Effects Of Earlier Intervention On The Preservation Of Pancreatic β-cells In Db/db Mice

Objectives:To explore the effects of different timings and intervention methods on the preservation of pancreaticβ-cells in db/db mice. To make comparison of effects ofβ-cell preservation between interventions on prediabetes and newly onset diabetes or cross-sectional comparison between different interventions.Methods:1. Ninety-eight male,3-week old db/db mice which were confirmed as euglycemia were randomly assigned into two intervention model, early and earlier intervention. The interventions started at age 4 week referred to earlier intervention (treatments starting from age 4 week, T4), whereas interventions started at age 6 week referred to early intervention (treatments starting from age 6 week, T6). The mice fed with normal chow set as control group. Four groups of mice received liraglutide (300μg/kg wt bid), pioglitazone (0.02%PIO in mice chow), calories restriction (70%of food available of control) and exercise treatment respectively. Glycosylated hemoglobin (HbAlc) were determined before and after interventions. Fasting blood glucose (FBG), body weight and 24h food intake were monitored weekly. All interventions ended when the mice were 12-week old and introperitoneai glucose tolerance lest (IPGTT) or insulin tolerance test (ITT) was performed before mice were sacrificed. Plasma TG, FFA, insulin, adiponectin, and glucagon were determined. Tissue slides from paraffin embedded pancreas were stained by HE or antibodies for insulin, glucagon, Brdu (for determiningβ-cell proliferation rate), PDX-1(a factor essential for insulin gene expression) and IRS-2 (a protein involved in insulin signal pathway ofβ-cells). Apoptosis of pancreaticβ-cells were detected by TUNEL assay. Expression of GRP78 and CHOP were quantified by real-time quantitative PCR and western blot for assessing endoplasmic reticulum stress (ERS). Gene expression of XBP-1/spliced XBP-1 was detected by PCR and electrophoresis. Expression of microRNA miR-34a and miR-375 were quantified by real-time quantitative PCR.2. Thirty five 8-week old male db/db mice were randomly assigned into 4 groups:control group, PIO group (0.02%PIO in mice chow), LIRA group (liraglutide 300μg/kg wt), and combined treatment group (liraglutide 300μg/kg wt+0.02%PIO in mice chow). The effects of combined interventions on glucose, lipid metabolism and pancreaticβ-cell preservation were assessed after 4 weeks intervention as described above. Results:1.1) HbA1c of mice (12 week of age) treated at 4 week of age were lower than mice treated at 6 week of age (control:7.3±0.3%, T4:4.9±0.8%, T6:5.5±0.9%, P<0.001 vs. control, P<0.001 vs. T6). AUCglu after IPGTT showed similar changes as HbA1c (control:4568±190 mmol/l*mi, T4:2306±727 mmol/l*mi, T6:3559±903 mmol/l*min, P<0.001 vs. control, P<0.001 vs. T6). There were differences among treatments in HbAlc:(LIRA4.9±0.5%, PIO 4.3±0.4%, CR 5.4±0.7%, EX 6.1±0.6%, P<0.001) and in AUCglu (LIRA:2558±639 mmol/l*min, PIO:1662±483 mmol/l*min, CR:3262±613 mmol/l*min; EX:3904±610 mmol/l*min, P<0.001).2) Fasting plasma insulin level were higher in treated groups (control:5.7±2.5ng/ml,T4 11.0±3.0ng/ml, T6: 9.1±4.6ng/ml P<0.01 vs. control, p<0.05 vs. T6). Regarding to various interventions, although fasting insulin levels in all intervention groups were slightly higher than control group, only a significant difference was found in mice with liraglutide intervention (P=0.001). Earlier intervention showed better preservation of glucose-stimulated insulin secretion assessed by AUCins by IPGTT (control:799±186 ng/ml*min, T4:1410±595 ng/ml*min, T6:1216±347 ng/ml*min, P<0.001 vs. control, P<0.05 vs. T6).3) The levels of plasma TG, FFA and glucagon were reduced by different magnitude with various interventions, while no differences were observed between time points that treatments started. Only pioglitazone increased adiponectin levels among interventions (9.5±1.6 vs.15.3±3.9mg/l, P<0.05).4) At 12 week of age, db/db mice without intervention showed hypertrophic islet with fewer and weaker insulin staining cells, and abnormally distributedα-cells. Interventions that started at 4 week of age had a better effect on the preservation of pancreaticβ-cells (Insulin stained area:control 27.0±1.5%, T4 50.8±6.4%, T6 44.5±8.1%, P<0.001). In PIO-treated mice, islets were smaller with well-stainedβ-cells and nearly normal distribution of a-cells; LIRA and CR failed to prevent islet from hypertrophy, but they preservedβ-cell mass and alleviated degranulation; EX only had modestly effects. Apoptosis were reduced with interventions except for EX-treated mice, in which LIRA shown the better effect. Increase of pancreaticβ-cell proliferation was observed in LIRA and CR groups.5) LIRA preserved PDX-1 expression more while PIO increased IRS-2 expression in islets.6) LIRA and PIO inhibited expression of pancreatic ERS markers GRP78 and CHOP.7) LIRA reduced pancreatic expression of miR-375 by 71% and miR-34a by 50%(P<0.05).(2) After 4 weeks, HbA1cwere decreased under all treatments (control:7.3±0.4, PIO:4.9±0.6, LIRA:5.5±0.4, combination:4.5±0.6) and combination therapy did better than PIO or LIRA alone. PIO in combination with LIRA improved glucose tolerance (reduction of area under curve of glucose by IPGTT of combination was 56%,P<0.001) and preserved insulin release response to glucose (augment of area under curve of insulin by IPGTT of combination was 91%, P<0.01), both of which were greater than either medicine alone. Combination treatment also reduced circulated FFA by 29%(PIO alone:22%; LIRA:no effect), TG by 49%(PIO alone:35%; LIRA alone:15%), and increased plasma adiponectin by 95%(PIO:80%; LIRA no effect) compared with control, more effectively than PIO or LIRA alone. Islet immunohistochemistry showed that insulin positive area were increased significantly by 1.7 folds of control (PIO:1.3 folds; LIRA:0.9 fold) and isletβ-cell proliferation rate were increased by 2 folds (PIO, no effect; LIRA:1.7 folds) in combination-treated group, which confirmed the greater preservation ofβ-cells by combination treatment of PIO and LIRA than either treatment alone.Conclusions:1. Earlier interventions when blood glucose is within normal range manifests greater effect on preservation of isletβ-cells than early interventions of type 2 diabetes.2. Liraglutide has dirrect effect on preservation of isletβ-cells but only partly compensates severe insulin resistance.3. Combined therapy improves glucose and lipid metabolism, preserves islet beta-cell function and stimulates beta-cell proliferation, which are greater than either liraglutide or pioglitazone treatment alone. Objectives.-To examine the protective effect of genipin from palmitate-induced cytotoxicity in HepG2 cells and investigate the underlying mechanism.Methods:HepG2 cells were treated respectively with bovine serum albumin (BSA), palmitate (lmmol/1), genipin(20μmol/l) or genipin+palmitate for 24h, the cell viability was assayed by Methyl thiazol tetrazolium (MTT) method. Lactate dehydrogenase enzyme (LDH) release was measured to assess cell damage. Flowcytometry (Annexin V-PI) and Hoechst staining were employed for determination of cell apoptosis after 16h-treatment. ERS markers GRP78 and CHOP mRNA expression were quantified by Real time PCR while XBP-1 splicing was showed by PCR and electrophoresis.Results:Compared with BSA, palmitate decreased cell viability while increased LDH release (P<0.05). It also significantly induced apoptosis of HepG2 cell (P<0.05). Expression of GRP78 and CHOP mRNA was up-regulated by palmitate (P<0.05), so was XBP-1 splicing. Compared with palmitate, genipin pretreatment increased cell viability (P<0.05), inhibited apoptosis (P<0.01), and reduced LDH release (P<0.01) of HepG2 cell. The expression of GRP78 and CHOP mRNA was decreased by genipin (P<0.01). Electrophoresis of XBP-1 PCR products showed less spliced XBP-1 in genipin pretreated cells than palmitate treated ones.Conclusions:The results suggest that genipin protects HepG2 cells from palmitate-induced cell apoptosis and death mediated by ER stress. Objectives:This study is aimed at characterizing the role of peroxisome proliferator activated receptors (PPAR) y or a in glucose metabolism and insulin resistance of human hepatoma cells.Methods:The model of insulin resistance was established with HepG2 cells cultured at high concentrations of palmitate. Insulin resistant HepG2 cells were treated with the PPAR-y agonist pioglitazone or the PPAR-a agonist WY14643. Quantification of glucose consumption and glycolysis products pyruvate and lactate were performed. Quantitative RT-PCR was employed to analyze mRNA expression of PPAR-γ/a and glycolysis related genes.Results:Palmitate treatment decreased glucose consumption of HepG2 cells. Pioglitazone increased glucose consumption of both normal and insulin resistant HepG2 cells. It induced an up-regulation of glycolysis gene expression and strongly increased glycolysis leading to an elevated pyruvate and lactate production. WY14643 slightly increased glucose consumption of insulin resistant HepG2 cells, but had no effects on glycolysis gene expression.Conclusions:Altogether these results show that PPAR-y or PPAR-a can enhance glucose metabolism in HepG2 cells through different mechanisms.