In this study, the effects of atorvastatin treatment on pancreatic β cell function in obese C57BL/6 J mice and its possible mechanism were investigated. After 58 days of treatment, GIR and insulin stimulation ratio of atorvastatin treated mice were improved compared with control mice. Furthermore, these mice had greater insulin positive β cell area. Besides, pancreas ER stress markers were down-regulated. In vitro results suggest a protective role of atorvastatin against cholesterol-induced apoptosis of NIT-1 cells. All the results indicate the beneficial effects of atorvastatin on β-cell function.
The hyperglycemic clamp has been demonstrated to be a reliable technique to evaluate β-cell sensitivity to glucose . Because the glucose level is held constant, the glucose infusion rate is an index of glucose metabolism. Defect of initial phase of insulin secretion is the earliest detectable abnormality in diabetes mellitus. The insulin stimulation ratios of first and second phases were both enhanced after atorvastatin treatment, indicating a preserved β cell function.
Up to date, many researchers have showed the beneficial effects of atorvastatin on insulin resistance were due to amelioration of inflammation [7, 18, 19]. In hyperglycemic clamp test, the insulin at 0 min in atorvastatin group was significantly lower (p < 0.05 vs. con), and we confirmed the result by examining insulin level again when the animals were sacrificed. The decreased fasting insulin implied the insulin resistance was ameliorated and this is accordant to results reported above. It is possible that the decreased demand of insulin could relieve the work burden of ER to synthesize and secrete insulin. Hence, the ER stress state was alleviated based on lipid-independent effect.
We observed that 30 mg/kg atorvastatin improved the lipid profile of obese C57BL/6 J mice. Plasma TG and pancreatic TG were both decreased (P < 0.05, P < 0.001 vs. Con ). TG is reported to activate ER stress, and ER stress impairs insulin sensitivity by decreasing the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) . Thus, the lipid-lowering effect of atorvastatin may lead to amelioration of ER stress and insulin resistance and eventually preserved the β-cell function. Moreover, the LXR-β, which plays a central role in regulation of cholesterol metabolism was also up-regulated in pancreas.
On the other hand, atorvastatin may improve β cell function through accelerating the pancreas proliferation as shown by the immunofluorescence results. PDX-1 mRNA and protein level in pancreas were both increased significantly. PDX-1 is important for pancreatic development and β-cell function. Pdx1+/- mice showed worsening glucose tolerance and insulin secretion and the islets were more susceptible to apoptosis .
Another possible reason of preserved β cell function could be the improvement of endothelial function. Islet endothelium plays an important role in providing oxygen and nutrients to endocrine cells, trans-endothelial rapid passage of secreted insulin into the circulation and blood glucose sensing and regulation [22, 23]. Endothelial dysfunction has been shown in patients with type 1 and type 2 diabetes mellitus. Improved metabolic control in diabetic patients is associated with near restoration of endothelial function . As statins were shown to increase the expression of eNOS and iNOS [25, 26], and may increase the NO production leading to vascular relaxation. Endothelium function may therefore be improved. Atorvastatin was reported to improve the regeneration of β-cell mass due to increase of intro-islet endothelial cells .
As the strong association between the ER stress and diabetes [27, 28], we specifically investigated whether atorvastatin exerted its effect on pancreatic β cells by modulating the ER stress. Once the ER stress is present, the UPR will be triggered to cope with stress conditions. There are three sensing proteins, inositol-requiring 1α (IRE1α), PERK and activating transcription factor 6 (ATF6) . As for the PERK pathway, PERK phosphorylates eIF2α, and this will cause more efficiently translation of ATF4. Chop is the downstream protein of PERK–eIF2α–ATF4 pathway and mainly induce the apoptosis caused by the ER stress in the UPR . Moreover, Chop-/- mice had improved glycemic control and expanded beta cell mass . In this study, we found that eIF2α–ATF4-Chop pathway was turned down after atorvastatin treatment. However, whether ATF6 and IRE1α pathways are involved needs to be investigated in further study.
The cholesterol induced apoptosis model of NIT-1 cells provides us a tool to investigate the effects of atorvastatin. Recently, cholesterol has been proved to induce the ER stress and apoptosis in macrophages . As type 2 diabetes is accompanied with inflammation, we mimicked the ER stress state by loading cholesterol onto NIT-1 cells. We found that cholesterol suppressed the viability of NIT-1 cells, which was attenuated by atorvastatin in a dose-dependent manner. Flow cytometry test further demonstrated that atorvastatin ameliorated cholesterol-induced apoptosis of NIT-1 cells. CHOP was shown to down-regulate the anti-apoptotic protein of Bcl-2 , the preserved Bcl-2 expression in NIT-1 cells is accordant with the depression of CHOP expression in pancreas. Besides, atorvastatin alone did not negatively affect NIT-1 cell viability and increased viability at high concentration. This result could interpret the increased insulin positive β-cell area observed in C57 mice.
However, some clinical trials have revealed the deterioration of glucose metabolism of statins . And FDA has expanded advice on statin risks of possibility of developing type 2 diabetes. The possibility that patients who have cardiovascular disease are already at high risk of developing diabetes can’t be expelled. Another explanation is that long term aggressive therapy with statins could induce the adverse effects.
In this study, aggressive dose of atorvastatin was used. The dose of 30 mg/kg/d of atorvastatin in mice is equivalent to 170 mg/d in a 70 Kg human calculated based on the body surface area (BSA) [34, 35]. This is more than the highest dose of 80 mg/d which is recommended. For adults with diabetes, the American Diabetes Association recommends aggressive use of statin in the treatment of diabetic dyslipidemia . In the REVERSAL trial, aggressive lipid lowering with atorvastatin (80 mg/d) showed beneficial effects on halting the atherosclerosis progression (-0.4%) compared with baseline, and the effect is superior to simvastatin 40 mg . Meanwhile, antioxidant and anti-inflammation benefits of atorvastatin 80 mg were also observed in MIRACL and ASAP trails [38, 39]. Besides, the aggressive (80 mg/d) and moderate (10 mg/d) lipid-lowering therapy with atorvastatin was compared in the DALI trail . Consequently, Fasting TG was reduced by 35% with aggressive therapy and by 25% with moderate therapy. Thus, atorvastatin 80 mg/d is of better effects on lipid file alterations compared with 10 mg/d.