Albert Souza Peixoto

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.

Pseudomonas aeruginosa relies on antioxidant enzymes to withstand host-derived oxidative stress during infection. Here, we investigated the role of bacterial glutathione peroxidase (GPx) in survival and virulence of the PA14 strain under inflammatory conditions associated with hyperuricemia. Wild-type (WT) and Δgpx strains were exposed to isolated hypochlorous acid (HOCl), hydrogen peroxide (H₂O₂), tert-butyl hydroperoxide, and urate hydroperoxide (HOOU) or challenged with human neutrophils in the presence or absence of oxidative burst modulators. In vivo, normouricemic and potassium oxonate-induced hyperuricemic mice were intranasally infected with WT or Δgpx strains to assess bacterial burden, inflammation, oxidative damage, and survival. Deletion of gpx increased bacterial susceptibility to all oxidants, particularly HOOU, and significantly reduced survival in activated neutrophils. In mice, Δgpx infection resulted in reduced pulmonary bacterial burden, attenuated neutrophil infiltration, lower oxidative damage, and markedly improved survival compared to WT infection, an effect exacerbated by hyperuricemia in WT-infected animals. These findings demonstrate that GPx detoxifies organic hydroperoxides generated during inflammation, enhancing P. aeruginosa resistance to oxidative killing and promoting virulence. Collectively, our results highlight the importance of redox regulation in bacterial pathogenesis and identify GPx as a potential target for redox-based anti-virulence strategies against multidrug-resistant P. aeruginosa.

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.

Cold acclimation and PPARγ agonism combined markedly increase brown and white adipose tissue total UCP1 content and mRNA levels of thermogenesis-related proteins. Higher UCP1 protein levels did not result in higher energy expenditure. The high thermogenic capacity induced by PPARγ agonism in cold-exposed animals markedly increases animals' body temperature in response to the β3-adrenergic agonist CL316,243.

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.

Invertase (β-fructofuranosidase, EC 3.2.1.26) catalyzes sucrose hydrolysis into glucose and fructose and it is one of the simplest carbohydrases. These enzymes occur widely in nature and their presence has been reported in microorganisms and plants. Since yeasts are the main industrial source, most researches concerning this enzyme have focused on invertase extracted from such source. This study extracted and characterized inverted intracellular (Inv-I) and extracellular (Inv-E) of Kluyveromyces marxianus CCMB 322 isolated in the baiano semi-arid region. Kluyveromyces marxianus CCMB 322 produces intracellular and extracellular invertase with different characteristics. The optimum activity was achieved at approximately pH 3.9 and 45ºC, in Inv-I and Inv-E. The invertases produced by K. marxianus CCMB 322 showed thermal stability similar to that found in other studies. The Km and Vmax values of the Inv-I enzyme were 61.12mM and 5.56 µmol/mL.min-1, but the Km and Vmax values of the Inv-E enzyme were 76.5mM and 0.364 µmol/mL.min-1. Inverted from K. marxianus is a higher affinity for sucrose compared to enzymes from other sources.