Tiago Eugênio Oliveira da Silva

Background.: Activation of de novo lipogenesis (DNL) is a major hallmark of steatotic liver disease (SLD), a highly prevalent group of diseases ranging from a harmless steatosis to more severe steatohepatitis, cirrhosis, and hepatocellular carcinoma. We interrogated herein the early alterations in liver proteome, transcriptome, lipidome, inflammation, fibrosis, and oxidative status in a mouse model of progressive SLD induced by constitutive activation of PI3K-AKT signaling, the major inducer of DNL. Methods.: Male, 8 weeks-old mice bearing phosphatase and tensin homolog ( Pten ) deletion and therefore constitutive activation of PI3K-AKT in hepatocytes (albumin-Cre) and littermate controls were evaluated for liver steatosis, injury, lipid peroxidation, metabolism, proteome, lipidome and transcriptome either upon feeding with a chow or high-fat diets supplemented or not with linoleic acid or after treatment with vehicle or acetyl-CoA carboxylase (ACC) inhibitor (ND-630, 20 mg/kg/day, i.p.) Results.: Mice bearing Pten deletion and therefore constitutive activation of PI3K-AKT in hepatocytes display steatohepatitis characterized by enhanced DNL, severe steatosis, hepatocyte ballooning, inflammation, fibrosis and hepatomegaly, which, unexpectedly, was associated with reduced lipid peroxidation, increased GSH content and enhanced rates of triacylglycerol turnover, fatty acid β-oxidation, tricarboxylic acid cycle flux, and mitochondrial respiration, altogether promoting a robust lipidome remodeling mainly defined by enrichment of DNL-derived fatty acids in detriment of a broad depletion of essential fatty acids (linoleic and α-linolenic acids) and cardiolipins. Dietary restoration of liver linoleic acid content mildly attenuated liver inflammation, without however affecting lipid peroxidation and improving steatosis and fibrosis, whereas pharmacological acetyl-CoA carboxylase (ACC) and DNL inhibition markedly reduced steatosis, inflammation and fibrosis, despite further lowering hepatic linoleic acid content. Conclusions.: Collectively, these findings support a harmful role of essential fatty acids depletion fomenting liver inflammation and identify DNL as a major culprit of steatohepatitis induced by constitutive PI3K-AKT activation. What is already known on this topic: Pten inactivation in hepatocytes leading to constitutive PI3K-AKT signaling and de novo lipogenesis activation (DNL) is a recurrent liver signature found in steatotic liver disease (SLD)-bearing patients, promoting disease progression through not completely defined molecular mechanisms. What this study adds: Pten inactivation in hepatocytes promotes liver steatosis, injury, inflammation and fibrosis, in spite of depleting essential fatty acids and cardiolipin, reducing lipid peroxidation, enhancing antioxidant capacity and mitochondrial mass and respiration, and eliciting lipid-related futile metabolic cycles. Dietary restoration of liver essential linoleic acid partially attenuates inflammation, but not steatosis and fibrosis induced by Pten inactivation in hepatocytes Pharmacological ACC and DNL inhibition mitigate the steatosis, inflammation and fibrosis induced by Pten inactivation in hepatocytes, but further deplete liver essential fatty acids. How this study might affect research, practice or policy: This study raises evidence supporting a harmful role of essential fatty acid depletion exacerbating liver inflammation. Pharmacological strategies that increase fatty acid oxidation including ACC inhibition may enhance, while dietary supplementation may mitigate essential fatty acid depletion. Despite protecting healthy cells from oxidative damage, the enhanced antioxidant capacity induced by Pten inactivation in hepatocytes may favor survival of hepatocytes bearing DNA mutations and tumorigenesis.

Non-selective hormone replacement with estradiol is known to improve metabolic homeostasis during menopause. However, this treatment is not recommended for individuals with genetic predisposition to hormone-responsive cancers. In contrast, selective activation of estrogen receptor beta (ERβ) has shown promising results, promoting antitumor effects and modulating metabolic outcomes, although the mechanisms in which these changes occur remain poorly understood. We investigated the effects of ERβ activation using diarylpropionitrile (DPN), a selective ERβ agonist, in both an in vivo model of post menopause and in vitro model of metabolic overload. Female Wistar rats were submitted to ovariectomy (OVX) and later treated with DPN. ERβ agonist treatment recovered fasting glucose and lipid profiles, improved pancreatic islet morphology, and reduced retroperitoneal white adipose tissue. Serum ketone bodies and free fatty acid levels were also recovered to control levels, suggesting modulation in liver lipid oxidation. DPN treatment also promoted substantial lipidome remodeling specifically in OVX animals, generating a lipid-buffering profile. To isolate the effects mechanistically, hepatocytes were submitted to nutrient overload and treated with DPN. In vitro , DPN also recovered ketone body secretion and promoted an increased dependence on complete fatty acid oxidation, as well as decreased metabolic flexibility, as assessed by modulated extracellular flux analysis. Overall, these findings demonstrate a new role of ERβ in the modulation of hepatic lipid and ketone metabolism, with positive metabolic outcomes in estradiol-deficient animals.

Hepatocellular carcinoma (HCC) markedly enhances liver secretion of fibroblast growth factor 21 (FGF-21), a hepatokine that increases brown and subcutaneous inguinal white adipose tissues (BAT and iWAT, respectively) uncoupling protein 1 (UCP-1) content, thermogenesis and energy expenditure. Herein, we tested the hypothesis that an enhanced BAT and iWAT UCP-1-mediated thermogenesis induced by high levels of FGF-21 is involved in HCC-associated catabolic state and fat mass reduction. For this, we evaluated body weight and composition, liver mass and morphology, serum and tissue levels of FGF-21, BAT and iWAT UCP-1 content, and thermogenic capacity in mice with Pten deletion in hepatocytes that display a well-defined progression from steatosis to steatohepatitis (NASH) and HCC upon aging. Hepatocyte Pten deficiency promoted a progressive increase in liver lipid deposition, mass, and inflammation, culminating with NASH at 24 weeks and hepatomegaly and HCC at 48 weeks of age. NASH and HCC were associated with elevated liver and serum FGF-21 content and iWAT UCP-1 expression (browning), but reduced serum insulin, leptin, and adiponectin levels and BAT UCP-1 content and expression of sympathetically regulated gene glycerol kinase (GyK), lipoprotein lipase (LPL), and fatty acid transporter protein 1 (FATP-1), which altogether resulted in an impaired whole-body thermogenic capacity in response to CL-316,243. In conclusion, FGF-21 pro-thermogenic actions in BAT are context-dependent, not occurring in NASH and HCC, and UCP-1-mediated thermogenesis is not a major energy-expending process involved in the catabolic state associated with HCC induced by Pten deletion in hepatocytes.

BAT and iWAT mTORC2 is inhibited by cold acclimation, but its residual activity is required for cold-induced increases in total UCP-1 content and thermogenic capacity, but not glucose uptake and mTORC1 activity. The impaired BAT and iWAT total UCP-1 content and thermogenic capacity induced by adipocyte mTORC2 deficiency are compensated by activation of muscle shivering in cold-acclimated mice.