The dipeptidyl peptidase-4 inhibitor vildagliptin has the capacity to repair ß-cell dysfunction and insulin resistance.

The aim of the present study was to determine whether the dipeptidyl peptidase (DPP)-4 inhibitor could repair pancreatic ß-cell dysfunction and insulin resistance. Ten subjects with type 2 diabetes who had never received DPP-4 inhibitor treatment were enrolled in the study. Just before and 3 months after twice-daily administration of vildagliptin (50 mg tablets), insulin secretion and insulin sensitivity were estimated using 2-compartment model analysis of C-peptide kinetics and insulin-modified minimal model parameters, respectively. The first-phase insulin secretion (CS1) was determined as the sum of the C-peptide secretion rate (CSR) from 0 to 5 min (normal range 6.8-18.5 ng/ml/min). The whole-body insulin sensitivity index (SI) was calculated using a minimal model software program (normal range 2.6-7.6×10(-4)/min/µU/ml). After vildagliptin treatment, reductions in mean (± SE) HbA1c were noted (43.28±1.53 vs. 40.98±1.77 mmol/mol; p=0.019). Vildagliptin treatment increased the area under the curve for the C peptide reactivity (CPR) (AUCCPR; 26.66±5.15 vs. 33.02±6.12 ng/ml · 20 min; p=0.003) and CS1 (0.80±0.20 vs. 1.35±0.38 ng/ml/min; p=0.037) in response to an intravenous glucose load. -Vildagliptin treatment significantly increased SI (0.46±0.27 vs. 1.21±0.48×10(-4)/min/µU/ml; p=0.037). The long-term administration of vildagliptin improved CS1 and Si suggesting that this drug has the capacity to repair impairments in pancreatic ß-cell function and insulin resistance in type 2 diabetes.

Efficacy and safety of the dipeptidyl peptidase-4 inhibitor sitagliptin compared with α-glucosidase inhibitor in Japanese patients with type 2 diabetes inadequately controlled on sulfonylurea alone (SUCCESS-2): a multicenter, randomized, open-label, non-inferiority trial.

We assessed the efficacy and safety of sitagliptin compared with α-glucosidase inhibitor (αGI) in 120 of Japanese patients with type 2 diabetes mellitus (T2DM) inadequately controlled on stable ≤2 mg/day glimepiride alone [mean hemoglobin A1c (HbA1c) 7.7%] by the randomized, active-controlled, non-inferiority trial. Patients were randomly assigned to receive additional sitagliptin or αGI for 24 weeks. The primary endpoint was change in HbA1c from baseline to week 12. After 12 weeks, sitagliptin reduced HbA1c by -0.44% (p < 0.001) relative to αGI. At 24 weeks, the reduction was almost identical between the groups (-0.091%, p = 0.47). Gastrointestinal disorders were more common with αGI than with sitagliptin, but only minor hypoglycaemia occurred in both groups at similar frequency. These data suggested that sitagliptin was not inferior to αGI for reduction of HbA1c in Japanese T2DM patients receiving glimepiride alone, and well tolerated with minimum risk of gastrointestinal symptoms and hypoglycaemia.

Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor.

The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the proband's lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.

Clinical and immunogenetic characteristics of fulminant type 1 diabetes associated with pregnancy.

OBJECTIVE: The objective of this study was to characterize the clinical and immunogenetic features of Japanese pregnancy-associated fulminant type 1 diabetes (PF). A group of patients with PF was compared with a group of patients of child-bearing age with fulminant type 1 diabetes that was not associated with pregnancy (NPF) in a nationwide survey conducted from 2000-2004. PATIENTS: The clinical characteristics of the 22 patients in the PF group were compared with those of the 48 patients in the NPF group. Human leukocyte antigen (HLA) class II DR and DQ genotyping of 17 PF and 20 NPF patients was performed. RESULTS: Arterial pH was significantly lower (P = 0.0366), and amylase values tended to increase in PF patients compared with NPF patients (P = 0.0515). In 22 PF patients, 18 developed disease during pregnancy (26.3 wk; range, 7-38), whereas four cases occurred immediately after delivery (10.5 d; range, 7-14 d). Twelve cases that developed during pregnancy resulted in stillbirth (67%), and five of the six fetal cases that survived were delivered by cesarean section. The haplotype frequency of HLA DRB1*0901-DQB1*0303 in PF was significantly higher than those in NPF (P = 0.0244) and controls (P = 0.0001), whereas that of DRB1*0405-DQB1*0401 in NPF was significantly higher than those in PF (P = 0.0162) and controls (P < 0.0001). CONCLUSIONS: The clinical symptoms of PF patients were more severe than those of NPF patients, and the prognosis of their fetuses was extremely poor. The type 1 diabetes-susceptible HLA class II haplotype is distinct in PF and NPF patients, suggesting that different HLA haplotypes underlie the presentation of PF or NPF.

Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells.

The effects of sulfonylureas and a biguanide on membrane-bound low Km cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 microM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 microM). Tolbutamide (0.5-10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. Km, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 microM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.

Effects of dithiothreitol on insulin-sensitive phosphodiesterase in rat fat cells.

Dithiothreitol activates the low-Km membrane-bound cyclic AMP phosphodiesterase when incubated with the enzyme in a cell-free system. To investigate the mechanism of its activation, we studied the effect of protease inhibitors. Isolated fat cells obtained from Sprague-Dawley rats were incubated in Krebs-Henseleit Hepes buffer, pH 7.4, at 37 degrees C with and without insulin (2 nM, 10 min). A crude microsomal fraction prepared by differential centrifugation was suspended in 0.25 M sucrose containing 10 mM Tes buffer, pH 7.5, with and without 2 mM dithiothreitol and protease inhibitors at 4 degrees C for 48 h. Dithiothreitol stimulated the phosphodiesterase, in a time-dependent manner. As little as 0.02 mM dithiothreitol activated the enzyme, and the maximally effective dose was 2-10 mM. Among the various protease inhibitors tested, antipain, leupeptin, chymostatin and E-64 were the most effective in preventing activation of the enzyme by dithiothreitol. Antipain also inhibited release of the enzyme from the bound fraction. These results suggest that activation of the low-Km phosphodiesterase by dithiothreitol may be provoked by stimulation of an endogenous thiol protease.

Mitogen-activated protein kinase and p70 ribosomal protein S6 kinase are not involved in the insulin-dependent stimulation of cAMP phosphodiesterase kinase in rat adipocytes.

To elucidate the mechanism of anti-lipolytic action of insulin in rat epididymal adipocytes, we explored the potential mechanism that might be involved in the hormone-dependent stimulation of cAMP phosphodiesterase (PDE) kinase. PDE kinase was assayed in a cell-free system. Both wortmannin and LY294002, highly specific inhibitors of phosphatidylinositol 3-kinase, almost completely blocked the hormonal effect not only on PDE kinase but also on mitogen-activated protein (MAP) kinase. Neither PD98059, a specific inhibitor of MAP kinase, nor rapamycin, a potent inhibitor of insulin-dependent stimulation of p70 ribosomal protein S6 kinase (p70S6K), had inhibitory effect on that of PDE kinase. These results are consistent with the view that (i) insulin-activated PDE kinase as well as MAP kinase and p70S6K are localized downstream of phosphatidylinositol 3-kinase, (ii) PDE kinase is distinct from either MAP kinase or p70S6K and (iii) PDE kinase does not exist downstream of either MAP kinase or p70S6K. It is suggested that PDE kinase and MAP kinase or p70S6K may be localized in separate branches of the cascade of insulin action. The branching point of the cascade could be either at or below the level of phosphatidylinositol 3-kinase.

Insulin resistance of fat cells from spontaneously diabetic KK mice. Analysis of insulin-sensitive phosphodiesterase.

In an attempt to determine the mechanism of insulin resistance in the presence of obesity, we examined effects of insulin on insulin-sensitive phosphodiesterase (PDE) in spontaneously diabetic KK mice. Isolated fat cells prepared from epididymal adipose tissue were incubated, with or without insulin, for 10 min. In the case of subcellular fractionation, only membrane-bound PDE was activated by insulin, as was noted in the case of rat fat cells. The specific activity was decreased in KK mice compared with control C57BL/6 mice. The dose-response curve, expressed as a percent of the maximal insulin effect, shifted to the right and the increase of ED50 indicated a decreased insulin sensitivity in the KK mice. The maximal insulin effect did not change, either when expressed as a percent of the basal enzyme activity or when expressed on a per cell basis. Specific binding of [125I]-insulin in fat cells increased in KK mice and curvilinear Scatchard plots showed an increase of the high-affinity sites. These data indicate that impairment of PDE activation in fat cells of KK mice relates to postreceptor defects and the uncoupling may result in a decreased sensitivity.

Is somatostatin a true local inhibitory regulator of insulin secretion?

Pancreatic somatostatin was depleted after oral administration of cysteamine to rats, yet the B-cells in the isolated islets were morphologically and functionally intact. Compared with islets from the rats not given cysteamine, the somatostatin-depleted islets released larger amounts of insulin during 1 h of incubation by glucose or 3-isobutyl-1-methylxanthine stimulation. Therefore, the possibility that pancreatic somatostatin may locally regulate the inhibitory effects of insulin secretion has to be considered.

Islet amyloid polypeptide/amylin in pancreatic beta-cell line derived from transgenic mouse insulinoma.

We examined the production and secretion of IAPP in a beta-cell line, MIN6, which is derived from an insulinoma obtained by targeted expression of the SV40 T-antigen gene in a transgenic mouse. RNA blot analysis revealed an abundance of IAPP and insulin II mRNA in the cells, findings comparable with those in the pancreas of a normal mouse. The presence of IAPP and insulin was confirmed immunohistochemically and by RIA. Analysis of the reverse-phase HPLC identified IAPP in cells with authentic mouse IAPP. Raising the glucose concentration from 5.6 to 25 mM failed to induce increments in IAPP and insulin II mRNAs. The cells secrete IAPP and insulin for short- and long-term incubations in response to concentration of glucose in the medium. These features resemble those of islet cells from normal animals. This beta-cell line will aid in analyzing the regulation of IAPP gene expression and the mechanisms of IAPP biosynthesis and secretion.

Increased sensitivity of insulin-sensitive phosphodiesterase to insulin in fat cells from streptozotocin diabetic rats.

The effects of insulin on insulin-sensitive phosphodiesterase were investigated in fat cells from control and streptozotocin diabetic rats. Isolated cells were incubated at 37 C for 10 min, with and without insulin. A crude microsomal fraction prepared by differential centrifugation was assayed for phosphodiesterase activity. The enzyme activities in diabetic rats were higher at 0-1 nM insulin than in control rats. The dose-response curve of insulin was biphasic and of the convex type in both groups. In diabetic rats, the curve shifted to the left, and half-maximal stimulation was obtained at 0.06 nM insulin compared with 0.16 nM insulin in control rats. Kinetic analyses of the enzyme from diabetic rats revealed much the same findings as obtained in the controls. Specific binding of insulin in fat cells from control and diabetic rats was 3% and 4.9%/2 X 10(5) cells, respectively, at 24 C for 60-min incubation. Scatchard analysis indicates that the overall binding affinity in diabetic cells was greater than that in the control cells. These results suggest that the insulin effector system related to phosphodiesterase activation is intact and has an increased sensitivity in fat cells from streptozotocin diabetic rats; there is also a good correlation with alteration of insulin binding to its receptors.

Dual mechanism involved in the hydrolysis of polyphosphoinositides in rat pancreatic islets.

We investigated insulin secretion and inositol phosphate formation in intact and permeabilized rat pancreatic islets, the objective being to elucidate mechanisms of activation of phospholipase-C in pancreatic islets. The intact islets prelabeled with myo-[3H]inositol were incubated in Krebs-Ringer bicarbonate buffer containing 10 mM LiCl and 1 mM myoinositol. Glucose, alpha-ketoisocaproate (KIC), and sulfated cholecystokinin (CCK8S) increased insulin secretion and formation of [3H]inositol phosphate, [3H]inositol bisphosphate, and [3H] inositol trisphosphate. Mannoheptulose, a glucokinase inhibitor, inhibited glucose-induced insulin secretion and [3H]inositol phosphate formation; however, it did not inhibit KIC- and CCK8S-induced secretion and formation. Both glucose- and KIC-induced insulin secretion and [3H]inositol phosphate formation were blocked by 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation in the mitochondria. The islets prelabeled with myo-[3H]inositol were permeabilized by digitonin and then incubated in intracellular mimicking medium containing 1 microM Ca2+ and 2.5 mM ATP. Glucose had no effect on [3H]inositol phosphate formation in the permeabilized islets, and CCK8S increased the formation of [3H]inositol phosphates. Thus, phospholipase-C in pancreatic islets is activated not only via ligand-receptor interaction in the plasma membrane in the case of hormone stimulation, but also by metabolic product(s) in the case of fuel stimulation.

Biphasic insulin response to high glucose and a role of protons and calcium.

In order to determine the role of protons and Ca++ in the biphasic insulin response to glucose, we studied the effect of monensin, a carboxylic ionophore, on the first phase and second phase of glucose-induced insulin release and Ca++ efflux from perifused rat pancreatic islets. The agent, 1-100 nM, dose dependently inhibited the insulin release from the islets incubated for 60 min in Krebs-Ringer bicarbonate buffer containing 16.7 mM glucose. Islet 14CO2 production rates from D-(U-14C)glucose were not affected by 10 or 100 nM monensin. Perifusion of the islets prelabeled with 45Ca++ demonstrated that 100 nM monensin had only a slight inhibitory effect on the first phase insulin response to 16.7 mM glucose and no effect on 45Ca++ efflux. This agent inhibited the second phase insulin release and depressed 45Ca++ efflux. When monensin was added at the start of the second phase release, the release was inhibited. When exposed to the agent before the stimulation by glucose, the first phase insulin release was observed, albeit significantly decreased, and the start of the insulin release and 45Ca++ efflux was delayed. The agent, added 30 min after the change to high glucose, immediately inhibited the insulin release. Thus, the first phase insulin release is mediated mainly through a mechanism which is not related to protons generated from glucose metabolism. Protons may be a crucial coupling factor in the second phase insulin release.

Changes in pancreatic somatostatin content in spontaneously diabetic mice, as determined by radioimmunoassay and immunohistochemical methods.

A specific RIA for somatostatin (SRIF) was used to determine the SRIF content of the pancreas and hypothalamus in spontaneously diabetic C57BL/KsJ dbdb and C57BL/6J obob mice. In addition, SRIF- and glucagon-containing cells were examined in the pancreatic islets with an immunohistochemical technique. In dbdb mice, immunoassayable pancreatic SRIF content was increased, as was the number of SRIF- or glucagon-containing cells. In obob mice, immunoassayable pancreatic SRIF content was also increased, but no increase was noted in the number of SRIF- or glucagon-containing cells. The hypothalamic SRIF content of either strain was not different from that of controls. These results regarding pancreatic SRIF content were in accord with some but not all previous reports. These differences may be related to the age of the mice or to the conditions in which they were bred. The pancreatic SRIF increase in both dbdb and obob mice may be attributable to hyperglucagonemia, hyperglycemia, or a decrease in insulin action. Further work is necessary to clearly delineate the mechanism.

Effect of calcium on the secretion of somatostatin and insulin from pancreatic islets.

Calcium ionophore A23187 (20 micrometer) evoked the secretion of somatostatin (SRIF) as well as insulin from isolated rat pancreatic islets in a medium containing a relatively low concentration of calcium (0.9 mM) and a low concentration of glucose (5.5 mM). A high level of extracellular calcium (7.5 mM) also had a stimulatory effect on SRIF and insulin release. On the other hand, in the presence of high glucose (16.7 mM), A23187 had different effects on D and B cells; insulin release was markedly suppressed by A23187, but SRIF secretion was significantly enhanced. A high concentration of glucose (16.7 mM) did not stimulate SRIF secretion at low extracellular calcium concentration (0.25 mM), at which level insulin release is significantly enhanced. These findings indicate that calcium may play an important role in the regulation of the secretion of SRIF as well as insulin and suggest that the B and D cells differ in their sensitivity to the calcium ion.

Effect of glucose on somatostatin secretion from isolated pancreatic islets of normal and streptozotocin-diabetic rats.

Pancreatic somatostatin (SRIF) secretion was examined using the RIA described in earlier paper. Ten isolated rat pancreatic islets were incubated for 30 min in 1 ml Krebs-Ringer bicarbonate buffer. Glucose (5.6 mM) caused a small but significant increase of SRIF secretion. The maximal secretion rate was observed at 16.7 mM glucose, and the half-maximal rate was seen at about 9.7 mM. Islets preincubated with 16.7 mM glucose released higher levels of SRIF and insulin during the subsequent incubation with 16.7 mM glucose than did islets preincubated with 2.8 mM glucose. Glucose-induced SRIF secretion was suppressed by epinephrine, but beta-adrenergic stimulation (epinephrine and phentolamine) produced an increase in SRIF secretion. Islets taken from rats 2 days after streptozotocin administration released minimal amounts of insulin. Basal and glucose-induced SRIF secretion from these islets, which had relatively unchanged SRIF contents and D cell numbers, equaled SRIF secretion from control rat islets. Islets taken from rats 6 weeks after streptozotocin administration, however, had increased SRIF content and D cell numbers, and they oversecreted SRIF. We conclude that pancreatic SRIF secretion can be induced by glucose and modulated by catecholamines and preexposure to high glucose, and the duration and severity of diabetes may be an important determinant of the changes in pancreatic D cell structure and function.

Hypersecretion of islet amyloid polypeptide from pancreatic islets of ventromedial hypothalamic-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 RIA 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 mM and 16.7 mM glucose (2.99 +/- 0.98 and 11.2 +/- 1.29 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 mM 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.

Relation between glucose-stimulated insulin secretion and intracellular calcium accumulation studied with a superfusion system of a glucose-responsive pancreatic beta-cell line MIN6.

The concept that cytosolic free calcium is the primary signal for insulin secretion is generally accepted, but studies with intact pancreatic beta-cells of the cytosolic free calcium concentration-insulin secretion relationship have produced contradictory and sometimes confusing data. We designed a superfusion system of a pancreatic beta-cell line, MIN6, loaded with fura-2, which allowed simultaneous measurement of cytosolic free calcium concentration and insulin secretion. MIN6 cells released insulin in response to high glucose, thus resembling events in normal islet cells. Cytosolic free calcium concentration and insulin secretion rapidly increased, and the increase was suppressed by mannoheptulose or by sodium azide. This increase was suppressed by lowering the temperature of the medium. Cytosolic free calcium concentration and the insulin secretion induced by leucine were not influenced by mannoheptulose but were inhibited by sodium azide. In RINm5F cells, cytosolic free calcium concentration and insulin release were slightly suppressed by glucose but were increased by ionomycin. There was a close relation between the rise in cytosolic free calcium concentration and insulin secretion in all cases. Our findings provide a direct evidence that a rise in cytosolic free calcium concentration depends on glucose metabolism and is a primary signal for insulin secretion.

Secretion of islet amyloid polypeptide in response to glucose.

The content of islet amyloid polypeptide (IAPP) in isolated rat pancreatic islets was determined by a radioimmunoassay. Reverse-phase high-performance liquid chromatography analysis revealed that a main peak of IAPP immunoreactivity in the extracts from the islets corresponded to a synthetic rat IAPP. Secretion of IAPP from the cells is regulated by the extracellular glucose concentration. Thus, IAPP may be a novel regulator for glucose homeostasis and changes in the secretion perhaps relate to insular amyloid deposits and impaired glucose tolerance in type 2 diabetes mellitus.

Effect of ACE gene on diabetic nephropathy in NIDDM patients with insulin resistance.

We investigated the influence of the angiotensin-converting enzyme (ACE) gene on the onset and/or progression of diabetic nephropathy in 62 Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM; type II diabetes). Because a number of factors are believed to be involved in the onset and/or progression of diabetic nephropathy, especially in patients with NIDDM, we selected the patients with well-matched risk factors, duration of disease, glycemic control, blood pressure, and others. All patients had normal renal function and none were receiving ACE inhibitors. Patients were divided into three groups according to albumin excretion rate (AER): group A, patients with an AER less than 15 microg/min (n = 29); group B, patients with an AER between 15 and 70 microg/min (n = 19); and group C, patients with an AER greater than 70 microg/min (n = 14). The glucose disposal rate was estimated using a euglycemic hyperinsulinemic clamp. We determined the mean glucose disposal rate in 132 patients with NIDDM (6.49 mg/kg/min). Patients with a glucose disposal rate less than the mean rate were considered to have a high degree of insulin resistance (n = 36). The presence of an insertion/deletion (I/D) polymorphism of the ACE gene was determined by the polymerase chain reaction method. Among patients with a high degree of insulin resistance, diabetic nephropathy was present in 2 of 11 patients with the II genotype of the ACE gene compared with 19 of 25 patients with the ID or DD genotype (P = 0.0024). The prevalence of diabetic nephropathy was greater in patients with both significant insulin resistance and the D allele (19 of 25) than in the remaining patients (14 of 37; odds ratio, 5.20). These results suggest that the ACE gene influences the onset and/or progression of diabetic nephropathy in patients with NIDDM with significant insulin resistance.