Lipid accelerating the fibril of islet amyloid polypeptide aggravated the pancreatic islet injury in vitro and in vivo

Abstract

Background: The fibrillation of islet amyloid polypeptide (IAPP) triggered the amyloid deposition, then enhanced the loss of the pancreatic islet mass. However, it is not clear what factor is the determinant in development of the fibril formation. The aim of this study is to investigate the effects of lipid on IAPP fibril and its injury on pancreatic islet.

Methods: The fibril form of human IAPP (hIAPP) was tested using thioflavin-T fluorescence assay and transmission electron microscope technology after incubated with palmitate for 5 h at 25 °C. The cytotoxicity of fibril hIAPP was evaluated in INS-1 cells through analyzing the leakage of cell membrane and cell apoptosis. Type 2 diabetes mellitus (T2DM) animal model was induced with low dose streptozotocin combined the high-fat diet feeding for two months in rats. Plasma biochemistry parameters were measured before sacrificed. Pancreatic islet was isolated to evaluate their function.

Results: The results showed that co-incubation of hIAPP and palmitate induced more fibril form. Fibril hIAPP induced cell lesions including cell membrane leakage and cell apoptosis accompanied insulin mRNA decrease in INS-1 cell lines. In vivo, Plasma glucose, triglyceride, rIAPP and insulin increased in T2DM rats compared with the control group. In addition, IAPP and insulin mRNA increased in pancreatic islet of T2DM rats. Furthermore, T2DM induced the reduction of insulin receptor expression and cleaved caspase-3 overexpression in pancreatic islet.

Conclusions: Results in vivo and in vitro suggested that lipid and IAPP plays a synergistic effect on pancreatic islet cell damage, which implicated in enhancing the IAPP expression and accelerating the fibril formation of IAPP.

Keywords: IAPP; Insulin; Lipid; Palmitate; Pancreatic islet; T2DM.

Fig. 1

Effects of palmitate on the fibrillation of hIAPP. a Relative thioflavin-T fluorescence intensity. b TEM image of hIAPP alone. c TEM image of hIAPP co-incubated with palmitate. * denotes p < 0.05 and ** denotes P < 0.01 compared to the hIAPP. n = 3. One-way ANOVA was used with post hoc test

Fig. 2

Effects of fibril hIAPP on INS-1 cells. a mRNA levels of insulin. b Membrane permeability evaluated by LDH leakage assay. c Representative images of WB for cleaved aspase-3 expression. d Relative quantitation of cleaved caspase-3 expression was performed. * denotes p < 0.05 and ** denotes P < 0.01 compared to the control group. n = 3. One-way ANOVA was used with post hoc test

Fig. 3

Body weight, fast glucose tolerance and insulin sensitivity after treating with high-fat diet, STZ injection, and STZ injection combined with high-fat diet. a Flow chart of animal experiment. b The extent of body weight gain duration of two months. * denotes p < 0.05 in the HD group vs. control group; ## denotes p < 0.01 in the STZ group vs. control group. n = 12. c Glucose tolerance is evaluated after 12 h fasting. * denotes p < 0.05 in the HD group vs. control group, n = 12. d Insulin sensitivity was evaluated after 12 h fasting, * denotes p < 0.05 in the HD group vs. control group. n = 12. Two-way ANOVA was used with post hoc test

Fig. 4

Blood biochemical parameters of rats after treating with high-fat diet, STZ injection, and STZ injection combined with high-fat diet for 2 months. a Plasma glucose. b Plasma triglyceride. c Plasma insulin. d Plasma rIAPP. * denotes p < 0.05 compared with the control group; ** denotes p < 0.01 compared with the control group. n = 12. Two-way ANOVA was used with post hoc test

Fig. 5

Morphology of pancreatic islet after treatment with STZ injection, high-fat diet, and STZ injection combined with high-fat diet in rats. a Representative images of H-E staining to show the islet by dashed lines. b Representative images of insulin immunoflurorescence staining to show the islet by dashed lines. c Representative images of amylin expression in the islet (outlined by dashed lines). d Levels of amylin expression calculated with ImageJ software in sixteen islets from four rats each group. e Representative images of TUNEL staining in the islet (outlined by dashed lines). f Numbers of TUNEL positive cells in the islet were calculated and normalized by unit area of pancreatic islet. * denotes p < 0.05 compared with the control group. n = 4. Two-way ANOVA was used with post hoc test

Fig. 6

Isolated pancreatic islet function was evaluated after treatment with STZ injection, high-fat diet, and STZ injection combined with high-fat diet in rats. a mRNA levels of amylin. b mRNA levels of insulin. c Representative images of WB for IRβ expression. Relative quantitation of IRβ expression was performed and shown in (d). e Representative images of WB for cleaved caspase-3 expression. f Relative quantitation of cleaved caspase-3 expression was performed. * denotes p < 0.05 compared with the control group; ** denotes p < 0.01 compared with the control group. n = 4. Two-way ANOVA was used with post hoc test