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.

Hepatitis B virus (HBV) is a major cause of chronic liver diseases, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. The role of HCV in associations with insulin signaling has been elucidated. However, the pathogenesis of HBV-associated insulin signaling remains to be clearly characterized. Therefore, we have attempted to determine the mechanisms underlying the HBV-associated impairment of insulin signaling.

Increased adiposity confers risk for systemic insulin resistance and type 2 diabetes (T2D), but mechanisms underlying this pathogenic inter-organ crosstalk are incompletely understood. We find PHLPP2 (PH domain and leucine rich repeat protein phosphatase 2), recently identified as the Akt Ser473 phosphatase, to be increased in adipocytes from obese mice. To identify the functional consequence of increased adipocyte PHLPP2 in obese mice, we generated adipocyte-specific PHLPP2 knockout (A-PHLPP2) mice. A-PHLPP2 mice show normal adiposity and glucose metabolism when fed a normal chow diet, but reduced adiposity and improved whole-body glucose tolerance as compared to Cre- controls with high-fat diet (HFD) feeding. Notably, HFD-fed A-PHLPP2 mice show increased HSL phosphorylation, leading to increased lipolysis in vitro and in vivo. Mobilized adipocyte fatty acids are oxidized, leading to increased peroxisome proliferator-activated receptor alpha (PPARα)-dependent adiponectin secretion, which in turn increases hepatic fatty acid oxidation to ameliorate obesity-induced fatty liver. Consistently, adipose PHLPP2 expression is negatively correlated with serum adiponectin levels in obese humans. Overall, these data implicate an adipocyte PHLPP2-HSL-PPARα signaling axis to regulate systemic glucose and lipid homeostasis, and suggest that excess adipocyte PHLPP2 explains decreased adiponectin secretion and downstream metabolic consequence in obesity.

The ubiquitin-proteasome system is a vital protein degradation system that is involved in various cellular processes, such as cell cycle progression, apoptosis, and differentiation. Dysregulation of this system has been implicated in numerous diseases, including cancer, vascular disease, and neurodegenerative disorders. Induction of cellular senescence in hepatocellular carcinoma (HCC) is a potential anticancer strategy, but the precise role of the ubiquitin-proteasome system in cellular senescence remains unclear. In this study, we show that the E3 ubiquitin ligase, TRIM22, plays a critical role in the cellular senescence of HCC cells. TRIM22 expression is transcriptionally upregulated by p53 in HCC cells experiencing ionizing radiation (IR)-induced senescence. Overexpression of TRIM22 triggers cellular senescence by targeting the AKT phosphatase, PHLPP2. Mechanistically, the SPRY domain of TRIM22 directly associates with the C-terminal domain of PHLPP2, which contains phosphorylation sites that are subject to IKKβ-mediated phosphorylation. The TRIM22-mediated PHLPP2 degradation leads to activation of AKT-p53-p21 signaling, ultimately resulting in cellular senescence. In both human HCC databases and patient specimens, the levels of TRIM22 and PHLPP2 show inverse correlations at the mRNA and protein levels. Collectively, our findings reveal that TRIM22 regulates cancer cell senescence by modulating the proteasomal degradation of PHLPP2 in HCC cells, suggesting that TRIM22 could potentially serve as a therapeutic target for treating cancer.

Tissue-specific transcriptional activity is silenced in mitotic cells but it remains unclear whether the mitotic regulatory machinery interacts with tissue-specific transcriptional programs. We show that such cross-talk involves the controlled interaction between core subunits of the anaphase-promoting complex (APC) and the ID2 substrate. The N-terminus of ID2 is independently and structurally compatible with a pocket composed of core APC/C subunits that may optimally orient ID2 onto the APCCDH1 complex. Phosphorylation of serine-5 by CDK1 prevented the association of ID2 with core APC, impaired ubiquitylation and stabilized ID2 protein at the mitosis-G1 transition leading to inhibition of basic Helix-Loop-Helix (bHLH)-mediated transcription. The serine-5 phospho-mimetic mutant of ID2 that inefficiently bound core APC remained stable during mitosis, delayed exit from mitosis and reloading of bHLH transcription factors on chromatin. It also locked cells into a "mitotic stem cell" transcriptional state resembling the pluripotent program of embryonic stem cells. The substrates of APCCDH1 SKP2 and Cyclin B1 share with ID2 the phosphorylation-dependent, D-box-independent interaction with core APC. These results reveal a new layer of control of the mechanism by which substrates are recognized by APC.

Hepatic γ-secretase regulates low-density lipoprotein receptor (LDLR) cleavage and degradation, affecting clearance of plasma triglyceride (TG)-rich lipoproteins (TRLs). In this study, we investigated whether γ-secretase inhibition modulates risk of Western (high-fat/sucrose and high-cholesterol)-type diet (WTD)-induced hepatic steatosis, dyslipidemia and atherosclerosis. We evaluated liver and plasma lipids in WTD-fed mice with hepatocyte-specific ablation of the non-redundant γ-secretase-targeting subunit Nicastrin (L-Ncst). In parallel, we investigated the effect of liver-selective Ncst antisense oligonucleotides (ASO) on lipid metabolism and atherosclerosis in wildtype (WT) and ApoE knockout (ApoE-/-) mice fed normal chow or WTD. WTD-fed L-Ncst and Ncst ASO-treated WT mice showed reduced total cholesterol and LDL-cholesterol (LDL-C), as well as reduced hepatic lipid content as compared to Cre- and control ASO-treated WT mice. Treatment of WTD-fed ApoE-/- mice with Ncst ASO markedly lowered total and LDL cholesterol, hepatic TG and attenuated atherosclerotic lesions in the aorta, as compared to control ASO-treated mice. L-Ncst and Ncst ASO similarly showed reduced plasma glucose as compared to control mice. In conclusion, inhibition of hepatic γ-secretase reduces plasma glucose, and attenuates WTD-induced dyslipidemia, hepatic fat accumulation and atherosclerosis, suggesting potential pleiotropic application for diet-induced metabolic dysfunction.

Patients with nonalcoholic steatohepatitis (NASH) have increased expression of liver monocyte chemoattractant protein-1 (MCP-1), but its cellular source and contribution to various aspects of NASH pathophysiology remain debated. We demonstrated increased liver CCL2 (which encodes MCP-1) expression in patients with NASH, and commensurately, a 100-fold increase in hepatocyte Ccl2 expression in a mouse model of NASH, accompanied by increased liver monocyte-derived macrophage (MoMF) infiltrate and liver fibrosis. To test repercussions of increased hepatocyte-derived MCP-1, we generated hepatocyte-specific Ccl2-knockout mice, which showed reduced liver MoMF infiltrate as well as decreased liver fibrosis. Forced hepatocyte MCP-1 expression provoked the opposite phenotype in chow-fed wild-type mice. Consistent with increased hepatocyte Notch signaling in NASH, we observed a close correlation between markers of Notch activation and CCL2 expression in patients with NASH. We found that an evolutionarily conserved Notch/recombination signal binding protein for immunoglobulin kappa J region binding site in the Ccl2 promoter mediated transactivation of the Ccl2 promoter in NASH diet-fed mice. Increased liver MoMF infiltrate and liver fibrosis seen in opposite gain-of-function mice was ameliorated with concomitant hepatocyte Ccl2 knockout or CCR2 inhibitor treatment. Hepatocyte Notch activation prompts MCP-1-dependent increase in liver MoMF infiltration and fibrosis.

As the obesity pandemic continues to expand, novel molecular targets to reduce obesity-related insulin resistance and Type 2 Diabetes (T2D) continue to be needed. We have recently shown that obesity is associated with reactivated liver Notch signaling, which, in turn, increases hepatic insulin resistance, opening up therapeutic avenues for Notch inhibitors to be repurposed for T2D. Herein, we tested the systemic effects of γ-secretase inhibitors (GSIs), which prevent endogenous Notch activation, and confirmed these effects through creation and characterization of two different adipocyte-specific Notch loss-of-function mouse models through genetic ablation of the Notch transcriptional effector Rbp-Jk (A-Rbpj) and the obligate γ-secretase component Nicastrin (A-Nicastrin).

Excess plasma triglycerides (TGs) are a key component of obesity-induced metabolic syndrome. We have shown that γ-secretase inhibitor (GSI) treatment improves glucose tolerance due to inhibition of hepatic Notch signaling but found additional Notch-independent reduction of plasma TG-rich lipoproteins (TRLs) in GSI-treated, as well as hepatocyte-specific, γ-secretase knockout (L-Ncst) mice, which suggested a primary effect on hepatocyte TRL uptake. Indeed, we found increased VLDL and LDL particle uptake in L-Ncst hepatocytes and Ncst-deficient hepatoma cells, in part through reduced γ-secretase-mediated low-density lipoprotein receptor (LDLR) cleavage and degradation. To exploit this novel finding, we generated a liver-selective Nicastrin ASO, which recapitulated glucose and lipid improvements of L-Ncst mice, with increased levels of hepatocyte LDLR. Collectively, these results identify the role of hepatic γ-secretase to regulate LDLR and suggest that liver-specific GSIs may simultaneously improve multiple aspects of the metabolic syndrome.

Obesity-induced nonalcoholic fatty liver disease (NAFLD) develops, in part, via excess insulin-stimulated hepatic de novo lipogenesis, which increases, paradoxically, in patients with obesity-induced insulin resistance. Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) terminates insulin signaling by dephosphorylating Akt; levels of PHLPP2 are reduced in livers from obese mice. We investigated whether loss of hepatic PHLPP2 is sufficient to induce fatty liver in mice, mechanisms of PHLPP2 degradation in fatty liver, and expression of genes that regulate PHLPP2 in livers of patients with NAFLD.