Hironaga Kuwahara

Genetic association between interleukin-10 gene promoter region polymorphisms and type 1 diabetes age-at-onset

This study investigated whether interleukin-10 (IL-10) gene promoter region polymorphisms are associated with susceptibility to or clinical presentation of type 1 diabetes. The frequency of -1082G/A, -819C/T, and -592C/A polymorphisms was analyzed in 128 Japanese patients with type 1 diabetes and in 107 healthy control subjects in a case-controlled study. The allelic and haplotypic frequencies of the IL-10 gene promoter region polymorphisms were similar in patients with type 1 diabetes and in control subjects. However, the -819T and -592A allele were associated with adult-onset (>18 years) of the disease (p = 0.037). Furthermore, the frequency of ATA haplotype was increased in adult-onset patients than that in early-onset patients (< or =18 years; p = 0.037). Among the genotypes comprising ATA haplotype, the frequency of ATA/ATA was significantly higher in adult-onset patients than in early-onset patients (p = 0.004). These results suggest that the IL-10 gene promoter polymorphisms are associated with the age-at-onset in Japanese patients with type 1 diabetes.

Autoantibodies to Insulin, Insulinoma-Associated Antigen-2, and Zinc Transporter 8 Improve the Prediction of Early Insulin Requirement in Adult-Onset Autoimmune Diabetes

OBJECTIVE The aim of this study was to identify the predictive marker for early insulin requirement in adult-onset autoimmune diabetes in the Japanese populations. DESIGN/PATIENTS We analyzed insulin autoantibodies (IAA), insulinoma-associated antigen-2 (IA-2) autoantibodies (IA-2icA), and zinc transporter 8 (ZnT8) autoantibodies (ZnT8A) by radioimmunoassay in 47 Japanese patients with adult-onset autoimmune diabetes who were identified by native GAD autoantibody (nGADA) screening of approximately 3000 non-insulin-requiring diabetes patients and 302 nGADA-negative type 2 diabetes patients. Furthermore, GAD65 autoantibody-specific epitopes were also analyzed using GAD65/GAD67 chimeric constructs. RESULTS The prevalence of IAA, IA-2icA, and ZnT8A in nGADA-positive patients was 26, 15, and 19%, respectively, which was significantly higher than that in nGADA-negative type 2 diabetes (2, 2, and 2%; P < 0.0001). Among nGADA-positive patients, 38% had one or more of IAA, IA-2icA, or ZnT8A, and 15% had two or more of these autoantibodies, compared with none of the nGADA-negative patients (P < 0.0001). Thirty-six percent of nGADA-positive patients subsequently required insulin therapy; and high nGADA titer (log-rank P = 0.003), middle epitope recognition of GAD65A (P = 0.002), and the presence of one or more of IAA, IA-2icA, or ZnT8A (P = 0.002) at diagnosis marked the risk for early requirement of insulin therapy. Multivariate logistic regression analysis showed the multiple islet autoantibodies to be independently associated with the risk for insulin requirement (odds ratio = 13.77; 95% confidence interval, 2.77-68.45; P = 0.001). CONCLUSIONS These results indicate that the determination of IAA, IA-2icA, and ZnT8A improves the prediction of a future insulin insufficiency in adult-onset autoimmune diabetes, which appears to be superior to GADA titer and GAD65A-specific epitopes.

Differences in the humoral autoreactivity to zinc transporter 8 between childhood- and adult-onset type 1 diabetes in Japanese patients

The aim of this study was to evaluate the humoral autoreactivity to zinc transporter 8 (ZnT8) depending on the clinical phenotype of type 1 diabetes (T1D). ZnT8 autoantibodies (ZnT8A) were determined by radioimmunoassay using carboxy-terminal ZnT8 constructs in 57 childhood-onset, 97 adult-onset, and 85 fulminant T1D. The ZnT8A frequency was higher in childhood-onset patients and decreased with increasing age of onset from 70% to 24% (P(trend)<0.005). None of the patients with fulminant T1D was positive for ZnT8A. There were at least two distinct ZnT8A epitope patterns associated with the aa325-restriction, childhood-onset patients have aa325-nonrestricted response more frequently compared to the adult-onset group (P<0.05). The level of ZnT8A was inversely associated with the copy number of HLA-DR4 allele (P<0.05). These results suggest differences in the humoral autoreactivity to ZnT8 depending on the clinical phenotype, which should provide strategy for autoantibody measurement in subjects to allow early diagnosis of autoimmune T1D.

Clinical and Genetic Characteristics of Autoimmune Polyglandular Syndrome Type 3 Variant in the Japanese Population

OBJECTIVE Type 1 diabetes (T1D) is commonly associated with autoimmune thyroid disease (AITD), and the occurrence of both T1D and AITD in a patient is defined as autoimmune polyglandular syndrome type 3 variant (APS3v). We aimed to clarify the differences in the clinical and genetic characteristics of APS3v patients and T1D patients without AITD [T1D/AITD(-)] in the Japanese population. DESIGN/PATIENTS Our subjects were 54 APS3v patients and 143 T1D/AITD(-) patients who were consecutively diagnosed at Nagasaki University Hospital from 1983 to the present. RESULTS A remarkable female predominance, a slow and older age onset of T1D, and a higher prevalence of glutamic acid decarboxylase autoantibodies were observed in APS3v patients compared to T1D/AITD(-) patients. The older onset age of T1D in APS3v patients was associated with a higher proportion of slow-onset T1D. Among the two major susceptible human leukocyte antigen (HLA) class II haplotypes in Japanese T1D, DRB1*0405-DQB1*0401, but not DRB1*0901-DQB1*0303, was associated with APS3v patients. Furthermore, DRB1*0803-DQB1*0601 was not protective in patients with APS3v. The frequencies of the GG genotype in +49G>A and +6230G>A polymorphism in the CTLA4 gene were significantly higher in T1D/AITD(-) patients, but not in APS3v patients, compared to control subjects. CONCLUSIONS In conclusion, we found notable differences in the clinical and genetic characteristics of APS3v patients and T1D/AITD(-) patients in the Japanese population, and the differences in the clinical characteristics between the two groups may reflect distinct genetic backgrounds including the HLA DRB1-DQB1 haplotypes and CTLA4 gene polymorphisms.

Zinc transporter 8 autoantibodies in fulminant, acute-onset, and slow-onset patients with type 1 diabetes

The aim of this study was to determine the prevalence and role of autoantibodies to zinc transporter 8 (ZnT8A) in three forms (fulminant, acute‐onset, and slow‐onset) of Japanese patients with type 1 diabetes.

Association of Interleukin-18 Gene Promoter Polymorphisms in Type 1 Diabetes and Autoimmune Thyroid Disease

Abstract: Type 1 diabetes is a heterogeneous autoimmune disease and is often associated with other organ‐specific autoimmune diseases, including autoimmune thyroid disease (AITD). IL‐18 is a potent proinflammatory cytokine capable of inducing IFN‐γ production that is associated with the development of type 1 diabetes and AITD. The gene for IL‐18 is located near Idd2 and has been reported to be associated with a susceptibility to type 1 diabetes. To test the putative involvement of IL‐18 gene polymorphism in predisposition to type 1 diabetes and AITD, we conducted a case‐control study in Japanese population. The SNPs at position −607 (C/A) and −137 (G/C) in the promoter region of the IL‐18 gene were analyzed by sequence‐specific PCR in 74 nondiabetic patients with AITD, 47 type 1 diabetic patients with AITD, and 114 normal controls. There was no significant increase in the genotype and allele frequencies not only in nondiabetic patients with AITD compared with normal controls, but also in type 1 diabetic patients with AITD compared with normal controls. The distribution of IL‐18 gene haplotypes was also similar between both patient groups and normal controls. These results suggest that polymorphisms of the IL‐18 gene are not associated with a susceptibility to AITD and type 1 diabetes coexistent with AITD in Japanese population.

Intracerebroventricular Administration of Insulin and Glucose Inhibits the Anorectic Action of Leptin in Rats

Obese individuals with glucose intolerance present with high serum levels of glucose, insulin, and leptin. These substances are potent inhibitors of feeding in the brain. Obese subjects still present with over-feeding despite elevation of the above factors. To elucidate the mechanism of this paradox, the effects of insulin and glucose on the anorectic action of leptin in the hypothalamus were examined. Adult male Sprague-Dawley rats (weighing 2850–320 g) were pretreated with intracerebroventricular injection of insulin, glucose, or saline, followed by leptin (7.5 μg) or phosphate-buffered saline (PBS) injection into the third cerebral ventricle (icv). The cumulative food intakes were measured 24 hr after leptin icv. The tyrosine phosphorylation of signal transducer and activator transcription factor 3 (STAT3) in the hypothalamus was determined by Western blotting. In rats pretreated with saline and stimulated with leptin (saline/LEPTIN group), food intake diminished to about 50% of that of the saline/PBS group (P < 0.005). Food intake in the insulin/LEPTIN group was significantly higher compared with the saline/LEPTIN group (P < 0.005) and reached the level seen in the saline/PBS group. Similar data were obtained in glucose pretreatment experiments. Insulin and glucose icv resulted in reduction of leptin-induced STAT3 tyrosine phosphorylation compared with saline. Infusion of insulin and glucose icv did not alter peripheral blood glucose levels in all groups. High insulin or glucose levels in the brain could result in leptin resistance as manifested by food intake, which is probably due to the attenuation of STAT3 phosphorylation downstream the leptin receptor.

Vanadate enhances leptin-induced activation of JAK/STAT pathway in CHO cells

Leptin, the product of the ob gene, is an adipocyte-derived hormone that plays a key role in the control of food intake and energy expenditure. Leptin acts through receptors that belong to a member of the class I cytokine receptor family. It has been demonstrated that the SH2 domain-containing tyrosine phosphatase 2 (SHP-2) negatively regulates STAT3-mediated transcriptional activation through long form leptin receptor (OBRb). Vanadate has been shown to be a potent and selective inhibitor of PTPase activity in vitro. In this study, we have demonstrated that vanadate increases leptin-induced JAK2 and STAT3 phosphorylation in CHO cells expressing OBRb. The increased leptin-dependent luciferase activity of SOCS3 gene was also seen in vanadate-treated cell. Furthermore, vanadate reversed the inhibitory effects of SOCS3 on leptin-induced STAT3 phosphorylation. The present findings suggest that PTP inhibitors including vanadate and vanadate-derived compounds could be used as a therapeutic agent in the treatment of obesity.

Ethanol inhibits leptin‐induced STAT3 activation in Huh7 cells

Leptin, an adipocyte‐derived hormone, regulates food intake and energy expenditure in the hypothalamus via its receptor, member of the class I cytokine receptor family. Leptin resistance has been observed in rodents and in humans. However, the mechanisms could not be explained in most cases of human obesity, except for rare cases with mutations in the leptin receptor. Recent reports demonstrated that ethanol inhibited the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activated by some members of the class I cytokine receptor family. In this study, we examined the effects of ethanol on the leptin‐induced JAK/STAT signaling pathway using human hepatoma cell lines transiently expressing long form of the leptin receptor. A 30 min pretreatment with ethanol dose‐dependently inhibited the leptin‐induced STAT3 phosphorylation. Furthermore, to determine the time course of ethanol inhibitory effects, the cells were incubated in 10 mM ethanol for various times. Partial inhibition of leptin‐induced STAT3 activation was seen after 1 min of treatment with ethanol and completely inhibited after 30 min pretreatment. SB 202190, a p38 mitogen‐activated protein kinase (MAPK) inhibitor, partly prevented this inhibition by ethanol of leptin‐induced STAT3 activation. These findings suggest that ethanol time‐ and dose‐dependently inhibits the leptin action, in part via p38 MAPK.

Stromal Cell-Derived Factor-1 Chemokine Gene Variant in Patients with Type 1 Diabetes and Autoimmune Thyroid Disease

Abstract: Type 1 diabetes is a heterogenous autoimmune disease and is frequently associated with other organ‐specific autoimmune diseases, including autoimmune thyroid disease (AITD). Type 1 diabetic patients with AITD are known to have clinical and immunological features distinct from patients without AITD. This study investigated whether stromal cell‐derived factor (SDF)‐1 gene polymorphism is associated with susceptibility to type 1 diabetes and AITD. SDF‐1 is a powerful chemokine that upregulates T‐cell migration and activation, and the gene for SDF‐1 is located near type 1 diabetes susceptibility locus IDDM10. The SDF1‐3′A variant (801 G to A in the 3′‐untranslated region) was determined by the PCR‐RFLP technique in 54 type 1 diabetic patients with AITD, 75 type 1 diabetic patients without AITD, 137 nondiabetic patients with AITD, and 106 healthy subjects in a case‐control study. No significant differences on the allele and genotype frequencies of the SDF1 gene polymorphism were found, not only in type 1 diabetic patients with AITD compared with normal controls but also between nondiabetic patients with AITD and healthy control subjects. These results suggest that the SDF1‐3′A variant is not associated with genetic susceptibility to type 1 diabetic patients and AITD.