Hironori Yamasaki

Association Between Liver Fibrosis and Insulin Sensitivity in Chronic Hepatitis C Patients

BACKGROUND:Several clinical studies have suggested a possible link between chronic hepatitis caused by hepatitis C virus (HCV) and the development of diabetes mellitus. We investigated the association between liver fibrosis and glucose intolerance in HCV-infected patients by measuring insulin sensitivity and β-cell function.METHOD:A total of 83 chronic HCV-infected patients were recruited into this study. We evaluated insulin sensitivity and β-cell function of all patients in a fasting state (homeostasis model assessment of insulin resistance [HOMA-R] and homeostasis model assessment of β-cell function [HOMA-β]) and after an oral load of 75 g glucose (whole-body insulin sensitivity index [WBISI] and Δ-insulin/Δ-glucose 30).RESULTS:In a multivariate analysis, severe fibrosis was the only independent factor associated with insulin resistance. There were significant differences in both HOMA-R (P = 0.0063) and WBISI (P = 0.0159) between patients with mild fibrosis (N = 34) and those with severe fibrosis (N = 49). Although HOMA-β was increased significantly in the subjects with severe fibrosis compared with those with mild fibrosis (P = 0.0169), Δ-insulin/Δ-glucose 30 showed no significant difference in stage of liver fibrosis, suggesting an uncertain association between liver fibrosis and β-cell function.CONCLUSION:Our findings suggest that the development of liver fibrosis is associated with insulin resistance in HCV-infected patients.

Effects of hyperglycaemia on sorbitol and myo-inositol contents of cultured embryos: treatment with aldose reductase inhibitor and myo-inositol supplementation

SummaryTo demonstrate the myo-inositol depletion hypothesis in hyperglycaemia-induced embryopathy, rat conceptuses of 9.5 days of gestation in the early head-fold stage were grown in vitro during neural tube formation for 48 h with increasing amounts of glucose. The effects of an aldose reductase inhibitor and the myo-inositol supplementation were also investigated. Sorbitol and myo-inositol contents were measured in separated embryos and extra-embryonic membranes including yolk sac and amnion at the end of culture. After addition of 33.3 mmol/l and 66.7 mmol/l glucose to the culture media, the myo-inositol content of the embryos was significantly decreased by 43.1% (p<0.05) and 64.6% (p < 0.01) of the control group, while a marked accumulation of sorbitol was observed (25 and 41 times that of the control). Although the addition of an aldose reductase inhibitor (0.7 mmol/l) to the hyperglycaemic culture media containing an additional 66.7 mmol/l glucose significantly reduced the sorbitol content of embryos to approximately one-eighth, the myo-inositol content of embryos remained decreased and the frequency of neural lesions was unchanged (23.1% vs 23.9%, NS). Supplementation of the myo-inositol (0.28 mmol/l) completely restored the myo-inositol content of the embryos and resulted in a significant decrease in the frequency of neural lesions (7.1% vs 23.9%, p < 0.01) and a significant increase in crown-rump length and somite numbers. Much less significantly, sorbitol accumulation was also observed in the extra-embryonic membrane in response to hyperglycaemia, neither hyperglycaemia nor the myo-inositol supplementation modified the myo-inositol contents of the extra-embryonic membrane. We conclude that the mechanism of hyperglycaemia-induced teratogenicity was mediated by the myo-inositol depletion of the embryo at a critical stage of organogenesis.

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.

Association between anti-ZnT8 autoantibody specificities and SLC30A8 Arg325Trp variant in Japanese patients with type 1 diabetes.

Aims/hypothesisWe analysed the association between humoral autoreactivity to zinc transporter-8 (ZnT8) and the SLC30A8 rs13266634 polymorphism (Arg325Trp), which is located at the most distal loop in the ZnT8 protein.MethodsAutoantibodies to ZnT8 were determined by RIA in 270 patients with type 1 diabetes using ZnT8 carboxy-terminal constructs (amino acids 268–369) carrying 325Trp(CW) and 325Arg(CR) and a hybrid construct (CW-CR). Forty-four ZnT8 autoantibody-positive sera with genomic DNA were used to examine the association between reactivity to ZnT8 constructs and the rs13266634 genotype.ResultsSeventy-five patients reacted to the CW-CR hybrid construct, whereas 37 and 36 patients reacted to the CW and CR constructs, respectively. All sera positive for either CW or CR autoantibodies were positive for CW-CR autoantibodies. Among 19 patients with a 325Arg(CC) genotype, 5% had CW-specific autoantibodies, 42% had CR-specific autoantibodies and 32% had dual reactivity. Conversely, 73% of 15 patients with the 325Trp(TT) genotype had CW-specific autoantibodies, no patients had CR-specific autoantibodies and 13% had dual reactivity. Nine of the ten patients (90%) with the CT genotype reacted with either CR or CW constructs. The titre of CR autoantibodies in patients carrying the C allele was significantly higher than that in TT homozygotes (p < 0.0001). In contrast, the titre of CW autoantibodies in patients carrying a T allele was significantly higher than that in CC homozygotes (p < 0.005). No evidence of an association between rs13266634 and type 1 diabetes was observed.Conclusions/interpretationThese results indicate that variant residue at amino acid 325 is a key determinant of humoral autoreactivity to ZnT8 and that the SLC30A8 genotype is an important determinant of autoantibody specificity.

Thyroid function is associated with carotid intima-media thickness in euthyroid subjects.

To investigate the relationship between thyroid function and carotid intima-media thickness (CIMT) in a relatively large general population with euthyroid status we initially enrolled 1772 Japanese adults (421 men and 1351 women) who participated in a medical screening program for the general population over 40 years old. To evaluate only euthyroid subjects without vascular diseases and/or its major risk factors, 1129 were excluded and 643 participants (175 men and 468 women) were included for further analysis. Simple and multivariate linear regression analyses were performed to evaluate free thyroxine (fT4) and thyroid-stimulating hormone (TSH) levels and other existing parameters, including carotid intima-media thickness. By multivariate linear regression analysis adjusted for age and sex, free thyroxine was significantly correlated with triglycerides (beta=0.07, p=0.015), carotid intima-media thickness (beta=-0.091, p=0.049), and thyroid-stimulating hormone (beta=-0.091, p=0.003). Thyroid-stimulating hormone was significantly correlated with high-density lipoprotein cholesterol (HDL-C) (beta=-0.001, p=0.015), HbA(1c) (beta=0.038, p=0.045), carotid intima-media thickness (beta=0.27, p=0.001), and free thyroxine (beta=-0.15, p=0.003). When adjusted for confounding factors, free thyroxine was significantly correlated only with carotid intima-media thickness (beta=-0.13, p=0.043) and thyroid-stimulating hormone was significantly correlated with HDL-C (beta=-0.001, p<0.001), HbA(1c) (beta=0.04, p=0.021), and carotid intima-media thickness (beta=0.29, p=0.001). We have demonstrated that carotid intima-media thickness is independently associated with thyroid function within the normal reference range, which suggests an increased cardiovascular risk in subjects with low normal thyroid function.

CTLA4 gene polymorphism correlates with the mode of onset and presence of ICA512 Ab in Japanese Type 1 diabetes

Recently, the association of CTLA4 gene polymorphism with type 1 diabetes and AITD has been reported in several populations. CTLA4 was originally reported to regulate T-cell activity and T-B cognate interaction. To investigate the role of CTLA4 in autoimmune diseases, we examined the correlation between CTLA4 gene polymorphism and the clinical characteristics of Japanese patients with type 1 diabetes, including the mode of onset of diabetes and presence of islet-specific autoantibodies (GAD, ICA 512 Ab) in the serum. We studied 111 patients with type 1 diabetes and 445 normal subjects. CTLA4 exon 1 position 49 (A/G: codon 17: Thr/Ala) polymorphism was defined, employing PCR-RFLP. Sixty-three (57%) patients had AITD. The allele frequencies of G and A in both 111 patients (G: 65%; A: 35%) and 63 patients (G: 62%; A: 38%) were not significantly different from the control subjects (G: 63%; A: 37%). Serum samples of 69 patients were obtained within a year after onset and used for pancreas specific autoantibodies analysis. These samples were also used for further analysis between CTLA4 gene polymorphism and clinical characteristics. The allele frequencies of G and A in patients who presented with diabetic ketoacidosis (DK+) (G: 75%; A: 25%) were significantly different from those in DK- patients (G: 50%, A: 50%, P = 0.003). Allele and genotype analyses showed significant differences between DK+ patients and control subjects (P = 0.014, P = 0.046, respectively). Allele frequencies of G and A were not significant between patients who were positive and negative for GAD Ab, but significant for ICA 512 Ab (G: 83%, A:17% versus G: 59%, A: 41%: positive patients versus negative patients, P = 0.004). Our results showed a significant correlation between CTLA4 gene polymorphism and ICA 512 Ab. Our results also indicated that CTLA4 gene polymorphism is associated with the onset mode of Japanese type 1 diabetes and the presence of ICA512 Ab. Further analysis of this polymorphism is necessary to fully understand the pathogenesis and progression of type 1 diabetes.

Differential effects of calcineurin inhibitors, tacrolimus and cyclosporin a, on interferon-induced antiviral protein in human hepatocyte cells†

The premise of our study is that selective inhibition of interferon (IFN) by calcineurin inhibitors contribute to the increased severity of hepatitis C virus (HCV) posttransplantation. Therefore, we examined the influence of calcineurin inhibitors in the human hepatocyte cell line on IFN‐α‐induced phosphorylation of Janus kinase (Jak) and signal transducers and activators of transcription (STAT), nuclear translocation of IFN‐stimulated gene factor 3 (ISGF‐3), IFN‐stimulated regulatory element (ISRE)‐contained promoter activity, and the expressions of antiviral proteins. Tacrolimus (Tac), but not cyclosporin A (CyA), had an inhibitory effect on IFN‐α‐induced double‐stranded ribonucleic acid (RNA)‐dependent protein kinase (PKR) in a dose‐dependent manner. STAT‐1 also acted in a similar fashion to PKR. IFN‐α combined with Tac attenuated the ISRE‐containing promoter gene activity as compared with IFN‐α alone. In contrast, its expression in pretreated CyA was slightly attenuated. In pretreated Tac, but not CyA, the levels of IFN‐α‐induced tyrosine phosphorylated STAT‐1 and ‐2 were clearly lower than those induced by IFN‐α alone. Tac and CyA did not decrease the IFN‐α‐induced JAK‐1 phosphorylation. The nuclear translocation rate of tyrosine phosphorylated STAT‐1 was inhibited by pretreatment of both Tac and CyA by western blotting and immunohistochemistry. In an HCV replicon system, pretreated Tac diminished the replication inhibitory effect of IFN‐α. In this study, we show that calcineurin inhibitors, especially Tac, are the negative regulators of IFN signaling in the hepatocyte; the greatest cause of such inhibition is the phosphorylation disturbance of STAT‐1, next to inhibition of the nuclear translocation of STAT‐1. In conclusion, disturbance of tyrosine phosphorylation of STAT‐1 resulted in diminished ISRE‐containing promoter activity and a decline in antiviral protein expression. Moreover, the replication of HCV was activated. This phenomenon is detrimental to IFN therapy after liver transplantation, and the selection of calcineurin inhibitors may warrant further discussion depending on the transplant situation. Liver Transpl 14:292–298, 2008.

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.

Effects of Cyclosporin A and Low Dosages of Steroid on Posttransplantation Diabetes in Kidney Transplant Recipients

Objective To investigate the adverse effects of cyclosporin A (CsA) on pancreatic beta-cell function in kidney transplant recipients. Research Design and Methods The study consisted of 73 patients without a history of diabetes mellitus who had undergone kidney transplantation in our clinic. Results We experienced a higher incidence of posttransplantation diabetes mellitus (PTDM) in patients receiving CsA and low dosages of methylprednisolone (6/20, 30%, P < 0.05) than in patients receiving conventional therapy of azathioprine methylprednisolone (4/53, 7.5%) since the introduction of CsA. In all 6 patients in the CsA-treated group, PTDM occurred within 3 mo after transplantation. The CsA level during the initial 3 mo posttransplant was significantly higher in diabetic than nondiabetic subjects, and the highest CsA level was observed shortly (1 mo) before the development of PTDM. After an average of 71 days of insulin therapy, there was complete remission of PTDM in 5 of 6 diabetic patients, with a corresponding decrease in CsA level. For the patients who were in remission for > 1 yr, a significant improvement of glucose intolerance was observed in association with a significantly higher insulin response to oral glucose load; however, their glycemic profile still showed a significantly higher plasma glucose concentration and a prolonged continuous elevation without initial peak of the insulin-response curve in contrast to the normal pattern found in nondiabetic subjects in the CsA-treated group. Conclusions This study suggests that CsA in combination with low dosages of steroid may have adverse effects on glucose metabolism, which may lead to effects similar to those in non-insulin-dependent diabetes mellitus.

Double deficiency in IL-17 and IFN-γ signalling significantly suppresses the development of diabetes in the NOD mouse

Aims/hypothesisT helper type (Th) 17 cells have been shown to play important roles in mouse models of several autoimmune diseases that have been classified as Th1 diseases. In the NOD mouse, the relevance of Th1 and Th17 is controversial, because single-cytokine-deficient NOD mice develop diabetes similarly to wild-type NOD mice.MethodsWe studied the impact of IL-17/IFN-γ receptor double deficiency in NOD mice on the development of insulitis/diabetes compared with IL-17 single-deficient mice and wild-type mice by monitoring diabetes-related phenotypes. The lymphocyte phenotypes were determined by flow cytometric analysis.ResultsIL-17 single-deficient NOD mice showed delayed onset of diabetes and reduced severity of insulitis, but the cumulative incidence of longstanding diabetes in the IL-17-deficient mice was similar to that in wild-type mice. The IL-17/IFN-γ receptor double-deficient NOD mice showed an apparent decline in longstanding diabetes onset, but not in insulitis compared with that in the IL-17 single-deficient mice. We also found that double-deficient NOD mice had a severe lymphopenic phenotype and preferential increase in regulatory T cells among CD4+ T cells compared with the IL-17 single-deficient mice and wild-type NOD mice. An adoptive transfer study with CD4+CD25− T cells from young non-diabetic IL-17 single-deficient NOD mice, but not those from older mice, showed significantly delayed disease onset in immune-deficient hosts compared with the corresponding wild-type mice.Conclusions/interpretationThese results indicate that IL-17/Th17 participates in the development of insulitis and that both IL-17 and IFN-γ signalling may synergistically contribute to the development of diabetes in NOD mice.