Mechanisms Of Lipotoxicity On β-cell Dedifferentiation And Protective Effects Of Endogenous GLP-1in Pancreatic Islet

Part Ⅰ Effects of lipotoxicity on the damage of β-cells and the dedifferentiation of pancreatic islet cellObjectives:To observe the effects of palmitate on cell viability and apoptosis to survive isolated pancreatic islets and explore the effects of high-fat diet on the status of β-cell function and differentiation in vivo.Methods:Pancreatic islets were isolated from overnight-fasted C57BL/6J with collagenase P and hand-picked using a stereomicroscope. After incubation with0.5mmol/L palmitate for the indicated durations (24h,48h and72h), islet viability and apoptosis were analyzed using the MTT assay and by examining histone-associated DNA fragments, respectively. After incubation with0.5mmol/L palmitate for48h, the insulin and PDX1mRNA and protein levels were determined using Q-PCR and immunoblotting, respectively. Long term high-fat diet was fed in C57BL/6J mice, to create a lipotoxicity animal model. Plasma insulin level was detected by ELISA. The intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) was performed at4weeks and8weeks. The area under curve of IPGTT and IPITT and HOMA-IR were calculated to evaluate the insulin sensitivity. Immunofluorescent staining was used to detect β-cell by FoxO1, Insulin and PDX1, a-cell by glucagon and stem cell by OCT4and Nanog. The proliferation and apoptosis of islet cells were detected by Ki67and Caspase-3, respectively.Results:After incubation of isolated mouse islets for24h,48h or72h, treatment with0.5mmol/1palmitate led to a significant decrease in islet viability and an increase in cell death in a time-dependent manner. Because exposure of cultured islet cells for72h was severely damaging, we focused on the effects of palmitate on P-cell-specific transcription factors after48h of treatment. Incubation with palmitate for48h did not significantly modify the expression of insulin mRNA. Nonetheless, both PDX1mRNA and protein levels decreased after48h of exposure. Plasma insulin levels were higher in high-fat diet group. High-fat diet enhanced insulin resistance which was shown by the calculated area under the curve of IPITT and IPGTT and HOMA-IR index, especially at8week (p<0.05). Immunofluorescence showed that high-fat diet reduced the protein expression of the β-cell markers FoxO1, insulin and PDX1, increased the a-cell mass and promoted the expression of stem cell markers OCT4and Nanog. The Ki67expression was upregulated in high-fat diet group, however, the overall number was still rare. The caspase-3expression was also raised, indicating the increase of islet cell apoptosis.Conclusions:Chronic exposure palmitate and high-fat diet damage islet cell viability cell and function and increased cell apoptosis. In high-fat diet group, the insulin resistance was enhanced. High-fat diet promoted islet cells dedifferentiation into stem cells. Part Ⅱ The activation of intra-islet GLP-1system by lipotoxicity-inducing oxidative stress and its mechanismObjectives:To evaluate the effects of lipotoxicity on endogenous glucagon-like peptide1(GLP-1) system and to explore the role of oxidative stress in pancreatic islet in vitro and in vivo.Methods:After incubation with0.5mmol/L palmitate for the indicated durations (24h,48h and72h), the GLP-1concentration was detected by ELISA in cell medium and cell lysates and the expression of prohormone convertase1/3(PC1/3) mRNA and protein were determined using Q-PCR and immunoblotting in cell lysates, respectively. The levels of ROS were detected by DCFH-DA assay and an antioxidant N-acetylcysteine (NAC) was used to further observe the alteration of PC1/3mRNA and GLP-1expression levels. After8weeks of being fed a HFD, plasma GLP-1level, the PC1/3mRNA in pancreas and the formation of pro-a were detected by ELISA, Q-PCR and immunofluorescent staining, respectively.Results:Importantly, incubation with0.5mmol/L palmitate for24h,48h or72h markedly induced GLP-1release into the medium by3.15-,6.55-and5.62-fold, respectively. Moreover, in line with the culture medium observation, the GLP-1concentration in cell lysates was elevated and showed an even larger increase. After prolonged exposure to palmitate, the mRNA and protein levels of PC1/3, the key enzyme of GLP-1generation, also increased. After8weeks of being fed a HFD, PC1/3expression and plasma GLP-1markedly increased. When pro-α cells (PC1/3-positive and insulin-negative a-cells) were quantified, an increased number of these cells (163) was noted in the HFD group compared with the LFD group (n=6)(48) in every20slices. Incubation with0.5mmol/L palmitate for24h,48h or72h markedly increased intra-islet ROS levels by approximately3.01-,5.12-and6.45-fold, respectively. In addition, pre-incubation with5mmol/L NAC, increased islet viability from59.56%to84.94%and decreased apoptosis by approximately64.84%in the presence of0.5mmol/L palmitate. Importantly, NAC significantly decreased the PC1/3mRNA levels and the GLP-1concentration in cell lysates, which might be due to the attenuation of β-cell injury. Nonetheless, elevated levels of PC1/3mRNA and GLP-1protein which induced by palmitate did not return to normal after NAC treatment.Conclusions:Chronic palmitate exposure or high-fat diet enhances the GLP-1levels and its Key enzyme PC1/3and in turn activates intra-islet GLP-1system. The inhibition of oxidative stress contributes to the survival of islet cell and partly reverses the activation of the GLP-1system. Those results demonstrate oxidative stress plays a role as the bridge between lipid overload and the intra-islet GLP-1system. Parts Ⅲ Protective effects of islet endogenous GLP-1by maintaining redox status and reducing inflammationObjective:To observe protective effects of islet endogenous GLP-1by altering the activation of GLP-1receptor and to explore its mechanism by measuring oxidative stress and inflammation in pancreatic islet in vitro and in vivo.Methods:After the exposure of palmitate and altering the activation of GLP-1R receptor by adding antagonist Exendin9-39or agonist liraglutide in isolated C57BL/6J mouse islets, islet cell viability, cell apoptosis, PDX1mRNA expression and ROS levels were detected by MTT, ELISA, Q-PCR and DCFH-DA assay, respectively. The effects of liraglutide on the mRNA expression were measured by Q-PCR, including NA(D)PH oxidase components (NOX4, p22phox and gp91phox), antioxidant genes (SOD2and Gpx-1) and inflammatory cytokines (TNF-a, IL-1β and IL-6). After C57BL/6J mice fed high-fat diet for8weeks and injected liraglutide for4weeks, the changes of islet structure and NF-κB signaling pathway were evaluated by immunofluorescence.Results:When combined with palmitate exposure for48h, Exendin-9-39treatment resulted in a progressive loss of islet cells that exhibited decreased viability from57.82to40.28%, higher apoptotic levels from0.48to0.72and reduced PDX1expression. While liraglutide significantly increased islet viability and decreased islet cell apoptosis nearly to control levels. Liraglutide also markedly upregulated PDX1mRNA expression7.70-fold, and this increase was attenuated by palmitate. Exendin-9-39also slightly increased the production of ROS and liraglutide had reverse effects. Liraglutide attenuated the palmitate-induced activation of NAD(P)H oxidase components (including NOX4, p22phox and gp91phox), upregulated the expression of mitochondrial-specific superoxide dismutase2(SOD2) and glutathione peroxidase1(Gpx-1) and suppressed palmitate-induced inflammatory factors (such as TNF-a, IL-1β and IL-6). In the HFD group, liraglutide also increased the mRNA expression of β-cell markers PDX1, Nkx6.1and GLUT2. Unlike the defined a-cell mantle and β-cell core characteristics of LFD islets, the HFD islets maintained a more scattered organization and a higher percentage of a-cells. Double immunostaining studies showed that p65protein expression levels were markedly near that of LFD in pancreatic islets of mice fed a HFD.Conclusion:Intra-islet GLP-1normalizes pancreatic islet structure and facilitates P-cell survival by maintaining the oxidative balance and inhibiting islet inflammation and the expression of inflammatory factors.