Jun Shirakawa

CD226 (DNAM-1) Is Involved in Lymphocyte Function–associated Antigen 1 Costimulatory Signal for Naive T Cell Differentiation and Proliferation

Upon antigen recognition by the T cell receptor, lymphocyte function–associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12–independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1–mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

Sitagliptin as a novel treatment agent for non-alcoholic Fatty liver disease patients with type 2 diabetes mellitus.

BACKGROUND/AIMS Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver injury, and is considered as the hepatic manifestation of metabolic syndrome. However, no effective drug therapy for NAFLD has been established yet. In the present study, we evaluated the efficacy of 4 months of treatment with sitagliptin in NAFLD patients with type 2 diabetes mellitus (DM). METHODOLOGY We evaluated 30 NAFLD patients with type 2 DM. NAFLD was diagnosed by ultrasonography. The patients were administered sitagliptin (50mg/body/day) for 4 months. RESULTS significant decreases of the plasma glucose and serum HbA1c, AST, ALT and γ-GTP levels were observed after 4 months of treatment with sitagliptin. CONCLUSIONS In this study, not only the parameters of diabetes, but also those of liver tests were improved by the treatment with sitagliptin. Our study demonstrated the efficacy of sitagliptin in NAFLD patients with type 2 DM, suggesting that a large-scale clinical trial is warranted in the future.

SerpinB1 Promotes Pancreatic β Cell Proliferation

Although compensatory islet hyperplasia in response to insulin resistance is a recognized feature in diabetes, the factor(s) that promote β cell proliferation have been elusive. We previously reported that the liver is a source for such factors in the liver insulin receptor knockout (LIRKO) mouse, an insulin resistance model that manifests islet hyperplasia. Using proteomics we show that serpinB1, a protease inhibitor, which is abundant in the hepatocyte secretome and sera derived from LIRKO mice, is the liver-derived secretory protein that regulates β cell proliferation in humans, mice, and zebrafish. Small-molecule compounds, that partially mimic serpinB1 effects of inhibiting elastase activity, enhanced proliferation of β cells, and mice lacking serpinB1 exhibit attenuated β cell compensation in response to insulin resistance. Finally, SerpinB1 treatment of islets modulated proteins in growth/survival pathways. Together, these data implicate serpinB1 as an endogenous protein that can potentially be harnessed to enhance functional β cell mass in patients with diabetes.

Metformin prevents liver tumorigenesis induced by high-fat diet in C57Bl/6 Mice

The prevalence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is increasing with the growing epidemics of obesity and diabetes. NAFLD encompasses a clinicopathologic spectrum of disease ranging from isolated hepatic steatosis to NASH, which is a more aggressive form of fatty liver disease, to cirrhosis and, finally, hepatocellular carcinoma (HCC). The exact mechanism behind the development of HCC in NASH remains unclear; however, it has been established that hepatic steatosis is the important risk factor in the development of HCC. Metformin has recently drawn attention because of its potential antitumor effect. Here, we investigated the effects of metformin on high-fat diet (HFD)-induced liver tumorigenesis, using a mouse model of NASH and liver tumor. Metformin prevented long-term HFD-induced liver tumorigenesis in C57Bl/6 mice. Of note, metformin failed to protect against liver tumorigenesis in mice that had already begun to develop NAFLD. Metformin improved short-term HFD-induced fat accumulation in the liver, associated with the suppression of adipose tissue inflammation. Collectively, these results suggest that metformin may prevent liver tumorigenesis via suppression of liver fat accumulation in the early stage, before the onset of NAFLD, which seems to be associated with a delay in the development of inflammation of the adipose tissue.

Protective Effects of Dipeptidyl Peptidase-4 (DPP-4) Inhibitor against Increased β Cell Apoptosis Induced by Dietary Sucrose and Linoleic Acid in Mice with Diabetes

Chronic exposure to high glucose and fatty acid levels caused by dietary sugar and fat intake induces β cell apoptosis, leading to the exacerbation of type 2 diabetes. Oleic acid and linoleic acid are two major dietary fatty acids, but their effects in diabetes are unclear. We challenged β cell-specific glucokinase haploinsufficient (Gck+/−) mice with a diet containing sucrose and oleic acid (SO) or sucrose and linoleic acid (SL) and analyzed β cell apoptosis. In Gck+/− but not wild-type mice, SL significantly decreased the β cell mass and β cell proportion in islet cells arising from increased apoptosis to a greater degree than did SO. The mRNA expression of SREBP-1c was significantly higher, and that of E-cadherin was significantly lower in the islets of Gck+/− mice fed SL compared with mice fed SO. We next evaluated monotherapy with desfluorositagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, in these mouse groups. DPP-4 inhibitor protected against β cell apoptosis, restored the β cell mass, and normalized islet morphology in Gck+/− mice fed SL. DPP-4 inhibition normalized the changes in the islet expression of SREBP-1c and E-cadherin mRNA induced by the SL diet. Furthermore, linoleic acid induced β cell apoptosis to a greater degree in the presence of high glucose levels than in the presence of low glucose levels in vitro in islets and MIN6 cells, whereas a GLP-1 receptor agonist prevented apoptosis. In conclusion, SL exacerbated β cell apoptosis in diabetic Gck+/− mice but not in euglycemic wild-type mice, and DPP-4 inhibition protected against these effects.

Glucokinase Activation Ameliorates ER Stress–Induced Apoptosis in Pancreatic β-Cells

The derangement of endoplasmic reticulum (ER) homeostasis triggers β-cell apoptosis, leading to diabetes. Glucokinase upregulates insulin receptor substrate 2 (IRS-2) expression in β-cells, but the role of glucokinase and IRS-2 in ER stress has been unclear. In this study, we investigated the impact of glucokinase activation by glucokinase activator (GKA) on ER stress in β-cells. GKA administration improved β-cell apoptosis in Akita mice, a model of ER stress–mediated diabetes. GKA increased the expression of IRS-2 in β-cells, even under ER stress. Both glucokinase-deficient Akita mice and IRS-2–deficient Akita mice exhibited an increase in β-cell apoptosis, compared with Akita mice. β-cell–specific IRS-2–overexpressing (βIRS-2-Tg) Akita mice showed less β-cell apoptosis than Akita mice. IRS-2–deficient islets were vulnerable, but βIRS-2-Tg islets were resistant to ER stress–induced apoptosis. Meanwhile, GKA regulated the expressions of C/EBP homologous protein (CHOP) and other ER stress–related genes in an IRS-2–independent fashion in islets. GKA suppressed the expressions of CHOP and Bcl2-associated X protein (Bax) and protected against β-cell apoptosis under ER stress in an ERK1/2-dependent, IRS-2–independent manner. Taken together, GKA ameliorated ER stress–mediated apoptosis by harmonizing IRS-2 upregulation and the IRS-2–independent control of apoptosis in β-cells.

Impact of the Dipeptidyl Peptidase-4 Inhibitor Vildagliptin on Glucose Tolerance and β-Cell Function and Mass in Insulin Receptor Substrate-2-Knockout Mice Fed a High-Fat Diet

Type 2 diabetes is characterized by diminished pancreatic β-cell mass and function. Glucagon-like peptide-1 has been reported to increase islet cell proliferation and reduce apoptosis of β-cells in rodents. In this study, we explored the effect of chronic administration of the dipeptidyl peptidase-4 inhibitor vildagliptin on glucose tolerance, β-cell function, and β-cell mass in Irs2-knockout (Irs2(-/-)) mice. Wild-type and Irs2(-/-) mice were fed a high-fat diet for 20 wk, with or without vildagliptin. In both genotypes of mice, vildagliptin significantly decreased the area under the curve (0-120 min) of blood glucose and increased the insulin response to glucose during the oral glucose tolerance test. In the oral glucose tolerance test performed 1 d after discontinuation of vildagliptin administration, the area under the curve (0-120 min) of blood glucose was still significantly decreased and the insulin response to glucose was significantly increased in the Irs2(-/-) mice treated with vildagliptin as compared with the values in the mice not treated with vildagliptin. Histochemical analysis of the pancreatic islets revealed significant increase of the β-cell mass and decrease in the proportion of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive β-cells but no significant increase of the bromodeoxyuridine incorporation in Irs2(-/-) mice treated with vildagliptin. Our results suggest that vildagliptin improved glucose tolerance and increased the β-cell mass by reducing β-cell apoptosis in the Irs2(-/-) mice, and that the reduction of β-cell apoptosis by vildagliptin was independent of the Irs2 expression in the cells.

Control of beta cell function and proliferation in mice stimulated by small-molecule glucokinase activator under various conditions

Aims/hypothesisWe investigated changes in the expression of genes involved in beta cell function and proliferation in mouse islets stimulated with glucokinase activator (GKA) in order to elucidate the mechanisms by which GKA stimulates beta cell function and proliferation.MethodsIslets isolated from mice were used to investigate changes in the expression of genes related to beta cell function and proliferation stimulated by GKA. In addition, Irs2 knockout (Irs2−/−) mice on a high-fat diet or a high-fat diet containing GKA were used to investigate the effects of GKA on beta cell proliferation in vivo.ResultsIn wild-type mice, Irs2 and Pdx1 expression was increased by GKA. In Irs2−/− mice, GKA administration increased the glucose-stimulated secretion of insulin and Pdx1 expression, but not beta cell proliferation. It was particularly noteworthy that oxidative stress inhibited the upregulation of the Irs2 and Pdx1 genes induced by GKA. Moreover, whereas neither GKA alone nor exendin-4 alone upregulated the expression of Irs2 and Pdx1 in the islets of db/db mice, prior administration of exendin-4 to the mice caused GKA to increase the expression of these genes.Conclusions/interpretationGKA-stimulated IRS2 production affected beta cell proliferation but not beta cell function. Oxidative stress diminished the effects of GKA on the changes in expression of genes involved in beta cell function and proliferation. A combination of GKA and an incretin-related agent might therefore be effective in therapy.

Proinflammatory Cytokines Induce Endocrine Differentiation in Pancreatic Ductal Cells via STAT3-Dependent NGN3 Activation

A major goal of diabetes research is to develop strategies that replenish pancreatic insulin-producing beta cells. One emerging strategy is to harness pancreatic plasticity-the ability of pancreatic cells to undergo cellular interconversions-a phenomenon implicated in physiological stress and pancreatic injury. Here, we investigate the effects of inflammatory cytokine stress on the differentiation potential of ductal cells in a human cell line, in mouse ductal cells by pancreatic intraductal injection, and during the progression of autoimmune diabetes in the non-obese diabetic (NOD) mouse model. We find that inflammatory cytokine insults stimulate epithelial-to-mesenchymal transition (EMT) as well as the endocrine program in human pancreatic ductal cells via STAT3-dependent NGN3 activation. Furthermore, we show that inflammatory cytokines activate ductal-to-endocrine cell reprogramming in vivo independent of hyperglycemic stress. Together, our findings provide evidence that inflammatory cytokines direct ductal-to-endocrine cell differentiation, with implications for beta cell regeneration.

LFA-1 decreases the antigen dose for T cell activation in vivo

Leukocyte adhesion molecule leukocyte function-associated antigen (LFA)-1 not only mediates intercellular binding but also delivers co-stimulatory signals in T cells. LFA-1 has been shown to decrease the threshold of TCR signal and an antigen dose required for T cell activation and proliferation in vitro. However, physiological significance of the role of LFA-1 in TCR signal has remained unclear. We examined whether LFA-1 decreased the antigen dose for T cell activation in vivo. We showed here that, although collagen-induced arthritis (CIA) could not be induced by immunization and challenge with a standard amount of type-II collagen in LFA-1-deficient mice, a higher dose of the antigen did induce CIA in the absence of LFA-1. We also showed that CD4+ T cells could be primed by immunization with a high, but not low, dose of ovalbumin antigen in LFA-1-deficient mice. These results suggest that LFA-1 decreases the threshold of TCR signal for T cell activation in vivo as well as in vitro. Further studies using TCR-transgenic LFA-1-deficient mice showed that LFA-1 cooperated with TCR in sustained Erk1/2 phosphorylation. Moreover, TCR could induce sustained Erk1/2 phosphorylation in the absence of LFA-1 when T cells were stimulated with a high, but not low, dose of antigen, suggesting that LFA-1 may cooperate with TCR in sustaining Erk1/2 phosphorylation.