Atorvastatin helps preserve pancreatic β cell function in obese C57BL/6 J mice and the effect is related to increased pancreas proliferation and amelioration of endoplasmic-reticulum stress

Abstract

Background: 3-Hydroxy-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitors or statins are competitive inhibitors of the rate-limiting enzyme in cholesterol biosynthesis. Currently, statins are used as first-line therapy in the treatment of diabetic dyslipidemia. However, effects of statins on β cell function remains unclear. This study aims to examine effects of atorvastatin treatment on pancreatic β cell function in obese C57BL/6 J mice and the possible mechanisms.

Methods: Diet-induced obesity (DIO) C57BL/6 J mice were treated with atorvastatin (30 mg/kg/day) for 58 days. β cell function was assessed by hyperglycemic clamp and the area of insulin-positive β cells was examined by immunofluorescence. Gene expression was assessed by RT-PCR, and endoplasmic reticulum (ER) stress related proteins were examined by Western blot. Additionally, cell viability and apoptosis of the cholesterol-loaded NIT-1 cells were investigated after atorvastatin treatment.

Results: Hyperglycemic clamp study revealed that glucose infusion rate (GIR) and insulin stimulation ratio in atorvastatin-treated DIO mice were markedly higher than control mice (P < 0.05, P < 0.01 vs. con), indicating preserved β-cell sensitivity to glucose. Lipid profiles of plasma triglyceride (TG), pancreas TG and plasma cholesterol (CHO) were improved. Pancreas weight and weight index were improved significantly after atorvastatin treatment (P < 0.05 vs. con). Immunofluorescence results showed that atorvastatin-treated mice had significantly larger insulin-positive β cell area (P < 0.05 vs. con). Furthermore, RT-PCR and western blot showed that the mRNA and protein expression of pancreatic and duodenal homeobox 1 (Pdx1) in the pancreas were upregulated (P < 0.001, P < 0.01 vs. con). Moreover, the expression level of ER stress markers of activating transcription factor 4 (ATF4), CCAAT-enhancer-binding protein homologous protein (CHOP) and phosphorylated eukaryotic initiation factor 2α (eIF2α) were downregulated in the pancreas of atorvastatin-treated mice (P < 0.001, P < 0.01, P < 0.01 vs. con). Besides, atorvastatin protected the pancreatic β cell line of NIT-1 from cholesterol-induced apoptosis. Western blot showed increased expression of anti-apoptotic protein of B-cell lymphoma 2 (Bcl-2).

Conclusion: Pancreatic β cell function of obese C57BL/6 J mice was preserved after atorvastatin treatment, and this improvement may be attributed to enhanced pancreas proliferation and amelioration of pancreatic ER stress.

Figure 1

Atorvastatin improves the β-cell sensitivity to glucose in obese C57 mice and decreases fasting insulin level. Pancreatic beta cell function was evaluated by hyperglycemic clamp. After sampling (t = 0 min) for the assay of the basal blood glucose and insulin, animals received intravenously glucose bolus followed by a constant infusion of glucose to maintain plasma glucose level at 14 mmol/l. (A) Plasma glucose level, (B) Glucose infusion rates (GIR), (C) Plasma insulin level, (D) Insulin stimulation ratio, and (E) fasting insulin level. Results are means ± S.E.M. (n = 4-5). *P < 0.05, **P < 0.01 vs. control.

Figure 2

Atorvastatin helps preserving β-cell area in obese C57 mice. (A) H&E staining of pancreatic islets. (B) Fluorescent staining of insulin-expressing β-cells (FITC; 20×) in non-obese, obese mice with and without atorvastatin treatmen. (C) Quantification of area of insulin-expressing β-cells. Results are means ± S.E.M. (n = 3). *P < 0.05 vs. control.

Figure 3

Atorvastatin upregulates PDX-1 and LXR-β expression and downregulates the protein expressions of ER stress markers. Total RNA was extracted from the pancreas of C57 mice and analyzed by quantitative real-time PCR. A comparative threshold cycle (CT) method was used for relative quantification of gene expression using beta-actin for normalization. Measurements were carried out in triplicate for each sample. (A) Relative mRNA levels of PDX-1 and LXR-β in pancreatic cells. Western blot analysis of pancreatic (B) PDX-1 (C) phosphorylated eIF2α (D) ATF4 (E) CHOP in C57 mice compared in three groups. Beta actin served as loading control. Data represented the mean of at least three independent experiments ± S.E.M. **p < 0.01, ***p < 0.001 vs. control.

Figure 4

Atorvastatin attenuates cholesterol-induced apoptosis of NIT-1 cells. (A) Effects of atorvastatin alone on NIT-1 cell viability. (n = 3–5) (B) Reserved β-cell relative viability after addition of different concentrations (10^-9 to 10^-5 M) of atorvastatin under treatment with 0.125 mM cholesterol for 12 h. Values are means ± S.E.M. (n = 3). (C) NIT-1 cell apoptosis with or without treatment of cholesterol and in addition of 10^-8 M atorvastatin incubation for 18 h. Results were detected by flow cytometry and quantification was made based on propidium iodide (PI) positive cells. (n = 3) (D) Protein expression of anti-apoptotic Bcl-2 in the pancreas. All the data were expressed as mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CHO group; ^##P < 0.01, ^###P < 0.001 vs. control.