Yoon Sin Oh

On the role of major vault protein in the resistance of senescent human diploid fibroblasts to apoptosis.

Major vault protein (MVP), the main component of vault complex, is overexpressed in many multidrug-resistant cancer cell lines, suggesting a possible role for MVP in cell signaling and survival. In this study, we have found that MVP is markedly increased in senescent human diploid fibroblasts (HDFs) as well as in aged organs. We examined whether MVP expression might be affected by apoptotic stress in an aging-dependent manner. We treated young and senescent HDFs with apoptosis-inducing agents such as H2O2, staurosporine and thapsigargin, and monitored MVP expression. We found that MVP expression is markedly reduced in young HDFs but not in senescent HDFs, in response to apoptotic stresses. Downregulation of MVP increased the sensitivity of senescent HDFs to apoptosis. Also, the level of antiapoptotic B-cell lymphoma protein-2 (Bcl-2) was significantly reduced and the accumulation of c-Jun increased in MVP knocked-down senescent HDFs. Moreover, treatment of MVP knocked-down senescent HDFs with SP600125, a specific c-Jun NH(2)-terminal kinase (JNK) inhibitor, restored the level of Bcl-2 protein. Taken together, these results suggest that MVP is important in the resistance of senescent HDFs to apoptosis by modulation of Bcl-2 expression by JNK pathway.

Failure of stress-induced downregulation of Bcl-2 contributes to apoptosis resistance in senescent human diploid fibroblasts.

We previously reported that senescent human diploid fibroblasts (HDFs) are resistant to apoptosis induced by H2O2 and staurosporine. We report here that senescent HDFs are resistant to thapsigargin-induced apoptosis as well. These agonists caused the reductions in mitochondrial membrane potential (MMP) and in the apoptosis inhibitory protein (B-cell lymphoma) only in young HDFs but not in senescent HDFs. In addition, downregulation of Bcl-2 increased the sensitivity of senescent HDFs to apoptosis induction, suggesting the significant role of Bcl-2 in apoptosis resistance of the senescent HDFs. We further found that P-cAMP response element-binding protein (CREB), a positive regulator of Bcl-2, decreased in stress-induced apoptosis of young HDFs but not in senescent HDFs, and that Bcl-2 was markedly reduced in CREB small interfering RNA (siRNA), transfected senescent HDFs. In addition, activity of protein phosphatase 2A (PP2A), which dephosphorylates p-CREB, significantly increased in young HDFs but not in senescent HDFs treated with H2O2, staurosporine or thapsigargin. Taken together, these results suggest that failure of stress-induced downregulation of Bcl-2 underlies resistance of senescent HDFs to apoptosis.

Interleukin‐6 treatment induces beta‐cell apoptosis via STAT‐3‐mediated nitric oxide production

Type 2 diabetes is characterized by progressive beta‐cell failure and apoptosis is probably the main form of beta‐cell death in this disease. It was reported that circulating levels of interleukin‐6 are elevated in type 2 diabetic patients, but whether this is involved in the pathogenesis of type 2 diabetes is still debated. In this study, we examined whether interleukin‐6 can induce beta‐cell damage in vitro and elucidated its mechanisms.

A potential role for skeletal muscle caveolin-1 as an insulin sensitivity modulator in ageing-dependent non-obese type 2 diabetes: studies in a new mouse model

Aims/hypothesisType 2 diabetes mellitus is a common age-dependent disease. We discovered that male offspring of non-diabetic C57BL/6 and DBA/2 mice, called JYD mice, develop type 2 diabetes when they grow old. JYD mice show characteristics of insulin resistance, hyperglycaemia and hyperinsulinaemia in old age without obesity. We postulated that the mechanism of age-dependent type 2 diabetes in this model relates to caveolin-1 status in skeletal muscle, which appears to regulate insulin sensitivity in the mice.MethodsWe compared insulin sensitivity in aged C57BL/6 and JYD mice using glucose and insulin tolerance tests and 18F-fluorodeoxyglucose positron emission tomography. We also determined insulin signalling molecules and caveolin proteins using western blotting, and altered caveolin-1 levels in skeletal muscle of C57BL/6 and JYD mice using viral vector systems, to examine the effect of this on insulin sensitivity.ResultsIn 30-week-old C57BL/6 and JYD mice, the basal levels of IRS-1, Akt and peroxisome proliferator-activated receptor-γ decreased, as did insulin-stimulated phosphorylation of Akt and insulin receptor β. However, caveolin-1 was only increased about twofold in 30-week-old JYD mice as compared with 3-week-old mice, whereas an eightfold increase was seen in C57BL/6 mice. Downregulation of caveolin-1 production in C57BL/6 mice caused severe impairment of glucose and insulin tolerance. Upregulation of caveolin-1 in aged diabetic JYD mice significantly improved insulin sensitivity with a concomitant increase of glucose uptake in the skeletal muscle.Conclusions/interpretationThe level of skeletal muscle caveolin-1 is correlated with the progression of age-dependent type 2 diabetes in JYD mice.

Betacellulin-Induced Beta Cell Proliferation and Regeneration Is Mediated by Activation of ErbB-1 and ErbB-2 Receptors

Background Betacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation. Methodology/Principal Findings The expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes. Conclusions/Significance These results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells.

Regulation of insulin response in skeletal muscle cell by caveolin status

Recent studies on the role of caveolin‐1 in adipocytes showed that caveolin has emerged as an important regulatory element in insulin signaling but little is known on its role in skeletal muscle cells. In this study, we demonstrate for the first time that caveolin‐1 plays a crucial role in insulin dependent glucose uptake in skeletal muscle cells. Differentiation of L6 skeletal muscle cells induce the expression of caveolin‐1 and caveolin‐3 with partial colocalization. However in contrast to adipocytes, phosphorylation of insulin receptor β (IRβ) and Akt/Erk was not affected by the respective downregulation of caveolin‐1 or caveolin‐3 in the muscle cells. Moreover, the phosphorylation of IRβ was detected not only in the caveolae but also in the non‐caveolae fractions of the muscle cells despite the interaction of IRβ with caveolin‐1 and caveolin‐3. These data implicate the lack of relationship between caveolins and IRβ pathway in the muscle cells, different from the adipocytes. However, glucose uptake was reduced specifically by downregulation of caveolin‐1, but not that of caveolin‐3. Taken together, these observations suggest that caveolin‐1 plays a crucial role in glucose uptake in differentiated muscle cells and that the regulation of caveolin‐1 expression may be an important mechanism for insulin sensitivity, implying the role of muscle cells for type 2 diabetes. J. Cell. Biochem. 99: 747–758, 2006.

Exendin-4 inhibits glucolipotoxic ER stress in pancreatic β cells via regulation of SREBP1c and C/EBPβ transcription factors.

Prolonged exposure to high glucose (HG) and palmitate (PA) results in increased ER stress and subsequently induces β-cell apoptosis. Exendin-4, a glucagon-like peptide-1 agonist, is known to protect β cells from toxicity induced by cytokines, HG, or fatty acids by reducing ER stress. However, the detailed molecular mechanisms for this protective effect are still not known. In this study, we investigated the role of exendin-4 in the inhibition of glucolipotoxicity-induced ER stress and β-cell apoptosis. Exendin-4 treatment protected INS-1 β cells from apoptosis in response to HG/PA (25 mM glucose+400 μM PA). HG/PA treatment increased cleaved caspase-3 and induced ER stress maker proteins such as PERK (EIF2AK3), ATF6, and phosphorylated forms of PERK, eIF2α, IRE1α (ERN1), and JNK (MAPK8), and these increases were significantly inhibited by exendin-4 treatment. HG/PA treatment of INS-1 cells increased SREBP1 (SREBF1) protein and induced its nuclear translocation and subsequently increased C/EBPβ (CEBPB) protein and its nuclear translocation. Exendin-4 treatment attenuated this increase. Knockdown of SREBP1c reduced the activation of C/EBPβ and also blocked the expression of ER stress markers induced by HG/PA treatment. Our results indicate that exendin-4 inhibits the activation of SREBP1c and C/EBPβ, which, in turn, may reduce glucolipotoxicity-induced ER stress and β-cell apoptosis.

Mechanistic insights into pancreatic beta-cell mass regulation by glucose and free fatty acids

Pancreatic islets are responsible for blood glucose homeostasis. Reduced numbers of functional (insulin-secreting) beta-cells in pancreatic islets underlies diabetes. Restoration of the secretion of the proper amount of insulin is a goal. Beta-cell mass is increased by neogenesis, proliferation and cell hypertrophy, and is decreased by beta-cell death primarily through apoptosis. Many hormones and nutrients affect beta-cell mass, and glucose and free fatty acid are thought to be the most important determinants of beta-cell equilibrium. A number of molecular pathways have been implicated in beta-cell mass regulation and have been studied. This review will focus on the role of the principle metabolites, glucose and free fatty acid, and the downstream signaling pathways regulating beta-cell mass by these metabolites.

Role of Bioactive Food Components in Diabetes Prevention: Effects on Beta-Cell Function and Preservation

Bioactive compounds found in fruits and vegetables can have anti-oxidant, anti-inflammatory, and anti-carcinogenic effects and can be protective against various diseases and metabolic disorders. These beneficial effects make them good candidates for the development of new functional foods with potential protective and preventive properties for type 1 and type 2 diabetes. This review summarizes the most relevant results concerning the effects of various bioactive compounds such as flavonoids, vitamins, and carotenoids on several aspects of beta-cell functionality. Studies using animal models with induced diabetes and diabetic patients support the hypothesis that bioactive compounds could ameliorate diabetic phenotypes. Published data suggest that there might be direct effects of bioactive compounds on enhancing insulin secretion and preventing beta-cell apoptosis, and some compounds might modulate beta-cell proliferation. Further research is needed to establish any clinical effects of these compounds.

Treatment with glucokinase activator, YH-GKA, increases cell proliferation and decreases glucotoxic apoptosis in INS-1 cells

Glucokinase (GK), an enzyme that phosphorylates glucose to form glucose-6-phosphate, has a role in regulating insulin secretion and proliferation in beta cells. GK activators (GKAs) have been developed as new therapies for type 2 diabetes. In this study, we evaluated the proliferation and anti-apoptotic actions of YH-GKA, a novel and potent GKA, in INS-1 pancreatic β-cells. YH-GKA treatment increased cell numbers at 3 mM glucose via upregulation of insulin receptor substrate-2 and subsequent activation of AKT/protein kinase B phosphorylation. YH-GKA also increased beta-catenin and cyclin D2 mRNA expression and inactivated GSK3β by increasing phosphorylation. These proliferative effects of YH-GKA were attenuated by IRS-2 downregulation. Moreover, YH-GKA reduced annexin-V-stained cells and expression levels of cleaved poly (ADP-ribose) polymerase and caspase-3 induced by glucotoxicity. YH-GKA inhibited apoptotic signaling via induction of ATP content, mitochondrial membrane potential, and citrate synthase activity and was correlated with changes of the mitochondrial function-related genes. YH-GKA also increased interaction between GK and voltage-dependent anion-selective channel protein. Our results suggest that the novel GKA, YH-GKA, promotes beta cell growth and prevents glucotoxic beta cell apoptosis. Therefore, YH-GKA may provide a therapy that compensates for beta cell loss in patients with type 2 diabetes.