The signaling mechanisms by which dietary fat and cholesterol signals regulate central pathways of glucose homeostasis are not completely understood. By using a hepatocyte-specific PKCβ-deficient (PKCβHep–/–) mouse model, we demonstrated the role of hepatic PKCβ in slowing disposal of glucose overload by suppressing glycogenesis and increasing hepatic glucose output. PKCβHep–/– mice exhibited lower plasma glucose under the fed condition, modestly improved systemic glucose tolerance and mildly suppressed gluconeogenesis, increased hepatic glycogen accumulation and synthesis due to elevated glucokinase expression and activated glycogen synthase (GS), and suppressed glucose-6-phosphatase expression compared with controls. These events were independent of hepatic AKT/GSK-3α/β signaling and were accompanied by increased HNF-4α transactivation, reduced FoxO1 protein abundance, and elevated expression of GS targeting protein phosphatase 1 regulatory subunit 3C in the PKCβHep–/– liver compared with controls. The above data strongly imply that hepatic PKCβ deficiency causes hypoglycemia postprandially by promoting glucose phosphorylation via upregulating glucokinase and subsequently redirecting more glucose-6-phosphate to glycogen via activating GS. In summary, hepatic PKCβ has a unique and essential ability to induce a coordinated response that negatively affects glycogenesis at multiple levels under physiological postprandial conditions, thereby integrating nutritional fat intake with dysregulation of glucose homeostasis.
Yaoling Shu, Faizule Hassan, Michael C. Ostrowski, Kamal D. Mehta
Xanthine oxidoreductase (XOR) is an enzyme that catalyzes hypoxanthine to xanthine and xanthine to uric acid, respectively. However, the underlying mechanisms of increased plasma XOR and its pathological roles in systemic diseases, such as atherosclerosis, are not fully understood. In this study, we found that changes in plasma XOR activity after bariatric surgery closely associated with those in liver enzymes, but not with those in BMI. In a mouse model of nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH), plasma XOR activity markedly increased. Besides, purine catabolism was accelerated in the plasma per se of NASH mice and human patients with high XOR activity. In our NASH mice, we observed an increased vascular neointima formation consisting of dedifferentiated vascular smooth muscle cells (SMCs), which was significantly attenuated by topiroxostat, a selective XOR inhibitor. In vitro, human liver S9–derived XOR promoted proliferation of SMCs with phenotypic modulation and induced ROS production by catabolizing hypoxanthine released from human endothelial cells. Collectively, the results from human and mouse models suggest that increased plasma XOR activity, mainly explained by excess hepatic leakage, was involved in the pathogenesis of vascular injury, especially in NAFLD/NASH conditions.
Yusuke Kawachi, Yuya Fujishima, Hitoshi Nishizawa, Takashi Nakamura, Seigo Akari, Takayo Murase, Takuro Saito, Yasuhiro Miyazaki, Hirofumi Nagao, Shiro Fukuda, Shunbun Kita, Naoto Katakami, Yuichiro Doki, Norikazu Maeda, Iichiro Shimomura
Alcohol-associated liver disease (ALD) represents a spectrum of histopathological changes, including alcoholic steatosis, steatohepatitis, and cirrhosis. One of the early responses to excessive alcohol consumption is lipid accumulation in the hepatocytes. Lipid ω-hydroxylation of medium- and long-chain fatty acid metabolized by the cytochrome P450 4A (CYP4A) family is an alternative pathway for fatty acid metabolism. The molecular mechanisms of CYP4A in ALD pathogenesis have not been elucidated. In this study, WT and Shp−/− mice were fed with a modified ethanol-binge, National Institute on Alcohol Abuse and Alcoholism model (10 days of ethanol feeding plus single binge). Liver tissues were collected every 6 hours for 24 hours and analyzed using RNA-Seq. The effects of REV-ERBα agonist (SR9009, 100 mg/kg/d) or CYP4A antagonist (HET0016, 5 mg/kg/d) in ethanol-fed mice were also evaluated. We found that hepatic Cyp4a10 and Cyp4a14 expression were significantly upregulated in WT mice, but not in Shp−/− mice, fed with ethanol. ChIP quantitative PCR and promoter assay revealed that REV-ERBα is the transcriptional repressor of Cyp4a10 and Cyp4a14. Rev-Erbα−/− hepatocytes had a marked induction of both Cyp4a genes and lipid accumulation. REV-ERBα agonist SR9009 or CYP4A antagonist HET0016 attenuated Cyp4a induction by ethanol and prevented alcohol-induced steatosis. Here, we have identified a role for the SHP/REV-ERBα/CYP4A axis in the pathogenesis of ALD. Our data also suggest REV-ERBα or CYP4A as the potential therapeutic targets for ALD.
Zhihong Yang, Rana V. Smalling, Yi Huang, Yanchao Jiang, Praveen Kusumanchi, Will Bogaert, Li Wang, Don A. Delker, Nicholas J. Skill, Sen Han, Ting Zhang, Jing Ma, Nazmul Huda, Suthat Liangpunsakul
Development of primary liver cancer is a multi-stage process. Detailed understanding of sequential epigenetic alterations is largely missing. Here, we performed Infinium Human Methylation 450k BeadChips and RNA sequencing analyses for genome-wide methylome and transcriptome profiling of cirrhotic liver (n=7), low- (n=4) and high-grade (n=9) dysplastic lesions, early (n=5) and progressed (n=3) hepatocellular carcinomas (HCC) synchronously detected in eight HCC patients with chronic hepatitis B infection. Integrative analyses of epigenetically driven molecular changes were identified and validated in two independent cohorts comprising 887 HCC. Mitochondrial DNA sequencing was further employed for clonality analyses and indicates multi-clonal origin in the majority of investigated HCC. Alterations in DNA methylation progressively increased from CL to dysplastic lesions and reached a maximum in early HCC. Associated early alterations identified by IPA pathway analyses involved apoptosis, immune regulation and stemness pathways, while late changes centered on cell survival, proliferation and invasion. We further validated putative 23 epi-drivers with concomitant expression changes and associated with overall survival. Functionally, Striatin 4 (STRN4) was demonstrated to be epigenetically regulated and inhibition of STRN4 significantly suppressed tumorigenicity of HCC cell lines. Overall, application of integrative genomic analyses defines epigenetic driver alterations and provides promising targets for novel therapeutic approaches.
Carolin Czauderna, Alicia Poplawski, Colm J. O´Rourke, Darko Castven, Benjamín Pérez-Aguilar, Diana Becker, Stefanie Heilmann-Heimbach, Margarete Odenthal, Wafa Amer, Marcel Schmiel, Uta Drebber, Harald Binder, Dirk A. Ridder, Mario Schindeldecker, Beate K. Straub, Peter R. Galle, Jesper B. Andersen, Snorri S. Thorgeirsson, Young Nyun Park, Jens U. Marquardt
The liver is the major source of glucose production during fasting under normal physiological conditions. However, the kidney may also contribute to maintaining glucose homeostasis in certain circumstances. To test the ability of the kidney to compensate for impaired hepatic glucose production in vivo, we developed a stable isotope approach to simultaneously quantify gluconeogenic and oxidative metabolic fluxes in the liver and kidney. Hepatic gluconeogenesis from phosphoenolpyruvate was disrupted via liver-specific knockout of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; KO). 2H/13C isotopes were infused in fasted KO and WT littermate mice, and fluxes were estimated from isotopic measurements of tissue and plasma metabolites using a multicompartment metabolic model. Hepatic gluconeogenesis and glucose production were reduced in KO mice, yet whole-body glucose production and arterial glucose were unaffected. Glucose homeostasis was maintained by a compensatory rise in renal glucose production and gluconeogenesis. Renal oxidative metabolic fluxes of KO mice increased to sustain the energetic and metabolic demands of elevated gluconeogenesis. These results show the reciprocity of the liver and kidney in maintaining glucose homeostasis by coordinated regulation of gluconeogenic flux through PEPCK-C. Combining stable isotopes with mathematical modeling provides a versatile platform to assess multitissue metabolism in various genetic, pathophysiological, physiological, and pharmacological settings.
Mohsin Rahim, Clinton M. Hasenour, Tomasz K. Bednarski, Curtis C. Hughey, David H. Wasserman, Jamey D. Young
Obesity is a risk factor for gallbladder cancer (GBC) development and correlates with shorter overall survival. Leptin, derived from adipocytes, has been suggested to contribute to the growth of cancer cells. However, the detailed mechanism of leptin in GBC drug resistance remains uninvestigated. In this study, it is clinically relevant that GBC patients with a higher BMI (BMI ≥ 24 kg/m2) (n=30) were associated with increased GBC risks, including survival. Moreover, obese NOD/SCID mice exhibited a higher circulating concentration of leptin, which is associated with GBC growth and attenuated gemcitabine efficacy. We further revealed that leptin can inhibit gemcitabine-induced GBC cell death through myeloid cell leukemia 1 (MCL1) activation. The transcription factor CCAAT/enhancer-binding protein delta (CEBPD) is responsive to activated signal transducers and activators of transcription 3 (pSTAT3) and contributes to MCL1 transcriptional activation upon leptin treatment. In addition, MCL1 mediates leptin-induced mitochondrial fusion and is associated with GBC cell survival. This study suggests the involvement of the pSTAT3/CEBPD/MCL1 axis in leptin-induced mitochondrial fusion and survival. It provides a new therapeutic target to improve the efficacy of gemcitabine in GBC patients.
Wei-Jan Wang, Hong-Yue Lai, Fei Zhang, Wan-Jou Shen, Pei-Yu Chu, Hsin-Yin Liang, Ying-Bin Liu, Ju-Ming Wang
Hepatocellular carcinoma (HCC) is the 6th-most common and the 4th-most deadly cancer worldwide. The development cost of new therapeutics is a major limitation in patient outcomes. Importantly, there is a paucity of preclinical HCC models in which to test new small molecules. Herein, we implemented novel patient-derived organoid (PDO) and patient-derived xenografts (PDX) strategies for high-throughput drug screening. Omacetaxine, an FDA-approved drug for chronic myelogenous leukemia (CML), was found to be a top effective small molecule in HCC PDOs. Next, omacetaxine was tested against a larger cohort of 40 human HCC PDOs. Serial dilution experiments demonstrated that omacetaxine is effective at low (nanomolar) concentrations. Mechanistic studies established that omacetaxine inhibits global protein synthesis, with a disproportionate effect on short-half-life proteins. High-throughput expression screening newly identified molecular targets for omacetaxine, including key oncogenes, such as PLK1. In conclusion, by using an innovative strategy, we report, for the first time, the effectiveness of omacetaxine in HCC. In addition, we newly elucidate key mechanisms of omacetaxine action. Finally, we provide a proof-of-principle basis for future studies applying drug screening PDOs sequenced with candidate validation in PDX models. Clinical trials could be considered to evaluate omacetaxine in patients with HCC.
Ling Li, Gilad Halpert, Michael G. Lerner, Haijie Hu, Peter Dimitrion, Matthew J. Weiss, Jin He, Benjamin Philosophe, Richard Burkhart, William R. Burns, Russell N. Wesson, Andrew MacGregor Cameron, Christopher L. Wolfgang, Christos Georgiades, Satomi Kawamoto, Nilofer S. Azad, Mark Yarchoan, Stephen J. Meltzer, Kiyoko Oshima, Laura M. Ensign, Joel S. Bader, Florin M. Selaru
BACKGROUND. The chemokine system of ligands and receptors is implicated in the progression of Alcohol-associated hepatitis (AH). Finding upstream regulators could lead to novel therapies. MOTHODS. The coordinated expression of chemokines in livers of healthy controls (HC) and patients with AH in two distinct cohorts of patients with various chronic liver diseases. Studies in cultured hepatocytes and in tissue-specific knockouts were used for mechanistic insight into a potential upstream regulator of chemokine expression in AH. RESULTS. Selected C-X-C chemokine members of the Interleukin-8 (IL-8) chemokine family and C-C chemokine CCl20 were highly associated with AH compared to HC, but not in patients with liver diseases of other etiologies (NAFLD or HCV). Our previous studies implicate Macrophage migration inhibitory factor (MIF) as a pleiotropic cytokine/chemokine with the potential to coordinately regulate chemokine expression in AH. LPS-stimulated expression of multiple chemokines in cultured hepatocytes was dependent on MIF. Gao-binge ethanol feeding to mice induced a similar coordinated chemokine expression in livers of wild-type mice; this was prevented in hepatocyte-specific Mif knockout (MifΔHep) mice. CONCLUSIONS. This study demonstrates that patients with AH exhibit a specific, coordinately expressed chemokine signature and hepatocyte-derived MIF might drive this inflammatory response.
Kyle L. Poulsen, Xiude D. Fan, Christopher D. Kibler, Emily Huang, Xiaoqin Wu, Megan R. McMullen, Lin Leng, Richard Bucala, Meritxell Ventura-Cots, Josepmaria Argemi, Ramon Bataller, Laura E. Nagy
Liver regeneration is critical to survival after traumatic injuries, exposure to hepatotoxins, or surgical interventions, yet the underlying signaling and metabolic pathways remain unclear. Here we show that hepatocyte-specific loss of the mitochondrial deacetylase SIRT3 drastically impairs regeneration and worsens mitochondrial function after partial hepatectomy. Sirtuins, including SIRT3, require nicotinamide adenine dinucleotide (NAD) as a cosubstrate. We previously showed that the NAD precursor nicotinamide riboside (NR) promotes liver regeneration, but whether this involves sirtuins has not been tested. Here we show that despite their NAD-dependence and critical roles in regeneration, neither SIRT3 nor its nuclear counterpart SIRT1 is required for NR to enhance liver regeneration. NR improves mitochondrial respiration in regenerating wild type or mutant livers and rapidly increases oxygen consumption and glucose output in cultured hepatocytes. Our data support a direct enhancement of mitochondrial redox metabolism as the mechanism mediating improved liver regeneration after NAD supplementation and exclude signaling via SIRT1 and SIRT3. Thus, we provide the first evidence for an essential role for a mitochondrial sirtuin during liver regeneration and insight into the beneficial effects of NR.
Sarmistha Mukherjee, James Mo, Lauren M. Paolella, Caroline E. Perry, Jade Toth, Mindy M. Hugo, Qingwei Chu, Qiang Tong, Karthikeyani Chellappa, Joseph A. Baur
Studies of human hepatitis B virus (HBV) immune pathogenesis are hampered by limited access to liver tissues and technologies for detailed analyses. Here, utilizing imaging mass cytometry (IMC) to simultaneously detect 30 immune, viral and structural markers in liver biopsies from patients with HBeAg+ chronic hepatitis B, we provide novel comprehensive visualization, quantitation and phenotypic characterizations of hepatic adaptive and innate immune subsets that correlated with hepatocellular injury, histological fibrosis and age. We further show marked correlations between adaptive and innate immune cell frequencies and phenotype, highlighting complex immune interactions within the hepatic microenvironment with relevance to HBV pathogenesis.
Daniel Traum, Yue J. Wang, Kathleen B. Schwarz, Jonathan Schug, David K.H. Wong, Harry L.A. Janssen, Norah A. Terrault, Mandana Khalili, Abdus S. Wahed, Karen F. Murray, Philip Rosenthal, Simon C. Ling, Norberto Rodriguez-Baez, Richard K. Sterling, Daryl T.Y. Lau, Timothy M. Block, Michael D. Feldman, Emma E. Furth, William M. Lee, David E. Kleiner, Anna S. Lok, Klaus H. Kaestner, Kyong-Mi Chang
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