Dong-Sik Ham

Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway

Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.

Id proteins facilitate self-renewal and proliferation of neural stem cells.

Members of helix-loop-helix (HLH) protein family of Id (inhibitor of differentiation) dimerize with bHLH transcription factors and function as negative regulators of differentiation during development. Most of inhibitory roles of Id proteins have been demonstrated in non-neural tissues, and their roles in the developing nervous system are not clearly demonstrated. In this study, we show that Id1, Id2, and Id3 increase self-renewing and proliferation potential of cortical neural stem cells (NSCs) while inhibiting neuronal differentiation. In electrophoretic mobility gel shift and luciferase assays, Id proteins interfered with binding of NeuroD/E47 complexes to the E-box sequences and inhibited E-box-mediated gene expression. Overexpression of Id proteins in NSCs increased both the number and the size of neurospheres in colony-forming assays. Expression of Hes1 and Hes5 was not increased by overexpression of Id proteins under the condition in which Nestin expression was increased. In utero electroporation of Id yielded higher numbers of Ki67-positive and Sox2-positive cells in the mouse embryonic brain. The study suggests Id proteins play independent roles in the maintenance of neural stem properties.

Generation of Functional Insulin-Producing Cells from Neonatal Porcine Liver-Derived Cells by PDX1/VP16, BETA2/NeuroD and MafA

Surrogate β-cells derived from stem cells are needed to cure type 1 diabetes, and neonatal liver cells may be an attractive alternative to stem cells for the generation of β-cells. In this study, we attempted to generate insulin-producing cells from neonatal porcine liver-derived cells using adenoviruses carrying three genes: pancreatic and duodenal homeobox factor1 (PDX1)/VP16, BETA2/NeuroD and v-maf musculo aponeurotic fibrosarcoma oncogene homolog A (MafA), which are all known to play critical roles in pancreatic development. Isolated neonatal porcine liver-derived cells were sequentially transduced with triple adenoviruses and grown in induction medium containing a high concentration of glucose, epidermal growth factors, nicotinamide and a low concentration of serum following the induction of aggregation for further maturation. We noted that the cells displayed a number of molecular characteristics of pancreatic β-cells, including expressing several transcription factors necessary for β-cell development and function. In addition, these cells synthesized and physiologically secreted insulin. Transplanting these differentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the reversal of hyperglycemia, and more than 18% of the cells in the grafts expressed insulin at 6 weeks after transplantation. These data suggested that neonatal porcine liver-derived cells can be differentiated into functional insulin-producing cells under the culture conditions presented in this report and indicated that neonatal porcine liver-derived cells (NPLCs) might be useful as a potential source of cells for β-cell replacement therapy in efforts to cure type I diabetes.

Both sitagliptin analogue & pioglitazone preserve the β-cell proportion in the islets with different mechanism in non-obese and obese diabetic mice

In this study, the effects of sitagliptin analogue (SITA) or pioglitazone (PIO) treatment on glucose homeostasis and Β-cell dynamics in animal models of type 2 diabetes--Akita and db/db mice were evaluated. After 4-6 weeks of treatment, both SITA and PIO were shown to lower non-fasting glucose levels and reduced glycemic excursion in the intraperitoneal glucose tolerance test. In addition, both drugs preserved normal islet structure and the proportion of Β-cells in the islets. Compared to the controls, SITA treatment induced a higher Β-cell proliferation rate in Akita mice and a lower rate of apoptosis in db/db mice, whereas PIO treatment induced a lower rate of apoptosis in db/db mice and reduced proliferation rates in Akita mice. In conclusion, both SITA and PIO appear to exert some beneficial effects on the islet structure in addition to glycemic control via different mechanisms that involve Β-cell dynamics in Akita and db/db mice. [BMB reports 2011; 44(11): 713-718].

Long-term Efficacy and Biocompatibility of Encapsulated Islet Transplantation With Chitosan-Coated Alginate Capsules in Mice and Canine Models of Diabetes.

Background Clinical application of encapsulated islet transplantation is hindered by low biocompatibility of capsules leading to pericapsular fibrosis and decreased islet viability. To improve biocompatibility, we designed a novel chitosan-coated alginate capsules and compared them to uncoated alginate capsules. Methods Alginate capsules were formed by crosslinking with BaCl2, then they were suspended in chitosan solution for 10 minutes at pH 4.5. Xenogeneic islet transplantation, using encapsulated porcine islets in 1,3-galactosyltransferase knockout mice, and allogeneic islet transplantation, using encapsulated canine islets in beagles, were performed without immunosuppressants. Results The chitosan-alginate capsules showed similar pore size, islet viability, and insulin secretory function compared to alginate capsules, in vitro. Xenogeneic transplantation of chitosan-alginate capsules demonstrated a trend toward superior graft survival (P = 0.07) with significantly less pericapsular fibrosis (cell adhesion score: 3.77 ± 0.41 vs 8.08 ± 0.05; P < 0.001) compared to that of alginate capsules up to 1 year after transplantation. Allogeneic transplantation of chitosan-alginate capsules normalized the blood glucose level up to 1 year with little evidence of pericapsular fibrotic overgrowth on graft explantation. Conclusions The efficacy and biocompatibility of chitosan-alginate capsules were demonstrated in xenogeneic and allogeneic islet transplantations using small and large animal models of diabetes. This capsule might be a potential candidate applicable in the treatment of type 1 diabetes mellitus patients, and further studies in nonhuman primates are required.

Suppression of Peroxisome Proliferator-Activated Receptor γ-Coactivator-1α Normalizes the Glucolipotoxicity-Induced Decreased BETA2/NeuroD Gene Transcription and Improved Glucose Tolerance in Diabetic Rats

Peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1alpha) is significantly elevated in the islets of animal models of diabetes. However, the molecular mechanism has not been clarified. We investigated whether the suppression of PGC-1alpha expression protects against beta-cell dysfunction in vivo and determined the mechanism of action of PGC-1alpha in beta-cells. The studies were performed in glucolipotixicity-induced primary rat islets and INS-1 cells. In vitro and in vivo approaches using adenoviruses were used to evaluate the role of PGC-1alpha in glucolipotoxicity-associated beta-cell dysfunction. The expression of PGC-1alpha in cultured beta-cells increased gradually with glucolipotoxicity. The overexpression of PGC-1alpha also suppressed the expression of the insulin and beta-cell E-box transcription factor (BETA2/NeuroD) genes, which was reversed by PGC-1alpha small interfering RNA (siRNA). BETA2/NeuroD, p300-enhanced BETA2/NeuroD, and insulin transcriptional activities were significantly suppressed by Ad-PGC-1alpha but were rescued by Ad-siPGC-1alpha. PGC-1alpha binding at the glucocorticoid receptor site on the BETA2/NeuroD promoter increased in the presence of PGC-1alpha. Ad-siPGC-1alpha injection through the celiac arteries of 90% pancreatectomized diabetic rats improved their glucose tolerance and maintained their fasting insulin levels. The suppression of PGC-1alpha expression protects the glucolipotoxicity-induced beta-cell dysfunction in vivo and in vitro. A better understanding of the functions of molecules such as PGC-1alpha, which play key roles in intracellular fuel regulation, could herald a new era of the treatment of patients with type 2 diabetes mellitus by providing protection from glucolipotoxicity, which is an important cause of the development and progression of the disease.

Antifibrotic effect of rapamycin containing polyethylene glycol-coated alginate microcapsule in islet xenotransplantation

Islet microencapsulation is an attractive strategy for the minimization or avoidance of life‐long immunosuppression after transplantation. However, the clinical implementation of this technique is currently limited by incomplete biocompatibility. Thus, the aim of the present study was to demonstrate the improved biocompatibility of rapamycin‐containing polyethylene glycol (Rapa–PEG)‐coating on alginate microcapsules containing xenogeneic islets. The Rapa–PEG‐coating on the alginate layer was observed using scanning electron microscopy (SEM) and the molecular cut‐off weight of the microcapsules was approximately 70 kDa. The viabilities of the alginate‐encapsulated and Rapa–PEG‐coated alginate‐encapsulated islets were lower than the viability of the naked islets just after encapsulation, but these the differences diminished over time in culture dishes. Rapa–PEG‐coating on the alginate capsules effectively decreased the proliferation of macrophage cells compared to the non‐coating and alginate coating of xenogeneic pancreas tissues. Glucose‐stimulated insulin secretion did not significantly differ among the groups prior to transplantation. The random blood glucose levels of diabetic mice significantly improved following the transplantation of alginate‐encapsulated and Rapa–PEG‐coated alginate‐encapsulated islets, but there were no significant differences between these two groups. However, there was a significant decrease in the number of microcapsules with fibrotic cell infiltration in the Rapa–PEG‐coated alginate microcapsule group compared to the alginate microcapsule group. In conclusion, Rapa–PEG‐coating might be an effective technique with which to improve the biocompatibility of microcapsules containing xenogeneic islets. Copyright

The Paradoxical Effects of AMPK on Insulin Gene Expression and Glucose‐Induced Insulin Secretion

The activation of AMP‐activated protein kinase (AMPK) is known to repress the expression of the insulin gene and glucose‐stimulated insulin secretion (GSIS). However, the mechanisms by which this occurs, as well as the effects of AMPK activation on glucolipotoxicity‐induced β‐cell dysfunction, have not been elucidated. To investigate the effects of 5‐amino‐4‐imidazolecarboxamide ribonucleotide (AICAR) and peroxisome proliferator‐activated receptorγ‐coactivator‐1α (PGC‐1α) on β‐cell‐specific genes under glucolipotoxic conditions, we performed real‐time PCR and measured insulin secretion by primary islets. To study these effects in vivo, we administered AICAR for 10 days (1 mg/g body weight) to 90% pancreatectomized hyperglycemic mice. The exposure of isolated rat and human islets to glucolipotoxic conditions and the overexpression of PGC‐1α suppressed insulin and NEUROD1 mRNA expression. However, the expression of these genes was preserved by AICAR treatment and by PGC‐1α inhibition. Exposure of isolated islets to glucolipotoxic conditions for 3 days decreased GSIS, which was also well maintained by AICAR treatment and by PGC‐1α inhibition. The administration of AICAR to 90% pancreatectomized hyperglycemic mice improved glucose tolerance and insulin secretion. These results indicate that treatment of islets with an AMPK agonist under glucolipotoxic conditions protects against glucolipotoxicity‐induced β‐cell dysfunction. A better understanding of the functions of molecules such as PGC‐1α and AMPK, which play key roles in intracellular fuel regulation, could herald a new era for the treatment of patients with type 2 diabetes mellitus by providing protection against glucolipotoxicity. J. Cell. Biochem. 117: 239–246, 2016.

Successful xenotransplantation with re‐aggregated and encapsulated neonatal pig liver cells for treatment of mice with acute liver failure

Hepatocyte transplantation is a promising therapy for acute liver failure. Cell therapy using xenogeneic sources has emerged as an alternative treatment for patients with organ failure due to the shortage of transplantable human organs. The purpose of this study was to improve the survival of mice with acute liver failure by transplanting encapsulated neonatal pig re‐aggregated liver cells (NPRLC).

Rapamycin suppresses the expansion and differentiation of porcine neonatal pancreas cell clusters.

Background. The role of rapamycin in pancreas stem cells remains to be clearly elucidated. Herein, we evaluated the effects of rapamycin on porcine neonatal pancreas cell clusters (NPCCs), which primarily comprised pancreatic precursors, and attempted to find an intracellular mechanism about the harmful effects of rapamycin. Methods. Porcine NPCCs were treated with rapamycin in a monolayer, and the apoptosis and proliferation were determined via caspase-3 assay and H3-thymidine uptake analysis. The expression of transcription factors was assessed via reverse-transcriptase polymerase chain reaction and Western blotting. For the in vivo study, the porcine NPCCs were transplanted into the kidney subcapsules of normal nude mice and treated with rapamycin. Results. Rapamycin treatment significantly reduced the number of &bgr; cells, glucose-stimulated insulin secretion, and the insulin contents in the monolayer-cultured porcine NPCCs. Furthermore, rapamycin treatment increased the apoptosis and inhibited the proliferation of &bgr; cells in the culture dishes. The expressions of the insulin, pancreatic and duodenal homeobox-1, and NeuroD/Beta2 genes were down-regulated via rapamycin treatment. The expression of insulin-like growth factor-II was significantly down-regulated, but the expression of Foxo1 was simultaneously inversely increased, and the translocation of Foxo1 from the cytoplasm to the nucleus was induced by rapamycin treatment. Moreover, rapamycin treatment induced a marked reduction in the relative volume and absolute mass of &bgr; cells in the porcine NPCCs grafts at 8 weeks after transplantation in the normal nude mice. Conclusions. Here, we demonstrate that rapamycin treatment suppresses the expansion and differentiation of porcine NPCCs, and the alteration of Foxo1 and insulin-like growth factor-II gene expression might be the crucial factors.