Shogo Funakoshi

Analysis of factors influencing pancreatic β-cell function in Japanese patients with type 2 diabetes: Association with body mass index and duration of diabetic exposure

AIMS To elucidate the clinical factors affecting beta-cell function, serum C-peptide immunoreactivity (CPR) levels of patients with type 2 diabetes were analyzed. METHODS Seven hundred Japanese patients with type 2 diabetes were enrolled. beta-Cell function was evaluated by fasting CPR (FCPR), 6 min after intravenous injection of 1mg glucagon (CPR-6 min), and the increment of CPR (DeltaCPR). Simple regression analysis between FCPR, CPR-6 min, and DeltaCPR and measures of variables and stepwise multiple regression analysis were carried out. RESULTS Years from diagnosis and BMI were the major independent variables predicting beta-cell function. Years from diagnosis was negatively correlated with CPR-6 min (P<0.0001, r=-0.271), and decrease in CPR-6 min was 0.050 ng/(ml year). BMI was positively correlated with CPR-6 min (P<0.0001, r=0.369). When subjects were divided according to BMI, the decrease in CPR-6 min per year in the high-BMI group (0.068 ng/(ml year)) was greater than that in the low-BMI group (0.035 ng/(ml year)). CONCLUSION A linear decline in endogenous insulin secretion over more than several decades of diabetes was confirmed by this cross-sectional study. The duration of diabetes exposure and BMI are thus major factors in beta-cell function in Japanese patients with type 2 diabetes.

Utility of indices using C‐peptide levels for indication of insulin therapy to achieve good glycemic control in Japanese patients with type 2 diabetes

Aims/Introduction:  Type 2 diabetes is progressive in that therapy must be altered over time, which is partly as a result of the progressive loss of pancreatic β‐cell function. To elucidate the relationship between residual endogenous insulin secretion and the necessity of insulin therapy to achieve good glycemic control, indices using serum C‐peptide immunoreactivity (CPR) were analyzed in patients with type 2 diabetes.

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

Aims/hypothesisNa+/K+-ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na+/K+-ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined.MethodsIsolated islets from Wistar rats and diabetic Goto–Kakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2′,7′-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed.ResultsOuabain caused a rapid Tyr418 phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets.Conclusions/interpretationThese results indicate that Src plays a role in the signal-transducing function of Na+/K+-ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucose-induced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.

Exome sequencing identifies a new candidate mutation for susceptibility to diabetes in a family with highly aggregated type 2 diabetes

The aim of this study was to investigate the genetic background of familial clustering of diabetes using genome-wide linkage analysis combined with exome sequencing. We recruited a Japanese family with a 3-generation history of diabetes. The family comprised 16 members, 13 having been diagnosed with diabetes. Nine members had been diagnosed before the age of 40. Linkage analysis was performed assuming an autosomal dominant model. Linkage regions were observed on chromosomes 4q34, 5q11-q13, and 12p11-q22 and the logarithm of odds (LOD) scores were 1.80. To identify the susceptibility variants, we performed exome sequencing of an affected family member. We predicted that the familial clustering of diabetes is caused by a rare non-synonymous variant, and focused our analysis on non-synonymous variants absent in dbSNP131. Exome sequencing identified 10 such variants in the linkage regions, 7 of which were concordant with the affection status in the family. One hundred five normal subjects and 67 lean diabetes subjects were genotyped for the 7 variants; the only variant found to be significantly more frequent in the diabetes subjects than in the normal subjects was the N1072K variant of the early endosome antigen 1 (EEA1) gene (0 in normal subjects and 4 in diabetes subjects, p=0.022). We therefore propose that the N1072K variant of the EEA1 gene is a candidate mutation for susceptibility to diabetes in the Japanese population.

GCKR mutations in Japanese families with clustered type 2 diabetes

OBJECTIVE The aim was to investigate the genetic background of familial clustering of type 2 diabetes. SUBJECTS AND METHODS We recruited Japanese families with a 3-generation history of diabetes. Genome-wide linkage analysis was performed assuming an autosomal dominant model. Genes in the linkage region were computationally prioritized using Endeavour. We sequenced the candidate genes, and the frequencies of detected nucleotide changes were then examined in normoglycemic controls. RESULTS To exclude known genetic factors, we sequenced 6 maturity onset diabetes of the young (MODY) genes in 10 familial cases. Because we detected a MODY3 mutation HNF1A R583G in one case, we excluded this case from further investigation. Linkage analysis revealed a significant linkage region on 2p25-22 (LOD score=3.47) for 4 families. The 23.6-Mb linkage region contained 106 genes. Those genes were scored by computational prioritization. Eleven genes, i.e., top 10% of 106 genes, were selected and considered primary candidates. Considering their functions, we eliminated 3 well characterized genes and finally sequenced 8 genes. GCKR ranked highly in the computational prioritization. Mutations (minor allele frequency less than 1%) in exons and the promoter of GCKR were found in index cases of the families (3 of 18 alleles) more frequently than in controls (0 of 36 alleles, P=0.033). In one pedigree with 9 affected members, the mutation GCKR g.6859C>G was concordant with affection status. No mutation in other 7 genes that ranked highly in the prioritization was concordant with affection status in families. CONCLUSIONS We propose that GCKR is a susceptibility gene in Japanese families with clustered diabetes. The family based approach seems to be complementary with a large population study.

Analysis of factors influencing postprandial C‐peptide levels in Japanese patients with type 2 diabetes: Comparison with C‐peptide levels after glucagon load

Aims/Introduction:  Postprandial serum C‐peptide levels are readily determined in clinical practice and have a good correlation with serum C‐peptide levels after glucagon load; the measurement is often used as an index of endogenous insulin secretion. However, the factors affecting postprandial serum C‐peptide levels remain to be evaluated.

Plasma mannose level, a putative indicator of glycogenolysis, and glucose tolerance in Japanese individuals

Mannose is a monosaccharide constituent of glycoproteins and glycolipids. Experiments in rats have shown previously that the plasma mannose level decreases after glucose load, but does not decrease in diabetic rats, and that hepatic glycogenolysis is a source of this plasma mannose; however, these results are not fully elucidated in humans. Plasma mannose levels before/after glucose loading in humans with various degrees of glucose intolerance were examined to analyze their association with clinical factors.