Volume 134, Issue 1, Pages 268-280 (January 2008) PTEN Down-Regulation by Unsaturated Fatty Acids Triggers Hepatic Steatosis via an NF-κBp65/mTOR-Dependent Mechanism  Manlio Vinciguerra, Christelle Veyrat–Durebex, Moulay Ahmed Moukil, Laura Rubbia–Brandt, Françoise Rohner–Jeanrenaud, Michelangelo Foti  Gastroenterology  Volume 134, Issue 1, Pages 268-280 (January 2008) DOI: 10.1053/j.gastro.2007.10.010 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 PTEN and SHIP2 expression and Akt phosphorylation in the liver of ZDF rats (■, ZDF) and their lean littermates (□, Lean). (A) Representative blots of PTEN, SHIP2, and actin expression and quantifications of PTEN and SHIP2 protein levels (left) and PTEN mRNA (right). (B) Western blots and quantifications of the ratio of Akt phosphorylation over total Akt. Means ± SE are derived from 5 different animals for each strain (*P < .05 and **P < .01 vs lean rats). Gastroenterology 2008 134, 268-280DOI: (10.1053/j.gastro.2007.10.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 FFA-mediated insulin resistance and PTEN down-regulation. HepG2 cells were incubated for 24 hours with insulin (Ins, 10−7 mol/L), glucosamine (Glu, 25 mmol/L), or 50 μmol/L FFA (PoA, palmitoleic acid; pa, palmitic acid; la, linoleic acid) before measuring glycogen synthesis or Western analyses. (A) Glycogen synthesis after 60 minutes of stimulation with insulin (10−7 mol/L). □, unstimulated; ■, + insulin. (B) Western blots (left) and quantifications (right) of PTEN (□) and SHIP2 (■) expression and of the ratio of Akt and ERK1/2 phosphorylations over total Akt/ERK1/2. Results are means ± SE of 3 independent experiments. (#P < .05 vs unstimulated control cells; *P < .05 and **P < .01 vs insulin-stimulated control cells). Gastroenterology 2008 134, 268-280DOI: (10.1053/j.gastro.2007.10.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 OA-mediated PTEN down-regulation via NF-κBp65/mTOR activation. (A) Representative blots and quantifications of the expression/phosphorylation of signaling molecules in HepG2 cells stimulated for 1 hour with OA 50 μmol/L. (B) Effect of inhibitors (PD98059 10 μmol/L, rapamycin 200 nmol/L, LY294002 200 nmol/L, SP600125 20 μmol/L, BAY11-7082 1 μmol/L, and PTD-p65-P1 50 μmol/L), NF-κBmut1-450 and NF-κBmutS276A mutants on OA-induced PTEN down-regulation and Akt phosphorylation. Cells were incubated for 24 hours ± OA 50 μmol/L in the presence of inhibitors or transfected with NF-κBp65 mutants before Western analyses. Western blots (left) and quantifications (right) of PTEN expression and ratios of Akt phosphorylation over total Akt in cells treated with inhibitors or transfected with NF-κBp65 mutants. Results are means ± SE of 3 independent experiments (*P < .05 and **P < .01 vs unstimulated control cells). (B) □, unstimulated; ■, OA, 50 μmol/L. Gastroenterology 2008 134, 268-280DOI: (10.1053/j.gastro.2007.10.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 OA-induced NF-κBp65 phosphorylation and nuclear translocation and mTOR/NF-κBp65 interaction in HepG2 cells. (A) Western blots and quantifications of NF-κBp65(Ser276) and mTOR(Ser2448) phosphorylations in cells stimulated for 24 hours by 50 μmol/L OA in the presence of 1 μmol/L BAY11-7082 or 200 nmol/L rapamycin. Results are means ± SE of 3 independent experiments (*P < .05 vs control cells). (B and C) Representative confocal immunofluorescences and quantifications of NF-κBp65 nuclear translocation after 50 μmol/L OA or 20 nmol/L tumor necrosis factor α stimulation in cells pretreated or not with 200 nmol/L rapamycin. Red line: nuclei contours. Means ± SE are derived from greater than 100 cells/condition. □, No inhibitor; ■, rapamycin. (D) Western blots and quantifications of NF-κBp65 partition between nuclear (n) and cytosolic (c) fractions in cells treated for 1 hour with 50 μmol/L OA ± 200 nmol/L rapamycin or 1 μmol/L BAY11-7082. Glyceraldehyde-3-phosphate dehydrogenase (gapdh), cytosolic marker; nucleolin, nuclear marker. □, Untreated; , rapamycin; ■, BAY 11-7082. Results are means ± SE of 3 independent experiments. (*P < .05 vs unstimulated control cells). (E) Endogenous mTOR and NF-κBp65 co-immunoprecipitation in cells ± 50 μmol/L OA for 1 hour (upper panel). Specificity of mTOR/NF-κBp65 co-immunoprecipitation was assessed using the same immunoglobulin (Ig)G isotypes targeting ERK1/2 and Akt. Lower panel: co-immunoprecipitation of endogenous mTOR with ectopically expressed Flag–NF-κBp65. Specificity of the co-immunoprecipitations was assessed using mouse IgG isotypes. Blots are representative of 3–5 independent experiments. Gastroenterology 2008 134, 268-280DOI: (10.1053/j.gastro.2007.10.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 PTEN expression controls Tg accumulation in HepG2 cells. (A) Western analysis of PTEN-GFP and PTENG129E-GFP over expression, and siRNA-mediated PTEN depletion in HepG2 cells. (B) Tg content in OA-treated cells (50 μmol/L, 24 h) ectopically expressing GFP or PTEN-GFP, or transduced with scrambled (scr. siRNA) or PTEN siRNAs. □, Unstimulated; ■, OA, 50 μmol/L. (C) Representative images of Oil-Red O staining (oro) in OA-treated cells (50 μmol/L, 24 h) over expressing GFP or PTEN-GFP. Quantifications of GFP-positive areas stained by ORO are means ± SE of greater than 200 cells/condition. (D) Tg content in OA-treated cells (50 μmol/L, 24 h) over expressing GFP-PTEN and GFP-PTENG129E mutant. Untransfected cells, or cells over expressing GFP, were used as controls. (E) Tg content in OA-treated cells (50 μmol/L, 24 h) incubated with 200 nmol/L rapamycin or 1 μmol/L BAY11-7082. □, Unstimulated; ■, OA, 50 μmol/L. Results of Tg measurements in panels (B), (D) and (E) are means ± SE of 3 independent experiments (*P < .05, **P < .01, and ***P < .001 vs unstimulated control cells). Gastroenterology 2008 134, 268-280DOI: (10.1053/j.gastro.2007.10.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Effect of PTEN down-regulation on HepG2 lipid metabolism. (A) Reverse-transcription PCR analysis of genes involved in the lipid metabolism in OA-treated cells (50 μmol/L, 24 h) or cells depleted of PTEN by siRNAs. Untreated cells (ctl) or cells expressing scrambled siRNAs (scr. siRNA) were used as controls. □, CTL; ■, OA; , scr. siRNA; , PTEN siRNA. (B) 14C-oleate uptake in control (ctl) and PTEN-depleted cells (PTEN siRNA). □, CTL; , PTEN siRNA. (C) Release in the medium of 14C-oleate derivatives in control (ctl) and PTEN-depleted (PTEN siRNA) cells. Radioactivity in the medium was measured 24 hours after uptake of 14C-oleate for different times. □, CTL; , PTEN siRNA. (D) Mitochondrial β-oxidation in control (ctl) and PTEN-depleted (PTEN siRNA) cells assessed by measuring the release of radiolabeled CO2. □, Unstimulated; ■, OA, 50 μmol/L. Results are means ± SE of 3 independent experiments for each panel (*P < .05, **P < .01, and ***P < .001 vs control cells). Gastroenterology 2008 134, 268-280DOI: (10.1053/j.gastro.2007.10.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 (A) PTEN expression in the liver of HF-fed Wistar and Lou/C rats. Western analysis of PTEN, SHIP2, and actin expressions and quantifications of PTEN protein (left) and mRNA (right) levels are shown for Wistar and Lou/C rats under standard (Std) or HF diets. (B) Western blots and quantifications of expression and ratios of Akt phosphorylation over total Akt in the liver of Wistar and Lou/C rats under Std or HF diets. (C and D) Western blots and quantifications of the expression and ratios of NF-κBp65 and mTOR phosphorylation over total NF-κBp65/mTOR in the liver of (C) ZDF rats and their lean littermates (Lean), and (D) Wistar and Lou/C rats under Std or HF diets. (A, B, and D) Means ± SE are derived from 10 animals for each strain and diet (* and **P vs rats under Std diet). (C) Means ± SE of the quantifications are derived from 5 animals for each strain (* and **P vs lean rats). (D) □, Std diet; ■, HF diet. Gastroenterology 2008 134, 268-280DOI: (10.1053/j.gastro.2007.10.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 PTEN down-regulation in human steatotic livers. (A and B) Immunohistochemical detection of PTEN in paraffin-embedded sections of nonsteatotic (A) and steatotic (B) human livers. Note that fibroblasts in the Glisson’s capsule are PTEN negative (A) and that PTEN expression is specifically decreased in the cytoplasm and nucleus of steatotic hepatocytes containing large lipid droplets (white holes) as compared with nonsteatotic hepatocytes (eg, on the right of the micrograph in B). (C) RT-PCR analyses of PTEN expression in normal and steatotic liver biopsy specimens. Expression of SHIP2 and PEPCK are shown as controls. Means ± SE are derived from 10 liver biopsy specimens coming from different patients for each condition. (*P < .05 and **P < .01 vs normal livers). □, Normal livers; ■, steatotic livers. (D) Representative Western analysis of PTEN and Akt/NF-κBp65 expression/phosphorylation in 3 different biopsy specimens from normal and steatotic livers. (E) Model of steatosis triggered by PTEN down-regulation after FFA activation of NF-κBp65/mTOR–dependent signaling in hepatocytes. FFA stimulates the phosphorylation of mTOR in complex with NF-κBp65. NF-κBp65 is in turn phosphorylated, the mTOR/NF-κBp65 complex dissociates and NF-κBp65 shuttles into the nucleus and activates mechanisms downregulating PTEN mRNA expression. PTEN down-regulation induces Akt activation and alterations in lipid metabolism, with an overall balance resulting in steatosis. Gastroenterology 2008 134, 268-280DOI: (10.1053/j.gastro.2007.10.010) Copyright © 2008 AGA Institute Terms and Conditions