The common patatin-like phospholipase domain–containing protein 3 (PNPLA3) variant I148M predisposes to nonalcoholic liver disease but not its metabolic sequelae. We compared the handling of labeled polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFA) in vivo in humans and in cells harboring different PNPLA3 genotypes. In 148M homozygous individuals, triglycerides (TGs) in very low–density lipoproteins (VLDL) were depleted of PUFAs both under fasting and postprandial conditions compared with 148I homozygotes, and the PUFA/SFA ratio in VLDL-TGs was lower relative to the chylomicron precursor pool. In human PNPLA3-148M and PNPLA3-KO cells, PUFA but not SFA incorporation into TGs was increased at the expense of phosphatidylcholines, and under lipolytic conditions, PUFA-containing diacylglycerols (DAGs) accumulated compared with PNPLA3-148I cells. Polyunsaturated TGs were increased, while phosphatidylcholines (PCs) were decreased in the human liver in 148M homozygous individuals as compared with 148I homozygotes. We conclude that human PNPLA3-I148M is a loss-of-function allele that remodels liver TGs in a polyunsaturated direction by impairing hydrolysis/transacylation of PUFAs from DAGs to feed phosphatidylcholine synthesis.
Panu K. Luukkonen, Auli Nick, Maarit Hölttä-Vuori, Christoph Thiele, Elina Isokuortti, Susanna Lallukka-Brück, You Zhou, Antti Hakkarainen, Nina Lundbom, Markku Peltonen, Marju Orho-Melander, Matej Orešič, Tuulia Hyötyläinen, Leanne Hodson, Elina Ikonen, Hannele Yki-Järvinen
Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are liver manifestations of the metabolic syndrome and can progress to hepatocellular carcinoma (HCC). Loss of Growth Hormone (GH) signaling is reported to predispose to NAFLD and NASH through direct actions on the liver. Here, we report that aged mice lacking hepatocyte Jak2 (JAK2L), an obligate transducer of Growth Hormone (GH) signaling, spontaneously develop the full spectrum of phenotypes found in patients with metabolic liver disease, beginning with insulin resistance and lipodystrophy and manifesting as NAFLD, NASH and even HCC, independent of dietary intervention. Remarkably, insulin resistance, metabolic liver disease, and carcinogenesis are prevented in JAK2L mice via concomitant deletion of adipocyte Jak2 (JAK2LA). Further, we demonstrate that GH increases hepatic lipid burden but does so indirectly via signaling through adipocyte JAK2. Collectively, these data establish adipocytes as the mediator of GH-induced metabolic liver disease and carcinogenesis. In addition, we report a new spontaneous model of NAFLD, NASH, and HCC that recapitulates the natural sequelae of human insulin resistance-associated disease progression. The work presented here suggests a attention be paid towards inhibition of adipocyte GH signaling as a therapeutic target of metabolic liver disease.
Kevin C. Corbit, Camella G. Wilson, Dylan Lowe, Jennifer L. Tran, Nicholas B. Vera, Michelle Clasquin, Aras N. Mattis, Ethan J. Weiss
BACKGROUND Sorafenib has been shown to reduce the extent of immunosuppression in patients with hepatocellular carcinoma (HCC). The rationale of this investigation was to identify biomarkers that can predict treatment efficacy of sorafenib in HCC patients and to unravel the mechanism by which sorafenib impedes immune suppression mediated by distinct immunosuppressive cell subsets.METHODS With informed consent, blood samples were collected from 30 patients with advanced HCC, at baseline and 2 time points after initiation of sorafenib treatment. The frequency of PD-1+ T cells, ERK2 phosphorylation on flt-3+ Tregs and MDSCs, and T effector cell function were quantified by using flow cytometry.RESULTS Elevated levels of CD8+Ki67+ T cells producing IFN-γ were associated with improved progression-free survival and overall survival (OS). High frequencies of these T cells were correlated with significantly reduced risk of death over time. Patients with an increased pretreatment T effector/Treg ratio showed significant improvement in OS. ERK+flt-3+ Tregs and MDSCs were significantly decreased after sorafenib therapy. Increased numbers of baseline flt-3+p-ERK+ MDSCs were associated with survival benefit of patients.CONCLUSION A high baseline CD4+ T effector/Treg ratio is a potential biomarker of prognostic significance in HCC. CD8+Ki67+ T cells producing IFN-γ are a key biomarker of response to sorafenib therapy resulting in survival benefit. The immune modulation resulted from sorafenib-mediated blockade of signaling through the VEGF/VEGFR/flt-3 pathway, affecting ERK phosphorylation. These insights may help identify patients who likely would benefit from VEGFR antagonism and inform efforts to improve the efficacy of sorafenib in combination with immunotherapy.TRIAL REGISTRATION NCT02072486.FUNDING National Comprehensive Cancer Network Oncology Research Program from general research support provided by Bayer US LLC (NCCNSORA0002), National Cancer Institute grant P30CA016056, and pilot funds from Roswell Park Alliance Foundation.
Suresh Gopi Kalathil, Alan Hutson, Joseph Barbi, Renuka Iyer, Yasmin Thanavala
Nonalcoholic fatty liver disease (NAFLD) prevails in obesity and is linked to several health complications including dyslipidemia and atherosclerosis. How exactly NAFLD induces atherogenic dyslipidemia to promote cardiovascular diseases is still elusive. Here, we identify Tsukushi (TSK) as a hepatokine induced in response to NAFLD. We show that both endoplasmic reticulum stress and inflammation promote the expression and release of TSK in mice. In humans, hepatic TSK expression is also associated with steatosis, and its circulating levels are markedly increased in patients suffering from acetaminophen-induced acute liver failure (ALF), a condition linked to severe hepatic inflammation. In these patients, elevated blood TSK levels were associated with decreased transplant-free survival at hospital discharge, suggesting that TSK could have a prognostic significance. Gain- and loss-of-function studies in mice revealed that TSK impacts systemic cholesterol homeostasis. TSK reduces circulating HDL cholesterol, lowers cholesterol efflux capacity, and decreases cholesterol-to–bile acid conversion in the liver. Our data identify the hepatokine TSK as a blood biomarker of liver stress that could link NAFLD to the development of atherogenic dyslipidemia and atherosclerosis.
Mathilde Mouchiroud, Étienne Camiré, Manal Aldow, Alexandre Caron, Éric Jubinville, Laurie Turcotte, Inès Kaci, Marie-Josée Beaulieu, Christian Roy, Sébastien M. Labbé, Thibault V. Varin, Yves Gélinas, Jennifer Lamothe, Jocelyn Trottier, Patricia L. Mitchell, Frédéric Guénard, William T. Festuccia, Philippe Joubert, Christopher F. Rose, Constantine J. Karvellas, Olivier Barbier, Mathieu C. Morissette, André Marette, Mathieu Laplante
Bile acids play a major role in the regulation of lipid and energy metabolism. Here we propose the hepatic bile acid uptake transporter Na+ taurocholate co-transporting polypeptide (NTCP) as a target to prolong postprandial bile acid elevations in plasma. Reducing hepatic clearance of bile acids from plasma by genetic deletion of NTCP moderately increased plasma bile acid levels, reduced diet-induced obesity, attenuated hepatic steatosis, and lowered plasma cholesterol levels. NTCP-G protein-coupled bile acid receptor (TGR5) double knockout mice were equally protected against diet-induced-obesity as NTCP single knockout mice. NTCP knockout mice displayed decreased intestinal fat absorption and a trend towards higher fecal energy output. Furthermore, NTCP deficiency was associated with an increased uncoupled respiration in brown adipose tissue, leading to increased energy expenditure. We conclude that targeting NTCP-mediated bile acid uptake can be a novel approach to treat obesity and obesity-related hepatosteatosis by simultaneously dampening intestinal fat absorption and increasing energy expenditure.
Joanne M. Donkers, Sander Kooijman, Davor Slijepcevic, Roni F. Kunst, Reinout L.P. Roscam Abbing, Lizette C.J.M Haazen, Dirk R. de Waart, Johannes H.M. Levels, Kristina Schoonjans, Patrick C.N. Rensen, Ronald P.J. Oude Elferink, Stan F.J. Van de Graaf
Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.
Alessia De Caneva, Fabiola Porro, Giulia Bortolussi, Riccardo Sola, Michela Lisjak, Adi Barzel, Mauro Giacca, Mark A. Kay, Kristian Vlahoviček, Lorena Zentilin, Andrés F. Muro
Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFβ (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-β-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.
Davide Povero, Eva M. Pinatel, Aleksandra Leszczynska, Nidhi P. Goyal, Takahiro Nishio, Jihoon Kim, David Kneiber, Lucas de Araujo Horcel, Akiko Eguchi, Paulina M. Ordonez, Tatiana Kisseleva, Ariel E. Feldstein
The evolutionary conserved Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex is one of the crucial multiprotein complexes that facilitates endosomal recycling of transmembrane proteins. Defects in WASH components have been associated with inherited developmental and neurological disorders in humans. Here, we show that hepatic ablation of the WASH component Washc1 in chow-fed mice increases plasma concentrations of cholesterol in both LDLs and HDLs, without affecting hepatic cholesterol content, hepatic cholesterol synthesis, biliary cholesterol excretion, or hepatic bile acid metabolism. Elevated plasma LDL cholesterol was related to reduced hepatocytic surface levels of the LDL receptor (LDLR) and the LDLR-related protein LRP1. Hepatic WASH ablation also reduced the surface levels of scavenger receptor class B type I and, concomitantly, selective uptake of HDL cholesterol into the liver. Furthermore, we found that WASHC1 deficiency increases LDLR proteolysis by the inducible degrader of LDLR, but does not affect proprotein convertase subtilisin/kexin type 9–mediated LDLR degradation. Remarkably, however, loss of hepatic WASHC1 may sensitize LDLR for proprotein convertase subtilisin/kexin type 9–induced degradation. Altogether, these findings identify the WASH complex as a regulator of LDL as well as HDL metabolism and provide in vivo evidence for endosomal trafficking of scavenger receptor class B type I in hepatocytes.
Melinde Wijers, Paolo Zanoni, Nalan Liv, Dyonne Y. Vos, Michelle Y. Jäckstein, Marieke Smit, Sanne Wilbrink, Justina C. Wolters, Ydwine T. van der Veen, Nicolette Huijkman, Daphne Dekker, Niels Kloosterhuis, Theo H. van Dijk, Daniel D. Billadeau, Folkert Kuipers, Judith Klumperman, Arnold von Eckardstein, Jan Albert Kuivenhoven, Bart van de Sluis
Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, and potentially morbid, disease that affects one-third of the U.S. population. Normal liver safely accommodates lipid excess during fasting or carbohydrate restriction by increasing their oxidation to acetyl-CoA and ketones, yet lipid excess during NAFLD leads to hyperglycemia and, in some, steatohepatitis. To examine potential mechanisms, flux through pathways of hepatic oxidative metabolism and gluconeogenesis were studied using five simultaneous stable isotope tracers in ketotic (24-hour fast) individuals with a wide range of hepatic triglyceride contents (0-52%). Ketogenesis was progressively impaired as hepatic steatosis and glycemia worsened. Conversely, the alternative pathway for acetyl-CoA metabolism, oxidation in the tricarboxylic (TCA) cycle, was upregulated in NAFLD as ketone production diminished and positively correlated with rates of gluconeogenesis and plasma glucose concentrations. Increased respiration and energy generation that occurred in liver when β-oxidation and TCA cycle activity were coupled may explain these findings, inasmuch as oxygen consumption was higher during fatty liver and highly correlated with gluconeogenesis. These findings demonstrate that increased glucose production and hyperglycemia in NAFLD is not a consequence of acetyl-CoA production per se, but how acetyl-CoA is further metabolized in liver.
Justin A. Fletcher, Stanislaw Deja, Santhosh Satapati, Xiaorong Fu, Shawn C. Burgess, Jeffrey D. Browning
Pharmacologic inhibition of the renal sodium/glucose cotransporter-2 induces glycosuria and reduces glycemia. Given that SGLT2 inhibitors (SGLT2i) reduce mortality and cardiovascular risk in type 2 diabetes, improved understanding of molecular mechanisms mediating these metabolic effects is required. Treatment of obese but nondiabetic mice with the SGLT2i canagliflozin (CANA) reduces adiposity, improves glucose tolerance despite reduced plasma insulin, increases plasma ketones, and improves plasma lipid profiles. Utilizing an integrated transcriptomic-metabolomics approach, we demonstrate that CANA modulates key nutrient-sensing pathways, with activation of 5′ AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR), independent of insulin or glucagon sensitivity or signaling. Moreover, CANA induces transcriptional reprogramming to activate catabolic pathways, increase fatty acid oxidation, reduce hepatic steatosis and diacylglycerol content, and increase hepatic and plasma levels of FGF21. Given that these phenotypes mirror the effects of FGF21 to promote lipid oxidation, ketogenesis, and reduction in adiposity, we hypothesized that FGF21 is required for CANA action. Using FGF21-null mice, we demonstrate that FGF21 is not required for SGLT2i-mediated induction of lipid oxidation and ketogenesis but is required for reduction in fat mass and activation of lipolysis. Taken together, these data demonstrate that SGLT2 inhibition triggers a fasting-like transcriptional and metabolic paradigm but requires FGF21 for reduction in adiposity.
Soravis Osataphan, Chiara Macchi, Garima Singhal, Jeremy Chimene-Weiss, Vicencia Sales, Chisayo Kozuka, Jonathan M. Dreyfuss, Hui Pan, Yanin Tangcharoenpaisan, Jordan Morningstar, Robert Gerszten, Mary-Elizabeth Patti
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