Gyeong l Ryu

Long-Term Treatment of Glucagon-Like Peptide-1 Analog Exendin-4 Ameliorates Diabetic Nephropathy through Improving Metabolic Anomalies in db/db Mice

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone and is a new clinically available class of agents for improving of insulin resistance in both animals and humans with type 2 diabetes. These studies aimed to determine whether long-term treatment with a long-acting GLP-1 analog, exendin-4, delayed the progression of diabetes. Male db/db mice and db/m mice at 8 wk of age were treated with exendin-4 for 8 wk, whereas the control db/db mice received only vehicle. Urinary albumin excretion was significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in db/db mice that were treated with 0.5 nmol/kg exendin-4 and control db/db mice (P < 0.005). Intraperitoneal glucose tolerance test was improved in db/db mice that were treated with 1 nmol/kg exendin-4 compared with other groups (P < 0.05). Despite this, fasting blood glucose, glycated hemoglobin, and creatinine concentrations were not significantly different among db/db mice. Renal histology studies further demonstrated that glomerular hypertrophy, mesangial matrix expansion, TGF-beta1 expression, and type IV collagen accumulation and associated glomerular lipid accumulation were significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4. Furthermore, there were fewer infiltrating inflammatory cells and apoptotic cells in the glomeruli of db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in the other groups accompanied by an increase in the renal immunoreactivity of peroxisome proliferator-activated receptor alpha and GLP-1 receptor-positive cells and a decrease in 24-h urinary 8-hydroxy-deoxyguanosine levels (P < 0.01, respectively) along with decreases in lipid content. Taken together, exendin-4 treatment seems to ameliorate diabetic nephropathy together with improvement of the metabolic anomalies. These results suggest that exendin-4 could provide a therapeutic role in diabetic nephropathy that results from type 2 diabetes.

Protective Effects of Epicatechin Against the Toxic Effects of Streptozotocin on Rat Pancreatic Islets : In Vivo and in Vitro

Introduction Green tea catechins have diverse pharmacological effects such as anticarcinogenic and antioxidant activities. Aim To study the protective effects of green tea (−)-epicatechin (EC) against the toxic effects of streptozotocin (STZ), a selective &bgr; cell toxin, on pancreatic islets in vivo and in vitro. Methodology Rats were randomly divided into four groups: control, EC (30 mg/kg)–treated, STZ (60 mg/kg)–treated, and EC plus STZ (same doses; EC+STZ)–treated rats. EC was administered twice a day for 6 days, and a single injection of STZ was used. In EC+STZ–treated rats, EC was administered 6 hours prior to STZ since posttreatment with EC had no beneficial effects on fully developed diabetes in our unpublished study. Insulin and insulin mRNA were detected by immunohistochemical analysis and in situ hybridization, respectively, and physiologic parameters including blood glucose concentration were measured daily. Following isolation of the islets, insulin release, nitrite levels, and islet morphology were observed in the four groups: control, EC (0.8 m M)–treated, STZ (5 m M)–treated, and EC+STZ (same doses)–treated islets. Results In EC+STZ–treated rats, hyperglycemia and weight loss were not observed and islet morphology was well preserved compared with STZ-treated rats. Compared with STZ treatment alone, insulin release was increased and nitrite production was decreased in EC+STZ–treated islets. Conclusion EC appears to be helpful in protecting pancreatic islets against exposure to STZ in both in vivo and in vitro systems.

Antioxidant treatment may protect pancreatic beta cells through the attenuation of islet fibrosis in an animal model of type 2 diabetes

Islet fibrosis could be important in the progression of pancreatic beta cell failure in type 2 diabetes. It is known that oxidative stress is involved in the pancreatic fibrosis through the activation of pancreatic stellate cells. However, no study has investigated the in vivo effects of antioxidants on islet fibrogenesis in type 2 diabetes. In this study, antioxidants (taurine or tempol) were administered in drinking water to Otsuka Long-Evans Tokushima Fatty rats, an animal model of type 2 diabetes, for 16 weeks. An intraperitoneal glucose tolerance test revealed that the blood glucose levels after the glucose injection were decreased by the antioxidants. The insulin secretion after the glucose injection, which was markedly reduced in the rats, was also restored by the antioxidants. Beta cell mass and pancreatic insulin content were greater in the rats treated with the antioxidants than in the untreated rats. Beta cell apoptosis was attenuated in the rats by the antioxidants. Finally, islet fibrosis and the activation of pancreatic stellate cells were markedly diminished in the rats by the antioxidants. Our data suggest that antioxidants may protect beta cells through the attenuation of both islet fibrosis and beta cell apoptosis in type 2 diabetes.

Inducible nitric oxide synthase-nitric oxide plays an important role in acute and severe hypoxic injury to pancreatic beta cells.

Background. Islet transplantation is a potential strategy to cure type 1 diabetes mellitus. However, a substantial part of the islet graft becomes nonfunctional due to several factors including hypoxia. However, the precise mechanism of cell damage is largely unknown in hypoxic exposure to pancreatic beta cells. The aim of the present study was to investigate whether acute and severe hypoxic injury could involve inducible nitric oxide synthase (iNOS)-nitric oxide (NO) signaling in beta cells. Methods. The rat beta cell line (INS-1) and primary rat islets were incubated in an anoxic chamber. Cell viability was determined by propium iodide staining or cell counting kit. The expression of iNOS mRNA and protein was examined using reverse-transcription polymerase chain reaction and Western blot analysis. NO production was measured as nitrite accumulation by Griess reagent method. Results. After hypoxic exposure, marked cell death occurred in INS-1 cells and rat islets, accompanied by increase in activated caspase-3 expression. NO production was increased in the culture medium in a time-dependent manner. Increase in expression of iNOS mRNA and protein was found. Pretreatment with a selective iNOS inhibitor, 1400W, significantly prevented cell death during hypoxia. In addition, hypoxia activated c-Jun N-terminal kinase (JNK) significantly, but the addition of 1400W inhibited hypoxia-induced JNK phosphorylation. Conclusions. Our data suggest that iNOS-NO plays an important role in acute and severe hypoxic injury to pancreatic beta cells. Therefore, iNOS-NO might be a potential therapeutic target for preserving beta cell survival in islet transplantation through prevention of hypoxia-mediated cell death.

Activation of AMP-activated protein kinase mediates acute and severe hypoxic injury to pancreatic beta cells.

In islet transplantation, a substantial part of the graft becomes nonfunctional for several reasons including hypoxia. AMP-activated protein kinase (AMPK) in mammalian cells is a regulator of energy homeostasis, and is activated by metabolic stresses such as hypoxia. However, the role of AMPK in hypoxic injury to pancreatic beta cells is not clear. When a rat beta cell line, INS-1 cell, was incubated in an anoxic chamber, phosphorylation of both AMPK and its downstream protein, acetyl-CoA carboxylase 2 increased with time. Adenovirus-mediated expression of constitutively active form of AMPK under normoxic conditions increased caspase-3 activation, suggesting induction of apoptosis. Reactive oxygen species production also increased with time during hypoxia. Pretreatment with compound C, an AMPK inhibitor, or N-acetyl-l-cysteine, an antioxidant, significantly lowered hypoxia-mediated cell death. These results suggest that AMPK, in association with oxidative stress, plays an important role in acute and severe hypoxic injury to pancreatic beta cells.

Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic β-Cell Injury.

Background Glucose toxicity that is caused by chronic exposure to a high glucose concentration leads to islet dysfunction and induces apoptosis in pancreatic β-cells. Heme oxygenase-1 (HO-1) has been identified as an anti-apoptotic and cytoprotective gene. The purpose of this study is to investigate whether HO-1 up-regulation when using metalloprotophyrin (cobalt protoporphyrin, CoPP) could protect pancreatic β-cells from high glucose-induced apoptosis. Methods Reverse transcription-polymerase chain reaction was performed to analyze the CoPP-induced mRNA expression of HO-1. Cell viability of INS-1 cells cultured in the presence of CoPP was examined by acridine orange/propidium iodide staining. The generation of intracellular reactive oxygen species (ROS) was measured using flow cytometry. Glucose stimulated insulin secretion (GSIS) was determined following incubation with CoPP in different glucose concentrations. Results CoPP increased HO-1 mRNA expression in both a dose- and time-dependent manner. Overexpression of HO-1 inhibited caspase-3, and the number of dead cells in the presence of CoPP was significantly decreased when exposed to high glucose conditions (HG). CoPP also decreased the generation of intracellular ROS by 50% during 72 hours of culture with HG. However, decreased GSIS was not recovered even in the presence of CoPP. Conclusion Our data suggest that CoPP-induced HO-1 up-regulation results in protection from high glucose-induced apoptosis in INS-1 cells; however, glucose stimulated insulin secretion is not restored.

Exendin‐4 induction of Egr‐1 expression in INS‐1 β‐cells: Interaction of SRF, not YY1, with SRE site of rat Egr‐1 promoter

Glucagon‐like peptide‐1 (GLP‐1) induces several immediate early response genes such as c‐fos, c‐jun, and early growth response‐1 (Egr‐1), which are involved in cell proliferation and differentiation. We recently reported that exendin‐4 (EX‐4), a potent GLP‐1 agonist, upregulated Egr‐1 expression via phosphorylation of CREB, a transcription factor in INS‐1 β‐cells. This study was designed to investigate the role of another transcription factors, serum response factor (SRF) and Yin Yang‐1 (YY1), in EX‐4‐induced Egr‐1 expression. EX‐4 significantly increased Egr‐1 mRNA and subsequently its protein level. EX‐4‐induced Egr‐1 expression was inhibited by pretreatment with a PKA inhibitor, H‐89, and an MEK inhibitor, PD 98059. The siRNA‐mediated inhibition of PKA and ERK1 resulted in significant reduction of EX‐4‐induced Egr‐1 expression. Promoter analyses showed that SRE clusters were essential for Egr‐1 transcription, and YY1 overexpression did not affect Egr‐1 promoter activity. EMSA results demonstrated that EX‐4‐induced transient increase in DNA–protein complex on SRE site, and that both SRF and phospho‐SRF were bound to this site. Treatment of either YY1 consensus oligonucleotide or YY1 antibody did not effect the change of density or migration of the DNA–protein complex. Collectively, EX‐4‐induced Egr‐1 expression is largely dependent on cAMP‐mediated extracellular signal‐regulated kinase activation, and EX‐4 induces Egr‐1 transcription via the interaction of SRF and phospho‐SRF to SRE sites. J. Cell. Biochem. 104: 2261–2271, 2008.

Oxidative stress plays a role in high glucose-induced activation of pancreatic stellate cells.

The activation of pancreatic stellate cells (PSCs) is thought to be a potential mechanism underlying islet fibrosis, which may contribute to progressive β-cell failure in type 2 diabetes. Recently, we demonstrated that antioxidants reduced islet fibrosis in an animal model of type 2 diabetes. However, there is no in vitro study demonstrating that high glucose itself can induce oxidative stress in PSCs. Thus, PSCs were isolated and cultured from Sprague Dawley rats, and treated with high glucose for 72 h. High glucose increased the production of reactive oxygen species. When treated with high glucose, freshly isolated PSCs exhibited myofibroblastic transformation. During early culture (passage 1), PSCs treated with high glucose contained an increased number of α-smooth muscle actin-positive cells. During late culture (passages 2-5), PSCs treated with high glucose exhibited increases in cell proliferation, the expression of fibronectin and connective tissue growth factor, release of interleukin-6, transforming growth factor-β and collagen, and cell migration. Finally, the treatment of PSCs with high glucose and antioxidants attenuated these changes. In conclusion, we demonstrated that high glucose increased oxidative stress in primary rat PSCs, thereby facilitating the activation of these cells, while antioxidant treatment attenuated high glucose-induced PSC activation.

Changes in IP3 receptor are associated with altered calcium response to cholecystokinin in diabetic rat pancreatic acini.

Objectives: Pancreatic acini of diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion by CCK is closely related to the second messenger inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium stores via the endoplasmic reticulum-located receptor IP3 (IP3R). Recently, we observed altered intracellular calcium response on CCK-8 stimulation in streptozotocin (STZ)- treated diabetic rat acini. Methods: To determine whether IP3R is involved in altered calcium response, we measured inositol phosphate (IP) formation and the expression and phosphorylation of type III IP3R protein in diabetic acini. Also, CCK receptor mRNA expression was examined to determine whether the changes in IP formation and IP3R protein phosphorylation in diabetic acini might result from the defect at the postreceptor level. Results: CCK-8–induced IP formation at all concentrations used was significantly reduced in diabetic acini, though IP formation was increased in a concentration-dependent manner. The expression of type III IP3R protein was significantly reduced in diabetic acini. Additionally, CCK-8–stimulated phosphorylation of type III IP3R protein was not observed in diabetic acini. However, the reduction of CCK receptor mRNA expression was not detected in diabetic acini. Conclusion: Our results indicate that altered calcium response to CCK-8 in diabetic acini might be associated with a post-CCK receptor defect including the changes in IP formation, type III IP3R protein expression, and phosphorylation of type III IP3R protein.

Decreased Expression and Induced Nucleocytoplasmic Translocation of Pancreatic and Duodenal Homeobox 1 in INS-1 Cells Exposed to High Glucose and Palmitate

Background Type 2 diabetes mellitus (T2DM) is often accompanied by increased levels of circulating fatty acid. Elevations in fatty acids and glucose for prolonged periods of time have been suggested to cause progressive dysfunction or apoptosis of pancreatic beta cells in T2DM. However, the precise mechanism of this adverse effect is not well understood. Methods INS-1 rat-derived insulin-secreting cells were exposed to 30 mM glucose and 0.25 mM palmitate for 48 hours. Results The production of reactive oxygen species increased significantly. Pancreatic and duodenal homeobox 1 (Pdx1) expression was down-regulated, as assessed by reverse transcription-polymerase chain reaction and Western blot analyses. The promoter activities of insulin and Pdx1 were also diminished. Of note, there was nucleocytoplasmic translocation of Pdx1, which was partially prevented by treatment with an antioxidant, N-acetyl-L-cysteine. Conclusion Our data suggest that prolonged exposure of beta cells to elevated levels of glucose and palmitate negatively affects Pdx1 expression via oxidative stress.