Glucagon-like peptide-1 stimulates insulin secretion by a Ca2+-independent mechanism in Zucker diabetic fatty rat islets of Langerhans

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca2+ concentration ([Ca2+]i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca2+]i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 micromol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca2+]i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca2+ influx through voltage-dependent Ca2+ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca2+-independent signaling pathways.     

Glucagon-like peptide-1 stimulates insulin secretion by a Ca-independent mechanism in Zucker diabetic fatty rat islets of Langerhans

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca²⁺]_i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 μmol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca²⁺]_i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca²⁺ influx through voltage-dependent Ca²⁺ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca²⁺-independent signaling pathways.     

Genetically obese rats with (SHR/N-cp) and without diabetes (LA/N-cp) share abnormal islet responses to glucose.

To assess the effect of hyperglycemia on the function of islets obtained from obese rats, the behavior of isolated islets from LA/N-corpulent (nondiabetic obese) and SHR/N-corpulent (diabetic obese) male rats was examined and compared. Islets from both genetic models showed a left-shifted glucose dose-response curve for insulin release (concentrations for half-maximal release, 5 to 6 mmol/L v 12 to 13 mmol/L in LA/N lean littermates and 3 mmol/L v 10 mmol/L in lean SHR/N). When insulin release was expressed per unit islet volume, the fourfold to fivefold enlarged islets from both obese diabetic and obese nondiabetic rats showed decreased insulin secretory response in high (16.5 to 28 mmol/L) glucose concentrations, although the decrease was more severe in the diabetic rats. Glucose-stimulated insulin release by islets from both models was relatively resistant to inhibition by 1.2 mmol/L mannoheptulose (eg, 82% +/- 3% inhibition in LA/N lean v 16% +/- 8% in LA/N obese), although nearly complete inhibition was observed with 16 mmol/L mannoheptulose (96% v 85%, NS). Islets of obese diabetic rats were also resistant to the calcium-channel blocker, verapamil, suggesting an abnormal pathway of stimulus-secretion coupling for glucose. Glucose oxidation to carbon dioxide was increased in both obese models at all glucose concentrations when expressed per islet. In data expressed per unit volume, the larger islets from the obese-nondiabetic rats showed a left-shifted dose-response curve with an unchanged maximum rate of glucose oxidation at high (16.5 mmol/L) glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acid inhibition of glucose-stimulated insulin secretion is enhanced in pancreatic islets from insulin-resistant rats.

A study was initiated to test two hypotheses. The first was the postulate that glucose-stimulated insulin secretion would be enhanced in pancreatic islets isolated from normal non-obese rats made insulin-resistant by dietary means. The second, related hypothesis was that glucose-stimulated insulin secretion by pancreatic islets isolated from insulin-resistant rats would be more vulnerable to inhibition following culture in the presence of fatty acids. For this purpose, insulin resistance was induced in normal Sprague-Dawley rats by feeding fat-enriched and fructose-enriched diets. The results indicate that islets isolated from either fat-fed or fructose-fed rats secreted significantly more insulin at a glucose concentration of 2.5 to 10.0 mmol/L. In addition, the mean maximal glucose (27 mmol/L)-stimulated insulin secretion rate was significantly lower (15.3 +/- 2.5 ng/islet/h) in islets from fructose-fed rats versus chow-fed rats (25.2 +/- 3.1 ng/islet/h) following culture for 48 hours in the presence of palmitate (0.125 micromol/L). These results support the view that glucose-stimulated insulin secretion is enhanced in islets from insulin-resistant rats, and that these islets are more vulnerable to the inhibitory effects of free fatty acid (FFA) on insulin secretion.     

Hypersecretion of IAPP from the islets of VMH-lesioned rats and obese Zucker rats.

To investigate the possible role of islet amyloid polypeptide (IAPP) in the development of type 2 diabetes mellitus, we examined the IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats and genetically obese Zucker rats, using a specific radioimmunoassay for IAPP. Obesity and hyperinsulinemia were observed in rats 21 days after VMH lesioning. IAPP content was increased in the islets of VMH-lesioned rats compared with findings in the sham-operated controls (100.9 +/- 6.6 vs 72.8 +/- 3.85 fmol/islet; P less than 0.01). Isolated islets of VMH-lesioned rats secreted larger amounts of IAPP in the presence of 2.8 and 16.7 mM glucose (2.99 +/- 0.98 and 11.2 +/- 0.29 fmol islet-1 3 h-1) than was noted in sham-operated rats (ND and 6.65 +/- 0.78 fmol islet-1 3 h-1). In the obese Zucker rats, aged 14 weeks, IAPP concentrations in the islets were elevated compared with lean rats (133.3 +/- 10.6 vs 84.4 +/- 8.5 fmol/islet; P less than 0.01). The isolated islets secreted larger amounts of IAPP in response to 2.8 and 16.7 mM glucose (2.83 +/- 0.88 and 15.81 +/- 1.35 fmol islet-1 3 h-1) than did those from lean control rats (0.36 +/- 0.19 and 12.49 +/- 1.20 fmol islet-1 3 h-1). These results strongly suggest that overproduction and hypersecretion of IAPP occur in animals with obesity and hyperinsulinemia.     

Insulin response of cultured islets from diabetic and nondiabetic BB rats.

This study examines the insulin response of pancreatic islets isolated from diabetic BB rats (BBD), nondiabetic BB rats (BBN), and Wistar rats to in vitro stimulation. After a 48-hour culture period, insulin release in response to glucose (17.8 mmol/L) either alone, with glucose-dependent insulinotropic polypeptide (GIP) +/- somatostatin (SS), or with Arg +/- SS was measured. A static incubation system was used. Insulin secretion from islets cultured in 4.4 mmol/L glucose (basal) did not differ between BBN and BBD rats (0.50% +/- 0.08%, 0.67% +/- 0.25% of total islet cell content [TCC], respectively). High glucose concentrations (17.8 mmol/L) stimulated a modest increase in insulin release from BBD and BBN islets (1.8% +/- 0.48% and 2.1% +/- 0.19% TCC, respectively). The addition of GIP (1 nmol/L) enhanced glucose-stimulated insulin secretion from BBN rat islets (2.9% +/- 0.42% TCC), but had no effect on BBD islets (2.04% +/- 0.57% TCC). Somatostatin (1 mumol/L) completely reversed the glucose- and/or GIP-stimulated insulin secretion from both BBN and BBD rat islets to basal levels (0.42% +/- 0.043%, 0.42% +/- 0.09% TCC, respectively). Arg (1 mmol/L) enhanced glucose-stimulated insulin secretion in both groups, although the greatest response was elicited from BBD rat islets (8.4-fold v 3.2-fold). Experiments comparing BB rats with Wistar rats demonstrated significant differences in the glucose-stimulated (17.8 mmol/L) insulin response of the islets. Islets taken from BBN and BBD were less responsive to glucose than those from Wistar rats. However, islets from BBD rats were hyperresponsive to Arg when compared with islets from Wistar rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lifelong sequential changes in glucose tolerance and insulin secretion in genetically obese Zucker rats (fa/fa) fed a diabetogenic diet

Life-long sequential changes in glucose tolerance and insulin secretion were investigated in genetically obese Zucker rats (fa/fa) fed a diabetogenic diet rich in lard and sucrose. Comparisons were made with lean littermates (Fa/-) receiving normal chow diet. At 3-month intervals, seven to nine lean and obese rats had two permanent venous catheters implanted, allowing stress- and pain-free sampling of blood before, during, and after substrate administration. Intravenous glucose, iv arginine, and oral glucose tolerance were tested. The obese rats progressively developed hyperglycemia and severe hyperinsulinemia; their basal glycemia reached 8.8 +/- 1.1 vs. 5.8 +/- 0.2 mmol/liter in the lean rats at 46 weeks of age; respective insulinemia was 287.7 +/- 61.9 and 18.1 +/- 2.8 mU/liter (mean +/- SD). In the obese rats a distinct loss in glucose tolerance was seen with progression of age in spite of rising stimulated insulin secretion, which suggests progressive development of insulin resistance without exhaustion of B-cell secretory capacity. Absence of insulin deficiency was also suggested by immunohistochemical staining of pancreatic tissue specimens from obese rats, which showed large populations of insulin-containing cells. Like the obese animals, lean rats exhibited a decrease in insulin sensitivity with age. Relating basal individual glycemia and insulinemia, a rise by 1 mmol/liter in glycemia was associated with a 8.8-fold rise in basal insulinemia in lean rats, but only with a 1.8-fold increase in obese rats. Similar correlations for stimulated glycemia and insulinemia suggest impaired glucose sensitivity of pancreatic B-cells in obese vs. lean rats. In conclusion, hyperglycemia and hyperinsulinemia in insulin-resistant obese Zucker rats on a diabetogenic diet are not characterized by quantitatively deficient B-cell secretory capacity, but, rather, by impaired B-cell sensitivity to glucose with qualitatively intact regulation of glycemia and insulinemia at elevated plasma concentrations.     

Beta cell compensation for insulin resistance in Zucker fatty rats: increased lipolysis and fatty acid signalling

Aims/hypothesis The aim of this study was to determine the role of fatty acid signalling in islet beta cell compensation for insulin resistance in the Zucker fatty fa/fa (ZF) rat, a genetic model of severe obesity, hyperlipidaemia and insulin resistance that does not develop diabetes.Materials and methods NEFA augmentation of insulin secretion and fatty acid metabolism were studied in isolated islets from ZF and Zucker lean (ZL) control rats.Results Exogenous palmitate markedly potentiated glucose-stimulated insulin secretion (GSIS) in ZF islets, allowing robust secretion at physiological glucose levels (5–8 mmol/l). Exogenous palmitate also synergised with glucagon-like peptide-1 and the cyclic AMP-raising agent forskolin to enhance GSIS in ZF islets only. In assessing islet fatty acid metabolism, we found increased glucose-responsive palmitate esterification and lipolysis processes in ZF islets, suggestive of enhanced triglyceride–fatty acid cycling. Interruption of glucose-stimulated lipolysis by the lipase inhibitor Orlistat (tetrahydrolipstatin) blunted palmitate-augmented GSIS in ZF islets. Fatty acid oxidation was also higher at intermediate glucose levels in ZF islets and steatotic triglyceride accumulation was absent.Conclusions/interpretation The results highlight the potential importance of NEFA and glucoincretin enhancement of insulin secretion in beta cell compensation for insulin resistance. We propose that coordinated glucose-responsive fatty acid esterification and lipolysis processes, suggestive of triglyceride–fatty acid cycling, play a role in the coupling mechanisms of glucose-induced insulin secretion as well as in beta cell compensation and the hypersecretion of insulin in obesity. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Effect of pertussis toxin on islet insulin secretion in obese (fa/fa) Zucker rats.

We used isolated islets of lean and obese Zucker rats to determine whether inhibitory pathways mediated by pertussis toxin-sensitive guanyl nucleotide-binding (Gi) proteins contribute to hyperinsulinemia in obese rats. Epinephrine (10(-4) M) and somatostatin (10(-7) M) inhibited insulin secretion by +/- 75% in lean and fa/fa rats. Overnight culture of islets with pertussis toxin (300 ng/ml) enhanced insulin release more in lean (+/- 120%) than obese (+/- 60%) rats. In lean rats incubation of pertussis toxin-treated islets with epinephrine resulted in lower immunoreactive insulin release (p = 0.0005) than pertussis toxin-treated islets without epinephrine. However, in obese rats pertussis toxin treatment reversed this inhibition. Pertussis toxin completely reversed inhibition by somatostatin in both phenotypes. Galanin had no effect on insulin secretion. Cellular cAMP content was similar in lean and obese rats. Inhibitory hormones had no effect on cAMP production. We conclude that islets of obese rats respond normally to inhibitors of insulin release. Reversal of somatostatin-induced inhibition by pertussis toxin indicates normal function of Gi in obese rats. A subtle difference in sensitivity to pertussis toxin between lean and obese islets was noted.     

Insulinotropic actions of exendin-4 and glucagon-like peptide-1 in vivo and in vitro

This study compares in vitro effects of exendin-4 and glucagon-like peptide (GLP)-1 on basal and glucose-stimulated insulin release from isolated rat islets and in vivo insulinotropic actions of exendin-4 and GLP-1 after an intravenous glucose challenge in rats. In static incubation of isolated islets, changing ambient glucose concentration from 3 mmol/L to 10 mmol/L stimulated insulin secretion 9.8 +/- 1.3-fold. The addition of exendin-4 or GLP-1 (1 nmol/L to 1 micromol/L) increased glucose-stimulated insulin secretion up to 5.8 +/- 1.6-fold and 3.3 +/- 1.0-fold, respectively, over basal rates (defined as no hormones added, 3 mmol/L glucose) and 19.6 +/- 2.3-fold and 15.0 +/- 3.1-fold at 10 mmol/L glucose. In dynamically perfused isolated islets exposed to 7.5 mmol/L glucose, insulin secretion increased 6.4 +/- 1.5-fold, and exendin-4 (20 nmol/L) or GLP-1 (20 nmol/L) increased this similarly by up to 13.5 +/- 2.8 and 12.7 +/- 3.9-fold,respectively. Anesthetized rats administered 5.7 mmol/kg intravenous glucose increased plasma insulin concentration 3.0-fold. Infusion of exendin-4 or GLP-1 increased this to a maximum of 7.6-fold and 5.3-fold, respectively. As with isolated islet studies, in vivo dose responses and concentration responses with exendin-4 and GLP-1 were bell-shaped. When insulinotropic effects were mapped onto the steady-state plasma concentrations associated with these infusion rates, both peptides exhibited bell-shaped concentration responses with peak insulinotropic effects occurring with plasma peptide concentrations of approximately 1 nmol/L in this model. In summary, exendin-4 and GLP-1 exhibited similar insulinotropic potencies (median effective dose [ED(50)]) when assessed on a concentration basis in in vitro and in vivo models, while exendin-4 exhibited greater efficacy (maximum response).     

Changes in islet capillary angioarchitecture coincide with impaired B-cell function but not with insulin resistance in male Otsuka-Long-Evans-Tokushima fatty rats: dimorphism of the diabetic phenotype at an advanced age

The Otsuka-Long-Evans-Tokushima fatty (OLETF) rat is a genetic model of spontaneous development of non-insulin-dependent diabetes mellitus (NIDDM) established as an inbred strain after 20 generations of selective breeding. Although they are thought to be genetically homogeneous, they show a dimorphism regarding the diabetic phenotype at an advanced age, with one remaining obese and modestly diabetic while the other becomes lean and overtly diabetic. To clarify the causes for this divergence, we examined the physical, biochemical, and histopathological features in rats at 50 weeks of age, including an analysis of islet angioarchitecture. Sixty-one of 85 male OLETF rats lost weight, while the remainder remained obese. Mean nonfasting plasma glucose in the lean group was 21.8+/-4.6 mmol/L, significantly higher versus the obese group (10.5+/-1.4 mmol/L) and the age-matched control Long-Evans-Tokushima-Otsuka (LETO) group (7.1+/-0.6 mmol/L). Morphological studies of the pancreas from the lean group showed enlarged multilobulated fibrotic islets with a paucity of B cells, whereas islets from the obese group appeared slightly enlarged and showed a relative abundance of B cells. The fine capillaries that form a network in the islets were extremely sparse in the lean group, resulting in a defective glomerular-like configuration, whereas those from the obese group were dense, forming a nearly typical glomerular-like configuration. Increased plasma insulin responses to oral and intravenous (i.v.) glucose and i.v. glucagon loads were nearly absent in the lean group, while they were evident in the obese group, although to a lesser extent compared with the LETO group. Mean insulin secretory output from the perfused pancreas in response to 11.1 mmol/L glucose in the lean group (3.5+/-2.2 pmol/20 min) was significantly lower versus the obese group (8.8+/-6.5 pmol/20 min) and LETO group (22.0+/-10.8 pmol/20 min). Similarly, pancreatic insulin content was significantly lower in the lean group (9.3+/-6.1 microg) versus the others (26.1+/-17.3 microg for obese and 41.1+/-24.8 microg for LETO). In vivo insulin-stimulated glucose uptake measured by a euglycemic clamp technique was significantly higher in the lean group compared with the obese group. These results demonstrate that the dimorphism regarding the diabetic phenotype in male OLETF rats at 50 weeks of age was due to differences in the number of islet B cells, which could be the result of a variation in the capacity for B-cell proliferation among male OLETF rats.     

Islet beta cell failure in the 60% pancreatectomised obese hyperlipidaemic Zucker fatty rat: severe dysfunction with altered glycerolipid metabolism without steatosis or a falling beta cell mass

Aims/hypothesis  The Zucker fatty (ZF) rat subjected to 60% pancreatectomy (Px) develops moderate diabetes by 3 weeks. We determined whether a progressive fall in beta cell mass and/or beta cell dysfunction contribute to beta cell failure in this type 2 diabetes model. Methods  Partial (60%) or sham Px was performed in ZF and Zucker lean (ZL) rats. At 3 weeks post-surgery, beta cell mass and proliferation, proinsulin biosynthesis, pancreatic insulin content, insulin secretion, and islet glucose and lipid metabolism were measured. Results  ZL-Px rats maintained normal glycaemia and glucose-stimulated insulin secretion (GSIS) despite incomplete recovery of beta cell mass possibly due to compensatory enhanced islet glucose metabolism and lipolysis. ZF-Px rats developed moderate hyperglycaemia (14 mmol/l), hypertriacylglycerolaemia and relative hypoinsulinaemia. Despite beta cell mass recovery and normal arginine-induced insulin secretion, GSIS and pancreatic insulin content were profoundly lowered in ZF-Px rats. Proinsulin biosynthesis was not reduced. Compensatory increases in islet glucose metabolism above those observed in ZF-Sham rats were not seen in ZF-Px rats. Triacylglycerol content was not increased in ZF-Px islets, possibly due to lipodetoxification by enhanced lipolysis and fatty acid oxidation. Fatty acid accumulation into monoacylglycerol and diacylglycerol was increased in ZF-Px islets together with a 4.5-fold elevation in stearoyl-CoA desaturase mRNA expression. Conclusions/interpretation  Falling beta cell mass, reduced proinsulin biosynthesis and islet steatosis are not implicated in early beta cell failure and glucolipotoxicity in ZF-Px rats. Rather, severe beta cell dysfunction with a specific reduction in GSIS and marked depletion of beta cell insulin stores with altered lipid partitioning underlie beta cell failure in this animal model of type 2 diabetes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. M. Prentki and J. Leahy contributed equally to this work.     

Effect of adrenalectomy on the development of a pancreatic islet lesion in fa/fa rats

Summary Adrenalectomy prevents development of obesity and hyperinsulinaemia in obese (fa/fa) Zucker rats, thereby implicating the hypothalamopituitary-adrenal axis in the pathogenesis of obesity. In this study glucose-induced insulin secretion and glucokinase activity were investigated in isolated islets from adrenalectomized and control obese and lean female rats. Islets from control fa/fa rats were more sensitive to glucose with a half-maximal effective concentration (EC₅₀) of 6.1±2.0 mmol · l^–1 compared with 10.6±2.7 mmol · l^–1 for adrenalectomized fa/fa rat islets. Adrenalectomy did not alter the islet sensitivity to glucose in the lean rats (EC₅₀ of 9.4±1.5 mmol · l^–1 and 9.3±2.0 mmol · l^–1 for adrenalectomized and control lean rats respectively). Mannoheptulose did not inhibit insulin secretion from control obese rats; however at concentrations of 1.0 mmol · l^–1 or more it significantly inhibited glucose-induced insulin secretion in adrenalectomized obese and lean, and control lean rat islets (p<0.05). In adrenalectomized fa/fa islets the glucokinase K_m was increased twofold compared with the control fa/fa rats (9.5±1.5 mmol · l^–1 vs 5.0±1.5 mmol · l^–1, respectively), but there was no significant change in glucokinase K_m in the lean rat islets after adrenalectomy. Mannoheptulose (10 mmol · l^–1) caused a significant reduction in glucose phosphorylation in disrupted islets of adrenalectomized fa/fa and lean, and of control lean rats, but not of control fa/fa rats. These data demonstrate that development of abnormal regulation of glycolysis in pancreatic islet beta cells of fa/ fa rats, as indicated by the insulin response to mannoheptulose and glucokinase activity, is dependent on an intact hypothalamo-pituitary-adrenal axis.Abbreviations ADX Adrenalectomy/adrenalectomized - CRH corticotrophin releasing hormone - DMEM Dulbecco's modified Eagle's medium - EC₅₀ half-maximal effective concentration - HPA hypothalamo-pituitary-adrenal - MH mannoheptulose - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid     

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.     

Evidence against a rate-limiting role of proinsulin processing for maximal insulin secretion in subjects with impaired glucose tolerance and beta-cell dysfunction

In subjects with impaired glucose tolerance (IGT) insulin secretion is impaired. Increased proinsulin/insulin (PI/I) ratios suggest that there is also reduced processing of proinsulin to insulin in this condition. The PI/I ratio in the insulin secretory granule is ideally assessed by plasma measurements in response to acute stimulation of insulin secretion. In the present study we tested the hypothesis that maximal stimulation of insulin secretion results in exhaustion of the proinsulin conversion pathway to insulin. We therefore determined the PI/I ratio in 11 normal glucose-tolerant subjects (NGT) and 11 subjects with IGT in response to glucose (squarewave hyperglycemic clamp, 10 mmol/L), glucagon-like peptide-1 (GLP-1; primed-continuous infusion), and arginine given during the continued GLP-1 infusion. In IGT, insulin levels were significantly lower during the first phase (144 +/- 20 vs. 397 +/- 119 pmol/L; P = 0.02), at the end of the GLP infusion (2142 +/- 350 vs. 5430 +/- 1091 pmol/L; P: = 0.002), and in response to arginine (3983 +/- 375 vs. 8663 +/- 1430 pmol/L; P = 0.005). In response to glucose, the minimum PI/I ratio was significantly higher in IGT (3.4 +/- 0.6%) than in NGT (1.4 +/- 0.5%; P = 0.02), suggesting defective proinsulin processing in this condition. In subjects with IGT, the PI/I ratio decreased significantly after GLP-1 priming (1.7 +/- 0.2%; P = 0.02) and after arginine given during GLP-1 (1.4 +/- 0.2%; P = 0.007) and was not significantly different from those values in NGT (1.3 +/- 0.2% and 1.3 +/- 0.2%, respectively; both P = NS). In conclusion, during maximal stimulation of insulin secretion in subjects with IGT, the PI/I ratio in plasma decreased significantly and was not different from that in normal controls. This strongly argues against the hypothesis that defective processing of proinsulin to insulin represents a major component of the beta-cell dysfunction in IGT.     

A link between insulin resistance and hyperinsulinemia: inhibitors of phosphatidylinositol 3-kinase augment glucose-induced insulin secretion from islets of lean, but not obese, rats

Wortmannin (5-100 nM), a specific phosphatidyinositol 3-kinase inhibitor, augmented 8 mM glucose-induced insulin secretion from control Sprague Dawley rat islets in a dose-dependent manner. This effect persisted after its removal from the perifusion medium; however, this augmenting effect was reduced by the calcium channel inhibitor nitrendipine or by lowering the glucose level to 3 mM. Wortmannin amplified insulin release induced by the combination of 6-8 mM glucose plus 1 microM carbachol; however, it had no effect on phorbol ester- or alpha-ketoisocaproate-induced insulin secretion. The potentiating action of wortmannin on 8 mM glucose-induced release was duplicated by LY294002. Wortmannin had no effect on glucose usage rates or inositol phosphate accumulation in [3H]inositol-prelabeled islets. Of particular significance, although 50 nM wortmannin potentiated 8 mM glucose-induced secretion from islets of lean Zucker control rats, the fungal metabolite had little effect on 8 mM glucose-induced release from islets of insulin-resistant Zucker fatty rats. These findings support the concept that the same biochemical process, inhibition ofphosphatidyinositol 3-kinase, that causes peripheral tissue insulin resistance enhances beta-cell sensitivity to glucose and produces a compensatory increase in insulin secretion from these cells. The efficacy of wortmannin depends on the in vivo status of the donor's insulin signaling pathways. This elegant biochemical control mechanism in beta-cells ensures the maintenance of glucose homeostasis despite a reduction in insulin action on peripheral tissues.     

Vasopeptidase inhibition prevents nephropathy in Zucker diabetic fatty rats

BACKGROUND: Blocking the renin-angiotensin system is an established therapeutic principle in diabetic nephropathy. We investigated whether inhibition of both neutral endopeptidase and ACE (vasopeptidase inhibition) can prevent functional and morphological features of nephropathy in the Zucker diabetic fatty (ZDF) rat, an animal model of type II diabetes. METHODS: Homozygous (fa/fa) ZDF rats (each n=15) aged 10 weeks were treated with placebo, ramipril (1 mg/kg/day in drinking water), or the vasopeptidase inhibitor AVE7688 (45 mg/kg/day in chow). Metabolic parameters and renal function (metabolic cages) were assessed at baseline (age 10 weeks), and at age 17, 27, and 37 weeks. Twenty heterozygous animals (fa/-) served as lean, nondiabetic controls. At age 37 weeks, the animals were sacrificed and the kidneys analyzed histopathologically. RESULTS: Overt diabetes mellitus (blood glucose >20 mmol/l) was established at age 17 weeks in all homozygous ZDF rats. In the placebo group, urinary protein excretion increased progressively from 8+/-1 (baseline) to 342+/-56 mg/kg/day (week 37) whereas diabetes and proteinuria were absent in the lean control group. Ramipril tended to reduce albuminuria and morphological damage (p=ns) but AVE7688 virtually prevented albuminuria (33+/-12 mg/kg/day, p<0.05 vs. ZDF placebo) and drastically reduced the incidence and severity of glomerulosclerosis and tubulointerstitial damage. CONCLUSIONS: In ZDF rats, development of diabetes mellitus is accompanied by functional and morphological kidney damage that resembles human diabetic nephropathy. Diabetic nephropathy can be prevented by chronic vasopeptidase inhibition.     

Effects of weight loss and reduced hyperglycemia on the kinetics of insulin secretion in obese non-insulin dependent diabetes mellitus

Impairment in pancreatic production of insulin, a cardinal feature of noninsulin dependent diabetes mellitus (NIDDM), was quantified and the kinetics of insulin secretion characterized in six obese individuals with NIDDM before and after weight loss (18.0 +/- 3.0 kg, mean +/- SEM) using a validated mathematical model that employs C-peptide as a marker of the in vivo rate of insulin secretion. The metabolic clearance of C-peptide, assessed by decay analysis after bolus injection of biosynthetic human C-peptide, was not changed by weight loss (0.143 +/- 0.009 L/min.m2 vs. 0.137 +/- 0.010 L/min.m2). Kinetic parameters from each individual's decay curve before and after weight loss were used to derive accurate rates of secretion during the basal (postabsorptive) state, an oral glucose tolerance test and two hyperglycemic clamps. Basal rates of insulin secretion declined 20 +/- 5 pmol/min.m2 (96 +/- 15 to 76 +/- 15 pmol/min.m2, P less than 0.05) concomitant with decreases of 6.9 +/- 0.9 mmol/L in fasting serum glucose (13.7 +/- 1.0 to 6.8 +/- 0.7 mmol/L, P less than 0.05), 60 +/- 14 pmol/L in serum insulin (134 +/- 30 to 74 +/- 15 pmol/L, P less than 0.05), and 0.15 +/- 0.03 pmol/ml in plasma C-peptide (0.67 +/- 0.11 to 0.52 +/- 0.08 pmol/ml, P less than 0.05) concentrations. As expected, weight loss resulted in improved glucose tolerance as measured by the glycemic profiles during the oral glucose tolerance test (P less than 0.05 analysis of variance). The insulin secretory response before weight loss showed a markedly reduced ability to respond appropriately to an increase in the ambient serum glucose. After weight loss, the pancreatic response was more dynamic (P less than 0.05, analysis of variance) and parralleled the moment-to-moment changes in glycemia. Insulin production above basal doubled (11.2 +/- 3.2 to 24.5 +/- 5.8 nmol/6h.m2, P less than 0.05) and peak rates of insulin secretion above basal tripled (55 +/- 16 to 157 +/- 32 pmol/min/m2, P less than 0.05). To assess the beta-cell response to glucose per se and the changes associated with weight reduction, two hyperglycemic clamps were performed at steady state glucose levels in the range characteristic of individuals with severe NIDDM. At a fixed glycemia of 20 mmol/L, average rates of insulin secretion increased almost 2-fold with treatment (161 +/- 41 to 277 +/- 60 pmol/min.m2, P less than 0.05). At an increment of 6 mmol/L glucose above prevailing fasting glucose levels, the average rate of insulin secretion increased 53% (120 +/- 21 to 183 +/- 39 pmol/min.m2, P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Relative hyperproinsulinemia as a sign of islet dysfunction in women with impaired glucose tolerance.

Proinsulin release is increased relative to insulin secretion in subjects with type 2 diabetes, indicative of islet dysfunction. However, it has not been conclusively shown whether there is an increased relative proinsulin release in subjects with impaired glucose tolerance (IGT), i.e. whether it precedes the development of diabetes. We therefore determined the proinsulin to insulin ratios in the fasting state and after acute stimulation of insulin secretion in 23 postmenopausal women, aged 61-62 yr (mean +/- SD, 61.7 +/- 0.5 yr). Ten women had normal glucose tolerance (NGT), and 13 had IGT. The groups were matched for insulin sensitivity and did not differ in body mass index. Proinsulin and insulin secretion were measured after arginine stimulation (5 g, i.v.) at three glucose levels (fasting, 14 mmol/L, and >25 mmol/L), and the acute insulin (AIR(arg)) and proinsulin responses (APIR(arg)) were calculated as the mean 2-5 min postload increase. At fasting glucose, levels of insulin, proinsulin, or the proinsulin/insulin ratio (13.6 +/- 5.0% vs. 11.1 +/- 2.7%; P = NS) did not differ between NGT and IGT. Although the AIR(arg) values were decreased in the IGT group at all glucose levels (P < 0.05), the absolute proinsulin levels and the APIRs(arg) were similar between IGT and NGT women. Therefore, the IGT women had higher proinsulin/insulin ratios at 14 mmol/L (10.7 +/- 4.4% vs. 6.4 +/- 1.8%; P = 0.006) and more than 25 mmol/L glucose (11.4 +/- 5.2% vs. 6.7 +/- 2.1%; P = 0.007). The IGT group had increased APIR(arg)/AIR(arg) at fasting (2.2 +/- 1.4% vs. 1.3 +/- 0.6%; P = 0.047) and more than 25 mmol/L glucose (3.5 +/- 1.6% vs. 2.3 +/- 0.7%; P = 0.037). We conclude that women with IGT exhibit increased relative proinsulin secretion, suggesting a defect in the intracellular proinsulin processing before diabetes develops.