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.

Fulminant type 1 diabetes as a high risk group for diabetic microangiopathy--a nationwide 5-year-study in Japan.

AIMS/HYPOTHESIS: The aim of the present study was to assess the development of microangiopathy in patients with fulminant type 1 diabetes, a novel subtype of type 1B diabetes. MATERIALS AND METHODS: In a nationwide survey, we followed 41 patients with fulminant type 1 diabetes and 76 age- and sex-matched patients with type 1A diabetes for 5 years. The following data were recorded every 12 months after the onset of diabetes: seven-point blood glucose concentrations, HbA1c level, urinary albumin excretion, serum C-peptide level, blood pressure, daily dosages of insulin, frequency of severe hypoglycaemic episodes, and neurological and fundoscopic examination. RESULTS: The 5-year cumulative incidence of microangiopathy was 24.4% in fulminant type 1 diabetes and 2.6% in type 1A diabetes. In longitudinal studies using the Kaplan-Meier method, the cumulative incidence of each form of microangiopathy was significantly higher in fulminant type 1 diabetes than in type 1A diabetes; retinopathy was 9.8% vs 0% (p=0.014), nephropathy 12.2% vs 2.6% (p=0.015) and neuropathy 12.2% vs 1.3% (p=0.010), respectively. Mean HbA1c levels were similar in the fulminant and type 1A diabetes groups during the follow-up periods. However, the mean M-value, mean insulin dosages and the frequency of severe hypoglycaemic episodes were significantly higher, and the mean postprandial C-peptide level was significantly lower in the fulminant type 1 diabetes group. CONCLUSIONS/INTERPRETATION: These data suggest that patients with fulminant type 1 diabetes are a high-risk subgroup for diabetic microangiopathy associated with the lack of endogenous insulin secretion from the onset of 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.

Different contribution of class II HLA in fulminant and typical autoimmune type 1 diabetes mellitus.

AIMS/HYPOTHESIS: Fulminant type 1 diabetes, which is characterised by a markedly acute onset of diabetes and an absence of islet-related autoantibodies, accounts for 20% of type 1 diabetes in Japan. We aimed to clarify the contribution of the HLA subtype to fulminant type 1 diabetes in Japanese. METHODS: We determined the serological subtypes of HLA-A, -DR and -DQ in 115 patients with fulminant type 1 diabetes, 98 patients with typical type 1A diabetes and 190 normal control subjects. RESULTS: The frequency of HLA-DR4, but not DR9, was significantly higher in fulminant type 1 diabetes, while those of HLA-DR1, DR2, DR5 and DR8 were significantly lower than those in controls. In contrast, DR9 but not DR4 was more frequent and DR2 was extremely rare in typical type 1A diabetes. Haplotype analysis revealed that DR4-DQ4 was significantly more frequent, and both DR2-DQ1 and DR8-DQ1 were less frequent in fulminant diabetes. In type 1A diabetes, DR2-DQ1 was extremely rare while DR9-DQ3 was significantly more frequent. In the combination analysis, the homozygotes of DR4-DQ4 in fulminant type 1 diabetes and DR9-DQ3 in typical type 1A diabetes indicated high odds ratios (13.3 and 13.3, respectively). CONCLUSIONS/INTERPRETATION: Our results suggest that class II HLA contributes to the development of fulminant type 1 diabetes. Susceptibility and resistance of the HLA subtype to type 1 diabetes are distinct between fulminant and typical autoimmune type 1 diabetes.

A patient with subcutaneous-insulin resistance treated by insulin lispro plus heparin.

Severe resistance to subcutaneous insulin but sensitivity to intravenous insulin persisted for 11 years in a 23-year-old diabetic woman. Several therapeutic trials revealed that (1) intravenous regular insulin improved her metabolic control; (2) continuous subcutaneous infusion (CSII) treatment with regular insulin or insulin lispro caused hyperglycemic period with hypoinsulinemia and hypoglycemic period with hyperinsulinemia alternately; (3) adding heparin to insulin lispro in CSII resulted in dramatic increase of serum insulin level and improvement of glycemic control; and (4) regular insulin plus heparin in CSII could not increase serum insulin level and thus the glycemic values was not improved. From these results, the patient followed the insulin lispro plus heparin protocol and obtained a better glycemic control without any adverse events. Effectiveness of this therapy may lead us to further understanding of pathophysiology of this syndrome.

Analysis of the insulin-sensitive phosphodiesterase 3B gene in type 2 diabetes.

We screened for mutations in the gene of insulin-sensitive phosphodiesterase 3B (PDE3B), which regulates antilipolytic actions of insulin via reduction of intracellular cyclic AMP levels, in Japanese patients with type 2 diabetes mellitus and lipoatrophic diabetes mellitus using single-stranded conformation polymorphism analysis and Southern analysis and investigated frequencies of variable number of tandem repeats. A silent polymorphism at the Arg463 codon (AGG-->AGA) in exon 4 was identified after examining all 16 exons and exon-intron splicing junctions of the gene. This polymorphism was found in 53 of 100 subjects with type 2 diabetes mellitus, 2 of 5 lipoatrophic diabetic patients and 24 of 50 control subjects, without any significant difference in allele frequency between groups. An EcoRI restriction fragment length polymorphism was identified in patients with type 2 diabetes mellitus and control subjects, again with no differences in occurrence. The allelic distribution of two polymorphic tandem repeats sequences in introns 5 and 12 of the gene did not differ significantly between patients with type 2 diabetes mellitus and control subjects. In conclusion, alterations in the PDE3B gene are unlikely to contribute importantly to the pathogenesis of type 2 diabetes mellitus or lipoatrophic diabetes mellitus in Japan.

Pathophysiologic phenotypes of Japanese subjects with varying degrees of glucose tolerance: using the combination of C-peptide secretion rate and minimal model analysis.

We tried to characterize the clinical features associated with glucose metabolism in the development of diabetes. Study subjects were glucose-tolerant subjects without a family history of diabetes (normal glucose tolerance [NGT]1 group, n = 15) and with a first-degree diabetes relative (NGT2, n = 9), 12 subjects with impaired glucose tolerance (IGT), and 13 subjects with type 2 diabetes mellitus (DM). The first phase C-peptide secretion (CS1), insulin sensitivity (Si), and glucose effectiveness (Sg) were assessed by the combination of C-peptide 2-compartment model and minimal model analyses. Using these parameters, each group was characterized: CS1 was decreased in NGT2 and IGT compared with NGT1 and further decreased in DM; Si was not different among NGT1, NGT2, and IGT, whereas Si was decreased in DM; CS1 x Si value was decreased in NGT2 compared with NGT1 and decreased in IGT, DM, progressively; Sg was decreased in IGT and DM compared with NGT1 and NGT2. CS1 x Si and Sg values could segregate each group distinctively, although it had a large variety of phenotypes. CS1 x Si value and Sg are assumed to represent the contributions of insulin-dependent and independent mechanisms to glucose tolerance, respectively, and thus, both mechanisms should play an important role in the characterization of pathophysiologic phenotypes of the subjects with various degrees of glucose tolerance.

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.

Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic beta cells.

Cytokines induce apoptosis in pancreatic beta cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-alpha) in the apoptosis of beta cells. Using the ribonuclease (RNase) protection assay and the reverse transcriptase-polymerase chain reaction (RT-PCR) method, we confirmed that TNF receptor 1 (TNFR1), TNFR1-associated death domain protein (TRADD), Fas receptor-associated intracellular protein with death domain (FADD), and FADD-like interleukin-1beta-converting enzyme (FLICE) were expressed in the pancreatic beta cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-alpha induced time- and dose-dependent apoptotic nuclear changes in these beta cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-alpha generated new 3'-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-alpha produced characteristic apoptotic patterns of DNA fragments formed by internucleosomal hydrolysis of static chromatin. In addition, C2-ceramides and natural ceramides dispersed in a solvent mixture of ethanol and dodecane induced characteristic features of apoptosis in MIN6 cells, mimicking TNF-induced DNA damage. We also determined endosomal ceramide production after TNF-alpha (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-alpha can cause apoptosis in pancreatic beta cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.

Increased activity and expression of MAP kinase in HCC model rats induced by 3'-methyl-4-dimethylamino-azobenzene.

BACKGROUND/AIMS: The ras-mitogen-activated protein kinase (MAPK) cascade plays an important role not only in the mitogenic signal transduction pathway but also in the development of cancer, and it is believed to be one of the important regulators in normal hepatocytes and hepatocellular carcinoma. The aim of this study was to determine the role of insulin receptor substrate-1 and the MAPK cascade in rats with hepatocellular carcinoma induced by 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB). METHODS: Liver cancer was induced in rats by feeding 3'-MeDAB, and the changes in expression of IRS-1 and MAPK were analyzed in tumorous, non-tumorous and control liver. RESULTS: Expression of insulin receptor substrate-1 (IRS-1) showed a 1.4-fold increase at protein level in the tumors (p<0.01), but the tyrosine phosphorylation of IRS-1 did not differ between the tumor and control liver. Expression of MAPK and its activity were elevated 4.5-7.5-fold (p<0.01) and 4.6-fold (p<0.01) in the tumor compared with control liver. In non-tumorous lesions from rats fed with 3'-MeDAB, expression of MAPK, but not IRS-1, increased significantly (p<0.01). Between tumorous and adjacent non-tumorous lesions, there was a significant difference in MAPK expression (p<0.05) and activities (p<0.05). CONCLUSIONS: The increased expression of MAPK may play an important role in the progression or initiation of HCC in this rat model.

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.

A missense mutation in the CD38 gene, a novel factor for insulin secretion: association with Type II diabetes mellitus in Japanese subjects and evidence of abnormal function when expressed in vitro.

Cyclic adenosine 5'diphosphate-ribose (cADPR) is thought to have a second messenger role in insulin secretion through mobilisation of Ca2+. As human lymphocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activity, it may be important in glucose-induced insulin secretion in islets. Thirty one randomly selected Japanese patients with Type II diabetes mellitus who had first-degree and/or second-degree relative(s) with Type II diabetes mellitus were screened for mutations of this gene using single-stranded conformation polymorphism. Two variant patterns in exon 3 and exon 4 of the CD38 gene were identified. The variant in exon 3 resulted in an amino acid substitution from Arg140 (CGG) to Trp (TGG). The Arg140Trp mutation was observed in 4 of 31 patients, and allele frequencies were significantly different in patients and the control subjects (p = 0.004). One patient with this mutation has two missense mutations on beta cell/liver glucose transporter (GLUT2) gene; her mother, who has impaired glucose tolerance, also has this mutation on the CD38 gene and one missense mutation on the GLUT2 gene. Enzyme activity studies using COS-7 cells expressing the Arg140Trp mutation showed a reduction in ADP-ribosyl cyclase and cADPR hydrolase activity of around 50%. The Arg140Trp mutation on CD38 thus appears to contribute to the development of Type II diabetes mellitus via the impairment of glucose-induced insulin secretion in the presence of other genetic defects.

Molecular cloning of rat Pax4: identification of four isoforms in rat insulinoma cells.

Molecular cloning of rat Pax4 cDNA from a rat insulinoma cell line, RINm5F, library by PCR-based cloning strategy revealed four isoforms of the protein. Analysis of tissue distribution using Northern blotting and RT-PCR showed specific expression of Pax4 mRNA in pancreatic islets and RIN cells. RT-PCR confirmed that the mRNAs of four isoforms are expressed in RIN cells. These Pax4 variants may regulate the transcriptional activity of Pax4 during the development of pancreatic islets.

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.

Expression of human islet amyloid polypeptide/amylin impairs insulin secretion in mouse pancreatic beta cells.

Non-insulin-dependent diabetes mellitus (NIDDM) is associated histopathologically with islet amyloid deposits of which a major component is islet amyloid polypeptide (IAPP)/amylin. We examined whether endogenous IAPP controls insulin secretion via a local effect within pancreatic islets and whether overexpression of this peptide contributes to pancreatic beta-cell dysfunction in this disease. Transgenic mice expressing human IAPP in pancreatic beta cell were used in this study. Human IAPP expression did not influence the mouse proinsulin mRNA level and insulin content. Glucose-induced insulin secretion was decreased in the isolated pancreatic islets of transgenic mice. MIN6, a glucose-responsive pancreatic beta-cell line, was transfected with human IAPP cDNA by a lipofectin method. Human IAPP expression was confirmed by RNA blot and immunohistochemical analysis. In two transfectants expressing the largest amount of human IAPP, insulin secretion was increased in response to glucose stimulation; however, the magnitude of the insulin response in cells transfected with human IAPP was smaller than in control clones. Insulin content was not influenced by the expression. We conclude that endogenous IAPP inhibits insulin secretion via an autocrine effect within pancreatic islets, and that the impaired insulin secretion in this disease may be partly caused by overexpression of IAPP.

Islet amyloid polypeptide/amylin contents in pancreas change with increasing age in genetically obese and diabetic mice.

To search for a possible relationship between islet amyloid polypeptide (IAPP)/amylin and the pathophysiology of non-insulin-dependent (type 2) diabetes mellitus (NIDDM), we examined the changes in IAPP contents in the pancreata of genetically obese and diabetic mice (C57BL/6J ob/ob and C57BL/KsJ db/db mice). In the male ob/ob mice, IAPP and insulin contents began to increase at 16 weeks and continued to increase. In the male db/db mice, IAPP content began to increase at 8 weeks of age and insulin content at 4 weeks. Both contents continued to increase until 16 weeks, but drastically decreased at 24 weeks. Immunohistochemical studies using anti-IAPP8-17 antibody showed the increase of islet cell mass and the heterogeneous immunoreactivity for IAPP in islet cells in the ob/ob mice at 24 weeks of age. In the db/db mice at the same age, the immunoreactivity was heterogeneous and weak in many islet cells. These results suggest that genetic factors that are important in the manifestation of NIDDM influence the capacity of beta-cells to synthesize and secrete IAPP, and that IAPP synthesis and secretion change in the course of the disease.