Vascular endothelial growth factor B inhibits insulin secretion in MIN6 cells and reduces Ca2+ and cyclic adenosine monophosphate levels through PI3K/AKT pathway

BACKGROUND Type 2 diabetes(T2 D) is characterized by insufficient insulin secretion caused by defective pancreatic β-cell function or insulin resistance,resulting in an increase in blood glucose.However,the mechanism involved in this lack of insulin secretion is unclear.The level of vascular endothelial growth factor B(VEGF-B) is significantly increased in T2 D patients.The inactivation of VEGF-B could restore insulin sensitivity in db/db mice by reducing fatty acid accumulation.It is speculated that VEGF-B is related to pancreatic β-cell dysfunction and is an important factor affecting β-cell secretion of insulin.As an in vitro model of normal pancreatic β-cells,the MIN6 cell line can be used to analyze the mechanism of insulin secretion and related biological effects.AIM To study the role of VEGF-B in the insulin secretion signaling pathway in MIN6 cells and explore the effect of VEGF-B on blood glucose regulation.METHODS The MIN6 mouse pancreatic islet β-cell line was used as the model system.By administering exogenous VEGF-B protein or knocking down VEGF-B expression in MIN6 cells,we examined the effects of VEGF-B on insulin secretion,Ca2+ and cyclic adenosine monophosphate(cAMP) levels,and the insulin secretion signaling pathway.RESULTS Exogenous VEGF-B inhibited the secretion of insulin and simultaneously reduced the levels of Ca2+ and cAMP in MIN6 cells.Exogenous VEGF-B also reduced the expression of phospholipase C gamma 1(PLCγ1),phosphatidylinositol 3-kinase(PI3 K),serine/threonine kinase(AKT),and other proteins in the insulin secretion pathway.Upon knockdown of VEGF-B,MIN6 cells exhibited increased insulin secretion and Ca2+ and cAMP levels and upregulated expression of PLCγ1,PI3 K,AKT,and other proteins.CONCLUSION VEGF-B can regulate insulin secretion by modulating the levels of Ca2+ and cAMP.VEGF-B involvement in insulin secretion is related to the expression of PLCγ1,PI3 K,AKT,and other signaling proteins.These results provide theoretical support and an experimental basis for the study of VEGF-B in the pathogenesis of T2 D.

LRP16 gene protects mouse insulinoma MIN6 cells against fatty acid-induced apoptosis through Akt/FoxO1 signaling

Background Pancreatic β cells are susceptible to fatty acid-induced apoptosis. The 17β-estradiol （E2） protects pancreatic βcells from apoptosis, mediated by the estrogen receptor-a （ERa）. The mRNA level and promoter activity of leukemia-related protein （LRP） 16 were significantly increased by E2 in E R-a and LRP 16 was a co-activator of ER-a. The aim of the study was to assess the effects of LRP16 on fatty acid-induced apoptosis in MIN6 cells. Methods Cells with over-expressing LRP16 were obtained by lipidosome transfection. Insulin content and glucose-stimulated insulin secretion （GSIS） were examined by radioimmunoassay. Western blotting was applied to detect protein expression. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） and flow cytometry. The forkhead boxO1 （FoxO1） subcellular localization was determined by immunocytochemical analysis. Results MIN6-LRP16 cells with overexpression of LRP16 were successfully established, and protein expression of LRP16 was 2.29-fold of that of control cells （MIN6-3.1, P 〈0.05）. Insulin content and GSIS in MIN6-LRP16 were substantially increased compared with those in control cells. When cells were stimulated with glucose, increased phosphorylation of extracellular signal-regulated kinase （ERK） 1/2 and serine-threonine kinase （Akt） were observed in MIN6-LRP16. When cells were under palmitate pressure, the TUNEL-positive rate in MIN6-LRP16 was （17.0±0.5）%, while it in MIN6-3.1 was （22.0±0.4）%. In palmitate-treated cells, attenuated Akt phosphorylation was observed, but the attenuation in Akt activity was partially restored in MIN6-LRP16 cells. Meanwhile, nuclear localization of FoxO1 in MIN6-LRP16 was apparently reduced compared with that in control cells. Conclusions LRP16 regulated insulin content and GSIS in MIN6 cells by ERK1/2 and Akt activated way. Meanwhile, LRP16 overexpression protected MIN6 cells from fatty acid-induced apoptosis by partially restoring Akt phosphorylation and inhibiting FoxO1 nuclear redistribution. Therefore, LRP16 played important roles not only in insulin content and GSIS but also in the antilipotoxic effect mediated by Akt/FoxO1 signaling.

Sangguayin preparation prevents palmitate-induced apoptosis by suppressing endoplasmic reticulum stress and autophagy in db/db mice and MIN6 pancreatic β-cells

Sangguayin preparation(SGY-P) is refined from the traditional Chinese medicinal compound Sangguayin, which"clears heat and promotes fluid" and "tonifies kidney and spleen" for "Xiaoke", commonly known as ‘Diabetes mellitus’ in clinics.Previous studies have shown that SGY-P could reduce insulin resistance and repair damaged pancreas in db/db mice, but the underlying mechanisms were unclear. Here, we investigated whether treatment with SGY-P could protect pancreatic β-cells from apoptosis and uncovered the underlying mechanisms. db/db mice were used to observe the hypoglycemic and islet protective effect in vivo. Apoptosis was induced in mouse insulinoma 6(MIN6) cells by palmitate, following which the cells were treated with SGY-P for elucidating the anti-apoptotic mechanism in vitro. Cell viability and nuclear morphology were detected by CCK-8 assay and Hoechst 33258 staining. The expression levels of apoptosis-, endoplasmic reticulum(ER) stress-, and autophagy-related proteins were measured by western blot. The results showed that SGY-P reduced fasting blood glucose, pancreatic pathological changes, and islet β-cell apoptosis in db/db mice. Palmitate-induced apoptosis in MIN6 cells was decreased by SGY-P treatment. Hence, SGY-P therapy exhibited a protective effect on pancreatic β-cells by decreasing the expression of cleaved caspase-3, cleaved PARP and Bax, and increasing Bcl-2 by suppressing ER stress(Bip/XBP1/IRE1α/CHOP/Caspase-12) and autophagy(LC3/p62/Atg5) pathways.2/Atg5 pathways.