar rats were pretreated with 3 ml triolein orally for 4 days in addition to their normal diet. A similar control group were allowed free access to normal laboratory food. When given an oral fat load (1 ml triolein) plasma gastric inhibitory polypeptide (GIP) and triglyceride levels were significantly higher in the fat pretreated group. Inhibition of fat-stimulated GIP release by exogenous insulin was demonstrated in the untreated control group (plasma GIP: 663±49 versus 853±92 ng/l, mean ± SEM p < 0.025), but pretreatment with an oral fat load abolished this effect (plasma GIP: 1008±95 versus 1116±100 ng/l, p NS). Plasma glucose levels were significantly higher in fat pretreated rats given oral fat and intraperitoneal insulin compared with untreated controls (plasma glucose nadir 2.6±0.48 versus 1.6±0.15 mmol/l, p < 0.05). Fat-pretreated rats showed significantly higher insulin and glucose levels compared with the untreated rats when given oral glucose (plasma insulin: 6.2±1.2 versus 2.5±0.59 n.g/l, p < 0.01; plasma glucose: 10.2±0.39 versus 8.9±0.41 mmol/l, p < 0.025). Pretreatment of rats on a high fat diet causes (1) increased GIP secretion in response to an oral fat load, (2) abolition of the feed-back inhibition of exogenous insulin on fat-stimulated GIP release, and (3) some degree of insulin resistance.     

Role of glucagon suppression on gluconeogenesis during insulin treatment of the conscious diabetic dog

Summary In seven insulin-deficient (<3 mU/l) pancreatectomised dogs, the direct and glucagon-related indirect effects of intraportal insulin infusion (350 U/kg-min; 12±1 mU/l) on glucose production were determined. Insulin was infused for 300 min during which time the plasma glucagon concentration was allowed to fall (314±94 to 180±63 ng/l) for 150 min before being replaced by an infusion intraportally at 2.6ng/kg-min (323±61 ng/l) for the remaining 150 min. Glucose production and gluconeogenesis were determined using arterio-venous difference and tracer techniques. Insulin infusion shut off net hepatic glucose output and caused the plasma glucose, blood glycerol and plasma non-esterified fatty acid levels to fall. It caused the hepatic fractional extraction of alanine (0.41 ±0.10 to 0.21±0.06) and lactate (0.32 ±0.09 to 0.04 ±0.03) to fall which increased their concentrations. When glucagon was replaced, all of these changes were fully or partly reversed with the exception of the changes in glycerol and nonesterified fatty acids. Indeed, 70% of the fall in hepatic glucose production and virtually 100% of the changes in lactate and alanine metabolism produced by basal insulin infusion were mediated by a fall in glucagon. However, the fall in hepatic uptake of glycerol was unaffected by changes in glucagon and thus gluconeogenesis from this substrate was inhibited by insulin per se probably as a result of reduced lipolysis. The latter effect of insulin may explain the incomplete restoration of hepatic glucose production when hyperglucagonaemia was re-established during insulin infusion.     

Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets

Summary Glucose-stimulated insulin secretion, glucose transport, glucose phosphorylation and glucose utilization have been characterized in the insulinoma cell line MIN6, which is derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells. Glucose-stimulated insulin secretion occurred progressively from 5 mmol/l glucose, reached the maximal level approximately seven-fold above the basal level at 25 mmol/l, and remained at this level up to 50 mmol/l. Glucose transport was very rapid with the half-maximal uptake of 3-O-methyl-d-glucose being reached within 15 s at 22 °C. Glucose phosphorylating activity in the cell homogenate was due mainly to glucokinase; the V_max value of glucokinase activity was estimated to be 255±37 nmol·h^–1·mg protein^–1, constituting approximately 80% of total phosphorylating activity, whereas hexokinase activity constituted less than 20%. MIN6 cells exhibited mainly the high K_m component of glucose utilization with a V_max of 289±18 nmol·h^–1·mg protein^–1. Thus, glucose utilization quantitatively and qualitatively reflected glucose phosphorylation in MIN6 cells. In contrast, MIN7 cells, which exhibited only a small increase in insulin secretion in response to glucose, had 4.7-fold greater hexokinase activity than MIN6 cells with a comparable activity of glucokinase. These characteristics in MIN6 cells are very similar to those of isolated islets, indicating that this cell line is an appropriate model for studying the mechanism of glucose-stimulated insulin secretion in pancreatic beta cells.