AMPK activation prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 signaling and endoplasmic reticulum stress response

Abstract

Lipid accumulation is a central event in the development of chronic metabolic diseases, including obesity and type 2 diabetes, but the mechanisms responsible for lipid accumulation are incompletely understood. This study was designed to investigate the mechanisms for excess nutrient-induced lipid accumulation and whether activation of AMP-activated protein kinase (AMPK) prevents the hepatic lipid accumulation in excess nutrient-treated HepG2 cells and high fat diet (HFD)-fed mice. Exposure of HepG2 cells to high levels of glucose or palmitate induced the endoplasmic reticulum (ER) stress response, activated sterol regulatory element-binding protein-1 (SREBP-1), and enhanced lipid accumulation, all of which were sensitive to ER stress inhibitor and gene silencing of eukaryotic initiation factor 2α. The increases in ER stress response and lipid accumulation were associated with activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Inhibition of mTORC1 signaling attenuated the ER stress response and lipid accumulation induced by high glucose or by deletion of tuberous sclerosis 2. In addition, AMPK activation prevented the mTORC1 activation, ER stress response, and lipid accumulation. This effect was mimicked or abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Finally, treatment of HFD-fed mice with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside inhibited the mTORC1 pathway, suppressed the ER stress response, and prevented insulin resistance and hepatic lipid accumulation. We conclude that activation of AMPK prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 and ER stress response.

Keywords: AMPK; ER stress; Lipid accumulation; SREBP; mTORC1.

Fig. 1.

High glucose and palmitate induce ER stress and lipid accumulation. (A–H) HepG2 cells were incubated in the presence or absence of high glucose (HG, 30 mM) for the indicated time. Levels of GRP78, P-PERK, and P-eIF2α (A and B) were analyzed by Western blotting and quantified by densitometry. *, P < 0.05 vs. control (Con); n = 5. (C and D) Protein levels of SREBP-1 in the nuclear fractions and FAS in cell lysates were analyzed by Western blotting and quantified by densitometry. *, P < 0.05 vs. control (Con); n = 5. (E) Relative levels of ACC1, ACLY, and SCD1 mRNA were determined by RT-PCR. *, P < 0.05 vs. Con; n = 3. (F) Lipid accumulation was determined by Oil red O staining. Images are representative of results of three independent experiments. Lipids were extracted and triglyceride (G) and cholesterol (H) levels were measured using a commercial kit. *, P < 0.05 vs. Con; n = 5. (I–L) HepG2 cells were incubated in the presence or absence of 0.4 mM palmitate (Pal) for indicated time. (I) Levels of ER stress markers GRP78, P-PERK, and P-eIF2α were determined by Western blotting. (J) Levels of SREBP-1 in the nuclear fractions and FAS in cell lysates were determined by Western blotting. Blots are representative of three independent experiments. Concentrations of triglyceride (K) and cholesterol (L) were assayed using a commercial kit. *, P < 0.05 vs. Con; n = 5. Values are expressed as mean ± standard errors of the mean (SEM).

Fig. 2.

Inhibition of ER stress abrogates high glucose-induced lipid accumulation. (A–D) HepG2 cells were incubated in the presence or absence of the indicated concentrations of tunicamycin (TM) for 24 h. (A) Expression of ER stress markers GRP78, P-PERK, and P-eIF2α was determined by Western analysis. (B) Expression of SREBP-1 in the nuclear fractions and FAS in cell lysates was determined by Western analysis. Blots are representative of at least three independent experiments. Triglyceride (C) and cholesterol (D) levels were measured using a commercial kit. *, P <0.05 vs. Con; n = 5. (E–H) HepG2 cells were treated with HG in the presence or absence of the indicated concentrations of PBA. (E and F) Levels of ER stress markers were analyzed by Western blotting and quantified by densitometry. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. (G and H) Levels of SREBP-1 in the nuclear fractions and FAS in cell lysates were analyzed by Western blotting and quantified by densitometry. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. (I–K) HepG2 cells were incubated in the presence or absence of HG and PBA (4 mM). (I) Lipid accumulation was measured by staining with Oil red O. Images are representative of results of three independent experiments. (J and K) Triglyceride (J) and cholesterol (K) levels were measured using a commercial kit. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. Values are expressed as mean ± SEM.

Fig. 3.

eIF2α mediates high glucose-induced lipid accumulation. HepG2 cells were transfected with control (C-siRNA) or eIF2α siRNA (eIF2α-si) for 24 h, and then cultured in the presence or absence of HG for 24 h. Expression of SREBP-1 in the nuclear fractions and levels of eIF2α and FAS in the cell lysates were examined by Western analysis (A) and quantified by densitometry (B). Concentrations of triglyceride (C) and cholesterol (D) were assayed using a commercial kit. *, P < 0.05 vs. Con; †, P < 0.05 vs. C-siRNA/HG; n = 5. Values are expressed as mean ± SEM.

Fig. 4.

Inhibition of mTORC1 signaling prevents high glucose-induced ER stress and lipid accumulation. (A) HepG2 cells were incubated in the presence or absence of high glucose (HG, 30 mM) for the indicated time. Phosphorylation of mTOR at Ser2448, of S6K at Thr389, and of 4EBP1 at Thr37 was determined by Western analysis and quantified by densitometry. *, P < 0.05 vs. Con; n = 5. (B) HepG2 cells were transfected with control (C-siRNA) or Rheb siRNA (Rheb-si) for 24 h, and then cultured in the presence or absence of high glucose for 24 h. Expression of Rheb, P-mTOR, and P-S6K in cell lysates was examined by Western blotting and quantified by densitometry. *, P < 0.05 vs. C-siRNA; †, P < 0.05 vs. HG/C-siRNA; n = 4. (C) HepG2 cells were pretreated with rapamycin (Rap) at the indicated concentrations and then exposed to HG for 24 h. Phosphorylation of mTOR, S6K, and 4EBP1 was examined by Western blotting. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. (D and E) HepG2 cells were pretreated with rapamycin (Rap) at the indicated concentrations and then incubated in the presence of HG for 24 h. Levels of ER stress markers GRP78, P-PERK, and P-eIF2α (D) were examined by Western analysis and quantified by densitometry. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. (E) Levels of SREBP-1 in the nuclear fractions and FAS in cell lysates were examined by Western analysis and quantified by densitometry. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. (F) HepG2 cells were pretreated with Rap and incubated in the presence of HG for 24 h. Triglyceride concentration was measured using a commercial kit. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. (G and H) Wild-type (WT) and TSC2^−/− MEFs were incubated in the presence or absence of 50 nM Rap for 24 h. Levels of ER stress markers (G) were determined by Western analysis and quantified by densitometry. *, P < 0.05 vs. Con; †, P < 0.05 vs. TSC2^−/− control; n = 5. (H) Levels of SREBP-1 in the nuclear fractions were determined by Western analysis and quantified by densitometry. *, P < 0.05 vs. Con; †, P < 0.05 vs. TSC2^−/− control; n = 5. Values are expressed as mean ± SEM.

Fig. 5.

AMPK activation attenuates high glucose-induced lipid accumulation through inhibition of mTORC1 signaling and ER stress. (A–D) HepG2 cells were incubated in the presence or absence of AICAR (AIC) at the indicated concentrations for 24 h. Phosphorylation of AMPK at Thr172 (A) and of mTOR and S6K (B) and expression of ER stress markers (C) were determined by Western analysis and quantitated by densitometry. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. (D) Expression of SEBP-1 in the nuclear fractions and FAS in cell lysates was determined by Western analysis and quantitated by densitometry. *, P < 0.05 vs. Con; †, P < 0.05 vs. HG; n = 5. (E) HepG2 cells were transfected with adenovirus encoding GFP, DN-AMPK, or CA-AMPK for 48 h, and incubated in the presence or absence of 2 mM AICAR for 24 h under high glucose conditions. Levels of SREBP-1 in the nuclear fractions and expression of FAS and P-AMPK in cell lysates were determined by Western analysis. *, P < 0.05 vs. Con; †, P < 0.05 vs. GFP/AIC; ‡, P < 0.05 vs. DN-AMPK/AIC; n = 5. (F) HepG2 cells were transfected with adenovirus encoding GFP and DN-AMPK for 48 h, and incubated in the presence or absence of 2 mM AICAR and presence or absence of high glucose for 24 h. Triglyceride concentrations were measured using a commercial kit. *, P < 0.05 vs. Con; †, P < 0.05 vs. GFP/HG; ‡, P < 0.05 vs. GFP/HG/AIC; n = 5. Values are expressed as mean ± SEM.

Fig. 6.

AMPK activation attenuates HFD-stimulated mTORC1 signaling and ER stress in vivo. Mice were fed a normal diet (Con) or HFD and treated with AICAR (AIC) or vehicle. (A) Body weights were monitored at the weeks indicated. n = 6; *, P < 0.05 vs. HFD; †, P < 0.05 vs. ND. (B) Daily caloric intake was calculated from the amount of ingested food in individually-caged mice. n = 6; *, P < 0.05 vs. Con. Phosphorylation of AMPK (C) (n = 6) and S6K (D) (n = 6) and protein levels of ER stress markers P-PERK and P-eIF2α (E) (n = 5) in liver homogenates were determined by Western analysis and quantified by densitometry. (F) Protein levels of SREBP-1 in the nuclear fractions were measured by Western blotting (n = 5) *, P < 0.05 vs. Con; †, P < 0.05 vs. HFD. Values are expressed as mean ± SEM.

Fig. 7.

Activation of AMPK reduces HFD-induced lipid accumulation and improves insulin sensitivity in vivo. Mice were fed a normal diet (Con) or HFD and treated with AICAR (AIC) or vehicle. (A) Relative levels of FAS and ACC1 mRNA in liver were determined by Q RT-PCR. *, P < 0.05 vs. Con; †, P < 0.05 vs. HFD; n = 6. (B) Representative images showing increased hepatic and visceral fat; n = 5 for each group. (C) Total fat mass was measured after the treatment. *, P < 0.05 vs. Con; †, P < 0.05 vs. HFD; n = 6. (D and E) Liver lipids were extracted and hepatic triglyceride and cholesterol levels were assayed using a commercial kit. *, P < 0.05 vs. Con; †, P < 0.05 vs. HFD; n = 6. (F) Fasting blood glucose levels were measured in tail vein blood samples using a glucometer. *, P < 0.05 vs. Con; †, P < 0.05 vs. HFD; n = 6. (G and H) Hyperinsulinemic–euglycemic clamps were performed over a 120-min period. Insulin sensitivity was evaluated based on average glucose infusion rate at equilibrium in a hyperinsulinemic–euglycemic clamp (3 mU insulin/kg/min). *, P < 0.05 vs. Con; †, P < 0.05 vs. HFD; n = 5. Values are expressed as mean ± SEM. (I) Proposed mechanism by which AMPK inhibits excess nutrient-induced lipid accumulation. Excess nutrients stimulate mTORC1 signaling, which activates ER stress, leading to activation of SREBP-1 and consequent lipid accumulation. Activation of AMPK prevents lipid accumulation by suppression of the mTORC1-ER stress pathway.