Yoshiharu Tokuyama

Secretion of islet amyloid polypeptide in response to glucose

The content of isolet 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.

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

Summary 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. [Diabetologia (1998) 41: 1024–1028]

Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic β cells

Abstract Cytokines induce apoptosis in pancreatic β cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-α) in the apoptosis of β 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-1β—converting enzyme (FLICE) were expressed in the pancreatic β cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-α induced time- and dose-dependent apoptotic nuclear changes in these β cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-α generated new 3′-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-α 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-α (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-α can cause apoptosis in pancreatic β cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.

Frequency of the G/G genotype of resistin single nucleotide polymorphism at -420 appears to be increased in younger-onset type 2 diabetes.

OBJECTIVE—Resistin is an adipocyte-secreted cytokine associated with insulin resistance in mice. We previously reported that the G/G genotype of a resistin single nucleotide polymorphism (SNP) at −420 increases type 2 diabetes susceptibility by enhancing its promoter activity. The aim of the present study was to determine the relevance of SNP −120 in a large number of subjects. RESEARCH DESIGN AND METHODS— We examined 2,610 type 2 diabetic case and 2,502 control subjects. The relation between SNP −420 and the age of type 2 diabetes onset was further analyzed by adding 237 type 2 diabetic subjects with age of onset ≤40 years. RESULTS—When analyzed without considering subject age, the SNP −420 genotype was not associated with type 2 diabetes. Since we reported that the onset of type 2 diabetes was earlier in G/G genotype, we analyzed the data using a trend test for age intervals of 10 years. The frequency of G/G genotype differed among age grades in type 2 diabetes (P = 0.037) and appeared to be higher in younger grades. In type 2 diabetes, G/G genotype was more frequent in subjects aged <40 years than in those aged ≥40 years (G/G vs. C/C, P = 0.003). In a total of 2,430 type 2 diabetic subjects with age of onset <60 years, the trend test showed that the G/G genotype had an increasing linear trend as the age grade of type 2 diabetes onset became younger (P = 0.0379). In control subjects, the frequency of C/G genotype showed an increasing linear trend with increasing age (P = 0.010). CONCLUSIONS—The G/G genotype frequency of resistin SNP −420 appears to be increased in younger-onset type 2 diabetic subjects.

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.

Islet amyliod polypeptide-derived amyloid deposition increases along with the duration of type 2 diabetes mellitus

To investigate the involvement of islet amyloid polypeptide (IAPP) and amyloid deposits in the pathophysiology of this disease, we studied the relationship between IAPP-derived amyloid deposition and the clinical features in type 2 diabetes mellitus. We examined pancreata obtained from 37 type 2 diabetic subjects and 12 non-diabetic ones by immunohistochemical techniques using two specific antibodies to IAPP. IAPP-derived deposits occurred in 1 of the 12 (8.3%) non-diabetic subjects and 28 of the 37 (75.7%) diabetics. When diabetic patients were divided into categories according to the presence of the deposits, the duration of the disease was significantly longer in patients with amyloid than that in the patients without it. The odds ratio of type 2 diabetes mellitus of at least 14-years-duration to the deposition was significantly high, and a body weight of at least 120% maximal ideal body weight was relatively high. In conclusion, IAPP-derived amyloid deposition increases along with the duration of type 2 diabetes mellitus and obesity may further enhance these deposits, hence hypersecretion of IAPP may be involved in the progression of this disease.

Predictors of glycemic control in Japanese subjects with type 2 diabetes mellitus

The purpose of this study was to investigate which pathophysiological and demographic characteristics of Japanese subjects with type 2 diabetes mellitus were associated with poor glycemic control and to propose a statistical model for predicting their glycemic control. A total of 220 subjects with type 2 diabetes mellitus were enrolled in this study. Frequently sampled intravenous glucose tolerance test was performed to determine the first-phase C-peptide secretion rate (CS1) and insulin sensitivity index. Multiple regression analysis in a stepwise manner was carried out to identify independent regulators of glycemic control. Upon stepwise linear regression analysis with hemoglobin A1c as a dependent parameter, fasting plasma glucose concentration (FPG), CS1, and onset age remained as predictors, explaining 41.0% of glycemic control. The young-onset group (onset age < or =48 years) had significantly higher hemoglobin A1c than the old-onset group (onset age >48 years) (P = .0148), although the present age was significantly older in the old-onset group; and there were no significant differences in duration of diabetes, treatment, body mass index, FPG, fasting insulin level, homeostasis model assessment of insulin resistance, CS1, and log(insulin sensitivity index) between them. Worsening factors of glycemic control in Japanese subjects with type 2 diabetes mellitus were elevated FPG, impaired first-phase insulin secretion, and young age of onset of the disease. Because glycemic control in the subjects with young-onset diabetes tends to be worse, early and aggressive intervention should be required for those with young-onset diabetes to prevent long-term complications.

Islet Amyloid Polypeptide/Amylin in Pancreatic β-Cell Line Derived From Transgenic Mouse Insulinoma

We examined the production and secretion of IAPP in a β-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 β-cell line will aid in analyzing the regulation of IAPP gene expression and the mechanisms of IAPP biosynthesis and secretion.

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.

The Dipeptidyl Peptidase-4 Inhibitor Vildagliptin has the Capacity to Repair β-Cell Dysfunction and Insulin Resistance

The aim of the present study was to determine whether the dipeptidyl peptidase (DPP)-4 inhibitor could repair pancreatic β-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 β-cell function and insulin resistance in type 2 diabetes.