Project description:The in vivo functions of mechanistic target of rapamycin complex 2 (mTORC2) and the signaling mechanisms that control brown adipose tissue (BAT) fuel utilization and activity are not well understood. Here, by conditionally deleting Rictor in the Myf5 lineage, we provide in vivo evidence that mTORC2 is dispensable for skeletal muscle development and regeneration but essential for BAT growth. Furthermore, deleting Rictor in Myf5 precursors shifts BAT metabolism to a more oxidative and less lipogenic state and protects mice from obesity and metabolic disease at thermoneutrality. We additionally find that Rictor is required for brown adipocyte differentiation in vitro and that the mechanism specifically requires AKT1 hydrophobic motif phosphorylation but is independent of pan-AKT signaling and is rescued with BMP7. Our findings provide insights into the signaling circuitry that regulates brown adipocytes and could have important implications for developing therapies aimed at increasing energy expenditure as a means to combat human obesity.
Project description:Previous studies demonstrate that Nrf2, a master regulator of antioxidative responses, is essential in mediating induction of many antioxidative enzymes by acute activation of the AhR. However, the role of Nrf2 in protecting against oxidative stress and DNA damage induced by sustained activation of the AhR remains unknown and was investigated herein. Tissue and blood samples were collected from wild-type (WT) and Nrf2-null mice 21 days after administration of a low-toxic dose (10 ?g/kg ip) of TCDD. Only Nrf2-null mice lost body weight after TCDD treatment; however, blood levels of ALT were not markedly changed in either genotype, indicating a lack of extensive necrosis. Compared to livers of TCDD-treated WT mice, livers of TCDD-treated Nrf2-null mice had: 1) degenerated hepatocytes, lobular inflammation, marked fat accumulation, and higher mRNA expression of inflammatory and fibrotic genes; 2) depletion of glutathione, elevation in lipid peroxidation and marker of DNA damage; 3) attenuated induction of phase-II enzymes Nqo1, Gsta1/2, and Ugt2b35 mRNAs, but higher induction of cytoprotective Ho-1, Prdx1, Trxr1, Gclc, and Epxh1 mRNAs; 4) higher mRNA expression of Fgf21 and triglyceride-synthesis genes, but down-regulation of bile-acid-synthesis genes and cholesterol-efflux transporters; and 5) trend of induction/activation of c-jun and NF-kB. Additionally, TCDD-treated Nrf2-null mice had impaired adipogenesis in white adipose tissue. In conclusion, Nrf2 protects livers of mice against oxidative stress, DNA damage, and steatohepatitis induced by TCDD-mediated sustained activation of the AhR. The aggravated hepatosteatosis in TCDD-treated Nrf2-null mice is due to increased lipogenesis in liver and impaired lipogenesis in white adipose tissue.
Project description:Consumption of trans-fatty acids (TFA), unsaturated fatty acids (FA) containing trans double bonds, is a risk factor for metabolic syndrome and steatohepatitis. Peroxisome proliferator-activated receptor ? (PPAR?) is a master regulator of hepatic lipid homeostasis. To examine the contribution of PPAR? to changes in liver phenotypes induced by TFA, two diets were used: a purified control diet and an isocaloric diet in which most of the soybean oil, a major source of FA in the diet, was replaced with TFA-rich shortening. The diets were fed to wild-type and Ppara-null mice for 2 months. Ppara-null mice fed a TFA-containing diet showed more severe hepatic steatosis and liver damage compared with similarly treated wild-type mice, as revealed by increased hepatic triglyceride (TG) contents and serum alanine aminotransferase activities. While the TFA-rich diet increased the hepatic expression of enzymes involved in de novo FA synthesis and decreased TG-hydrolyzing enzymes in both genotypes, the expression of FA-catabolizing enzymes was decreased in Ppara-null mice, resulting in more severe hepatosteatosis. Additionally, the expression levels of key contributors to inflammation, such as osteopontin, were increased, and nuclear factor-kappa B was activated in TFA-containing diet-fed Ppara-null mice. Enhanced inflammatory signaling in these mice was presumably mediated by toll-like receptor 2, with no accompanying inflammasome activation. Collectively, these results suggest a protective role for PPAR? in the pathological changes in the liver following TFA consumption. PPAR? might prevent TFA-containing diet-induced steatohepatitis.
Project description:The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.
Project description:The AMP-activated protein kinase (AMPK) is a highly conserved master regulator of metabolism, whose activation has been proposed to be therapeutically beneficial for the treatment of several metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). NAFLD, characterized by excessive accumulation of hepatic lipids, is the most common chronic liver disease and a major risk factor for development of nonalcoholic steatohepatitis, type 2 diabetes, and other metabolic conditions. To assess the therapeutic potential of AMPK activation, we have generated a genetically engineered mouse model, termed iAMPKCA, where AMPK can be inducibly activated in vivo in mice in a spatially and temporally restricted manner. Using this model, we show that liver-specific AMPK activation reprograms lipid metabolism, reduces liver steatosis, decreases expression of inflammation and fibrosis genes, and leads to significant therapeutic benefits in the context of diet-induced obesity. These findings further support AMPK as a target for the prevention and treatment of NAFLD.
Project description:Oxidative stress is implicated in the development of non-alcoholic steatohepatitis (NASH). The Nrf2-antioxidant response element pathway protects cells from oxidative stress. Studies have shown that global Nrf2 deficiency hastens the progression of NASH. The purpose of this study was to determine whether long-term hepatocyte-specific activation of Nrf2 mitigates NASH progression. Transgenic mice expressing a constitutively active Nrf2 construct in hepatocytes (AlbCre+/caNrf2+) and littermate controls were generated. These mice were fed standard or methionine-choline-deficient (MCD) diet, a diet used to induce NASH development in rodents. After 28 days of MCD dietary feeding, mice developed significant increases in steatosis, inflammation, oxidative stress, and HSC activation compared with those mice on standard diet. AlbCre+/caNrf2+ animals had significantly decreased serum transaminases and reduced steatosis when compared with the AlbCre+/caNrf2- animals. This significant reduction in steatosis was associated with increased expression of genes involved in triglyceride export (MTTP) and ?-oxidation (CPT2). However, there were no differences in the increased oxidative stress, inflammation, and HSC activation from MCD diet administration between the AlbCre+/caNrf2- and AlbCre+/caNrf2+ animals. We conclude that hepatocyte-specific activation of Nrf2-mediated gene expression decreased hepatocellular damage and steatosis in a dietary model of NASH. However, hepatocyte-specific induction of Nrf2-mediated gene expression alone is insufficient to mitigate inflammation, oxidative stress, and HSC activation in this nutritional NASH model.
Project description:Approximately half of all deaths from liver cirrhosis, the tenth leading cause of mortality in the United States, are related to alcohol use. Chronic alcohol consumption is accompanied by intestinal dysbiosis and bacterial overgrowth, yet little is known about the factors that alter the microbial composition or their contribution to liver disease. We previously associated chronic alcohol consumption with lower intestinal levels of the antimicrobial-regenerating islet-derived (REG)-3 lectins. Here, we demonstrate that intestinal deficiency in REG3B or REG3G increases numbers of mucosa-associated bacteria and enhances bacterial translocation to the mesenteric lymph nodes and liver, promoting the progression of ethanol-induced fatty liver disease toward steatohepatitis. Overexpression of Reg3g in intestinal epithelial cells restricts bacterial colonization of mucosal surfaces, reduces bacterial translocation, and protects mice from alcohol-induced steatohepatitis. Thus, alcohol appears to impair control of the mucosa-associated microbiota, and subsequent breach of the mucosal barrier facilitates progression of alcoholic liver disease.
Project description:The UPR is activated in the mouse retina expressing misfolded T17M rhodopsin (RHO) during autosomal dominant retinitis pigmentosa (ADRP) progression. Therefore, the goal of this study is to validate the UPR-induced caspase-7 as a new therapeutic target that modulates the UPR, reduces the level of apoptosis and protects the ADRP retina from retinal degeneration and light-induced damage. Mice were analyzed using ERG, SD-OCT and histology to determine the role of caspase-7 ablation. The results of these experiments demonstrate the significant preservation of photoreceptors and their function in T17M RHO CASP-7 retinas from P30 to P90 compared with control mice. These mice were also protected from the light-induced decline in the ERG responses and apoptosis. The RNA and protein analyses of T17M RHO+Csp7-siRNA, Tn+Csp7-siRNA 661W cells and T17M RHO CASP-7 retinas revealed that caspase-7 ablation reprograms the UPR and reduces JNK-induced apoptosis. This reduction is believed to occur through the downregulation of the mTOR and Hif1a proteins. In addition, decline in activated PARP1 was detected in T17M RHO CASP-7 retina. Altogether, our findings indicate that the targeting of caspase-7 in T17M RHO mice could be a feasible therapeutic strategy for advanced stages of ADRP.
Project description:Dietary arginine (Arg) supplementation has been proposed to have positive effects on the development of liver diseases. In the present study, we investigate if an oral Arg supplementation in diet protects mice fed a fructose, fat and cholesterol enriched Western-style diet (WSD) from the development of non-alcoholic steatohepatitis (NASH). Female C57BL/6J mice were fed a liquid control diet or a liquid WSD ± Arg (2.49 g/kg body weight/day) for 6 weeks. Indices of liver injury, glucose metabolism and intestinal permeability were determined. While Arg supplementation had no effects on body weight gain, fasting blood glucose levels were significantly lower in WSD+Arg-fed mice than in C+Arg-fed animals. WSD-fed mice developed liver steatosis accompanied with inflammation, both being significantly attenuated in WSD+Arg-fed mice. These effects of Arg supplementation went along with a protection against WSD-induced decreased tight junction protein levels in the upper parts of the small intestine, increased levels of bacterial endotoxin in portal plasma as well as increased hepatic toll-like receptor-4 mRNA and 4-hydroxynonenal protein adduct levels. In conclusion, Arg supplementation may protect mice from the development of NASH.
Project description:RATIONALE:An elevated level of plasma LDL (low-density lipoprotein) is an established risk factor for cardiovascular disease. Recently, we reported that the (pro)renin receptor ([P]RR) regulates LDL metabolism in vitro via the LDLR (LDL receptor) and SORT1 (sortilin-1), independently of the renin-angiotensin system. OBJECTIVES:To investigate the physiological role of (P)RR in lipid metabolism in vivo. METHODS AND RESULTS:We used N-acetylgalactosamine modified antisense oligonucleotides to specifically inhibit hepatic (P)RR expression in C57BL/6 mice and studied the consequences this has on lipid metabolism. In line with our earlier report, hepatic (P)RR silencing increased plasma LDL-C (LDL cholesterol). Unexpectedly, this also resulted in markedly reduced plasma triglycerides in a SORT1-independent manner in C57BL/6 mice fed a normal- or high-fat diet. In LDLR-deficient mice, hepatic (P)RR inhibition reduced both plasma cholesterol and triglycerides, in a diet-independent manner. Mechanistically, we found that (P)RR inhibition decreased protein abundance of ACC (acetyl-CoA carboxylase) and PDH (pyruvate dehydrogenase). This alteration reprograms hepatic metabolism, leading to reduced lipid synthesis and increased fatty acid oxidation. As a result, hepatic (P)RR inhibition attenuated diet-induced obesity and hepatosteatosis. CONCLUSIONS:Collectively, our study suggests that (P)RR plays a key role in energy homeostasis and regulation of plasma lipids by integrating hepatic glucose and lipid metabolism.