mTORC1-independent Raptor prevents hepatic steatosis by stabilizing PHLPP2

Abstract

Mechanistic target of rapamycin complex 1 (mTORC1), defined by the presence of Raptor, is an evolutionarily conserved and nutrient-sensitive regulator of cellular growth and other metabolic processes. To date, all known functions of Raptor involve its scaffolding mTOR kinase with substrate. Here we report that mTORC1-independent ('free') Raptor negatively regulates hepatic Akt activity and lipogenesis. Free Raptor levels in liver decline with age and in obesity; restoration of free Raptor levels reduces liver triglyceride content, through reduced β-TrCP-mediated degradation of the Akt phosphatase, PHLPP2. Commensurately, forced PHLPP2 expression ameliorates hepatic steatosis in diet-induced obese mice. These data suggest that the balance of free and mTORC1-associated Raptor governs hepatic lipid accumulation, and uncover the potentially therapeutic role of PHLPP2 activators in non-alcoholic fatty liver disease.

Figure 1. Free Raptor levels decline in aged and obese liver.

(a,b) Western blot from livers of young (8-week-old), adult (24-week-old) and ob/ob (8-week-old) male mice, following size-exclusion chromatography (a) and quantification of signal per fraction (b), normalized to total protein. (c,d) Western blot from livers of young, adult and ob/ob male mice, cross-linked with disuccinimidyl suberate (DSS) (c) and quantification of free Raptor signal (d), as a percentage of control (dimethylsulphoxide (DMSO)-treated liver lysate). (e,f) Western blot from hepatocytes deprived of amino acids or treated with rapamycin (e), or insulin (f), before disuccinimidyl suberate crosslinking. *P<0.05 as compared with the indicated control by two-way analysis of variance. All data are shown as the means±s.e.m. All blots are representative of three independent experiments, and samples within each group chosen randomly.

Figure 2. Rescue of free Raptor prevents aging- and obesity-dependent hepatic steatosis.

(a) Western blot from livers of adult Ad-GFP and Ad-Raptor male mice, following size-exclusion chromatography. Fractions 24 and 25 correspond to mTORC1-associated (∼800 kDa) Raptor, while 34 and 35 to free (∼150 kDa) Raptor. (b–d) Hepatic triglyceride (TG) in young or adult (n=6/group) (b), aged (10- to 12-month-old) (n=7 per group) (c), or DIO (n=5 per group) (d) Ad-GFP and Ad-Raptor male mice. (e–g) Plasma TG (e), hepatic fatty acid synthesis (f) and lipogenic gene expression (g) in young or adult, Ad-GFP and Ad-Raptor mice, killed after a 16 h fast followed by 4 h refeeding (n=6 per group). *P<0.05, **P<0.01 as compared with the indicated control by two-way analysis of variance. All data are shown as the means±s.e.m. All blots are representative of three independent experiments, and samples within each group chosen randomly.

Figure 3. Free Raptor reduces Akt hyperactivity by stabilizing PHLPP2.

(a,b) Western blots from livers of young or adult Ad-GFP and Ad-Raptor mice killed after a 16 h fast followed by 4 h refeeding. (c) Akt activity on recombinant GSK3β peptide, normalized to immunoprecipitated Akt levels. (d) Western blot from liver of adult Ad-GFP and Ad-Raptor mice killed after a 16 h fast followed by 4 h refeeding, normalized to β-actin. (e) Western blot from Hepa1c1c7 cells treated with Rapamycin (20 nM) or Torin1 (250 nM). (f) Western blot from primary hepatocytes transduced with Ad-shControl or Ad-shmTOR. (g) Western blot of cycloheximide (CHX, 50 mg ml⁻¹)-treated primary hepatocytes transduced with GFP or Raptor adenovirus. Quantification of PHLPP2, normalized to β-actin, relative to time 0 as 100%. (h,i) Western blot of Raptor (or GFP control)-transduced and/or HA/Ub-transfected primary hepatocytes following immunoprecipitation with anti-PHLPP2, with or without MG-132. *P<0.05 as compared with the indicated control by two-way analysis of variance. All blots are representative of three independent experiments, and samples within each group chosen randomly.

Figure 4. Rescue of lower PHLPP2 in aging or obesity reduces lipogenesis.

(a) Liver mRNA expression from young and adult mice (n=6 per each group). (b–d) Western blot from livers of young and adult (b), chow- and HFD-fed (c), or lean and ob/ob (d) mice, normalized to β-actin. (e–h) Western blots from liver (e), hepatic triglyceride (f), liver lipogenic gene expression (g) and plasma lipids (h) in adult, HFD-fed Ad-GFP or Ad-PHLPP2 mice (n=6–7/group). *P<0.05, **P<0.01 as compared with the indicated control by two-way analysis of variance. All data are shown as the means±s.e.m.

Figure 5. Reduced liver triglyceride (TG) by free Raptor is PHLPP2-dependent.

(a–d) Western blots from liver (a), hepatic TG (b), plasma TG (c) and liver lipogenic gene expression (d) of adult Ad-GFP and Ad-Raptor mice co-transduced with control (Ad-shControl), Ad-shPHLPP1 or Ad-shPHLPP2 adenoviruses, killed after a 16 h fast followed by 4 h refeeding (n=6 per group). (e) Hepatic TG from liver of adult Ad-GFP and Ad-Raptor mice co-transduced with control (GFP) or myrAkt adenovirus, killed after a 16 h fast followed by 4 h refeeding (n=6 per group). (f) GTT in adult, HFD-fed Ad-GFP or Ad-PHLPP2 mice (n=6–7 per group). *P<0.05, **P<0.01 as compared with the indicated control by two-way analysis of variance. All data are shown as the means±s.e.m. All blots are representative of three independent experiments, and samples within each group chosen randomly.

Figure 6. A proposed model of free Raptor-mediated regulation of hepatic lipogenesis.

Akt represents the common molecular hub of insulin regulation of hepatic glucose production and lipogenesis. In the post-prandial state, Akt-mediated FoxO nuclear exclusion occurs rapidly, after which Akt is dephosphorylated by PHLPP2 at Ser473 to terminate insulin action. In aging or obesity, loss of free Raptor destabilizes PHLPP2, leading to prolonged Akt activity and Srebp1c-dependent lipogenesis.