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. 2015 Nov 2;11(11):2089-2101.
doi: 10.1080/15548627.2015.1091139.

Chronic HMGCR/HMG-CoA reductase inhibitor treatment contributes to dysglycemia by upregulating hepatic gluconeogenesis through autophagy induction

Hye Jin Wang  1   2   3 , Jae Yeo Park  4   5 , Obin Kwon  3   6 , Eun Yeong Choe  1   2 , Chul Hoon Kim  3   6 , Kyu Yeon Hur  7 , Myung-Shik Lee  7   8 , Mijin Yun  9 , Bong Soo Cha  1   2   3 , Young-Bum Kim  10 , Hyangkyu Lee  4   5 , Eun Seok Kang  1   2   3
Affiliations

Chronic HMGCR/HMG-CoA reductase inhibitor treatment contributes to dysglycemia by upregulating hepatic gluconeogenesis through autophagy induction

Hye Jin Wang et al. Autophagy. .

Abstract

Statins (HMGCR/HMG-CoA reductase [3-hydroxy-3-methylglutaryl-CoA reductase] inhibitors) are widely used to lower blood cholesterol levels but have been shown to increase the risk of type 2 diabetes mellitus. However, the molecular mechanism underlying diabetogenic effects remains to be elucidated. Here we show that statins significantly increase the expression of key gluconeogenic enzymes (such as G6PC [glucose-6-phosphatase] and PCK1 (phosphoenolpyruvate carboxykinase 1 [soluble]) in vitro and in vivo and promote hepatic glucose output. Statin treatment activates autophagic flux in HepG2 cells. Acute suppression of autophagy with lysosome inhibitors in statin treated HepG2 cells reduced gluconeogenic enzymes expression and glucose output. Importantly, the ability of statins to increase gluconeogenesis was impaired when ATG7 was deficient and BECN1 was absent, suggesting that autophagy plays a critical role in the diabetogenic effects of statins. Moreover autophagic vacuoles and gluconeogenic genes expression in the liver of diet-induced obese mice were increased by statins, ultimately leading to elevated hepatic glucose production, hyperglycemia, and insulin resistance. Together, these data demonstrate that chronic statin therapy results in insulin resistance through the activation of hepatic gluconeogenesis, which is tightly coupled to hepatic autophagy. These data further contribute to a better understanding of the diabetogenic effects of stains in the context of insulin resistance.

Keywords: HMG-CoA reductase inhibitor; autophagy; diabetes; gluconeogenesis; statin.

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Figures

Figure 1.
Figure 1.
Statins upregulate the expression of gluconeogenic enzymes but not glycolytic enzymes in HepG2 cells. (A) Glycolysis and gluconeogenesis pathways. (B) Insulin inhibits gluconeogenesis and enhances glycolysis in this system. Statins (20 µM) upregulated not only mRNA levels of G6PC (C) and PCK1 (D), but also protein levels of G6PC and PCK1 (E). However statins had little effect on mRNA levels of GCK (F) and PKLR (G) in HepG2 cells. *, P <0.05; **, P<0.01 compared with control (CTL).
Figure 2.
Figure 2.
Statins induce autophagy in HepG2 cells. (A) HepG2 cells were transfected with the autophagy sensor GFP-LC3A then treated with rosuvastatin, fluvastatin, pravastatin, or atorvastatin (20 µM) for 24 h. Fluorescent images were obtained by confocal microscopy. (B) Columns in the histogram represent the number of LC3A puncta per cell. At least 6 random fields were chosen from each sample. (C) Results of western blot analysis showed increased LC3B-II levels in statin-treated cells. The Baf A1-treated group showed that statins induced autophagy flux. (D) After transfecting HepG2 cells with mRFP-GFP-LC3B, statins were added for 24 h. Fluorescent images were obtained by confocal microscopy. The GFP protein is unstable in low pH inside of the lysosome and thereby degraded. In contrast, RFP is more stable in acidic conditions and thereby it could maintain red fluorescence. (E) Columns in the histogram represent the ratio of mRFP and GFP LC3B puncta. *, P < 0.01 compared with control.
Figure 3.
Figure 3.
Statins increase autophagy-dependent gluconeogenesis in HepG2 cells. After treatment with 20 µM statins for 22 h, 50 µM chloroquine was added to HepG2 cells for 2 h, with statins. HepG2 cells treated with statins and CQ (A) G6PC and (B) PCK1 were analyzed with qRT-PCR. (C) Glucose production by HepG2 cells treated with statins and CQ. HepG2 cells treated with statins and 20 nM Baf A1. (D) G6PC and (E) PCK1 were analyzed with qRT-PCR. (F) Glucose production was measured. (G) After transfecting HepG2 cells with BECN1 shRNA, knockdown of BECN1 was confirmed by western blot analysis. Inhibition of autophagy by BECN1 knockdown decreased expression of G6PC (H) and PCK1 (I). *, P < 0.05, **, P < 0.01 compared with control cells or control cells transfected with scrambled shRNA.
Figure 4.
Figure 4.
The gluconeogenic effect of statins is attenuated in primary hepatocytes derived from liver-specific atg7-deficient mice. (A) Loss of Atg7 in liver tissue was confirmed by qRT-PCR. (B) Primary hepatocytes derived from wild-type and liver specific atg7-deficient mice were treated with statins (20 µM) for 24 h. Increased expression of G6pc and Pck1 was observed in wild-type hepatocytes but not in atg7-deficient hepatocytes. (C) Glucose output by cultured hepatocytes from wild-type and liver specific atg7-deficient mice after 24-h statin treatment. *, P < 0.05; **, P < 0.01 compared with wild-type hepatocytes.
Figure 5.
Figure 5.
Statin treatment increases body weight and fasting blood glucose levels and impairs pyruvate tolerance in high-fat diet-fed mice. (A) Mice were fed a high-fat diet with or without a statin (0.01%, w/w) for 16 wk. Mean body weight gain was significantly higher in statin-treated mice compared with untreated control mice. (B) Food intake did not differ among the groups. (C) Fasting blood glucose levels were elevated in statin-treated mice. (D) Results of the oral glucose tolerance test performed at 20 wk of age showed no differences among groups (pravastatin-treated, atorvastatin-treated, and untreated control mice). (E) Results of the insulin tolerance test showed attenuated insulin responses in statin-treated mice. (F) Results of the pyruvate tolerance test performed at 21 wk of age showed elevated blood glucose levels in pravastatin- and atorvastatin-treated mice. Statin treatments significantly decreased serum cholesterol (G), triglyceride (TG) (H), and free fatty acid (FFA) (I) levels. *, P < 0.05; **, P < 0.01 compared with untreated mice (control, n = 9 ; rosuvastatin, n = 7 ; fluvastatin, n = 7 ; pravastatin, n = 11 and atorvastatin, n = 11 ).
Figure 6.
Figure 6.
Statin treatment elevates expression of gluconeogenic enzymes but not glycolytic enzymes in the livers of statin-treated mice. Results of qRT-PCR showed that statins increase expression of G6pc (A) and Pck1 (B), which encode gluconeogenic enzymes. In contrast, expression of Gck (C) and Pklr (D), which encode glycolytic enzymes, did not differ between statin-treated and untreated control mice. *, P < 0.05; **, P < 0.01 compared with control.
Figure 7.
Figure 7.
Electron microscopy analysis of autophagosomes in the livers of statin-treated mice. (A) Hepatocytes of statin-treated mice showed prominent vacuolization and autophagosomes, as assessed by transmission electron microscopy. Arrows indicate double membranes of autophagosomes; scale bars: 2 µm at ×10,000 and 1 µm at ×30,000 magnification. (B) All statins significantly increased autophagic vacuole formation in mouse livers. *, P < 0.05; **, P < 0.01 compared with control.
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