Gene Expression In Hepatocytes Research Articles

Local and systemic actions of hepatic fatty acid oxidation

Mitochondrial long chain fatty acid beta‐oxidation is required for hepatic ketogenesis and gluconeogensis during fasting, starvation and times of low carbohydrate intake. The liver has a large capacity and dynamic range for fatty acid oxidation to facilitate ketogenesis and gluconeogenesis. To gain a better understanding of the roles of hepatic fatty acid oxidation on liver function, we generated mice with a hepatocyte‐specific knockout of Carnitine Palmitoyltransferase 2 (Cpt2L−/−), an obligate step in mitochondrial long‐chain fatty acid β‐oxidation. Mice with a loss in hepatic fatty acid oxidation were able to maintain systemic glucose homeostasis during a 24hr fast, but did not produce ketone bodies. Systemic energy homeostasis was largely maintained by adaptations in hepatic and systemic oxidative gene expression mediated in part by a robust hepatic transcriptional response. Open chromatin assessed by ATAC‐seq and histone H3K9 ChIP‐seq analysis revealed that many genes induced in fasted Cpt2L−/− mice exhibited Pparα regulatory sites. To understand the contribution of Pparα, we generated Pparα;Cpt2L−/− double knockout mice. The deletion of Pparα in Cpt2L−/− mice largely blunted this transcriptional response, demonstrating the requirement for Pparα. Triglyceride hydrolysis has been shown to be critical for Pparα‐mediated transcriptional control. To determine the requirement of hepatic triglyceride hydrolysis on the Cpt2L−/− transcriptional response, mice were generated with a liver‐specific defect in triglyceride hydrolysis (AtglL−/−) and fatty acid oxidation (AtglL−/−;Cpt2L−/− double knockout). The loss of Cpt2 or Atgl resulted in largely independent effects on hepatocyte morphology, intermediary metabolism and gene expression in response to fasting. However, high fat feeding revealed the interaction of Atgl and Cpt2 as the loss of only one of these genes results in steatosis but the loss of both components resulted in significant inflammation and fibrosis. These data suggest that defects in mitochondrial fatty acid oxidation induce an exaggerated fasting response that initiates lipid dependent transcription to mediate changes in local and systemic physiology.Support or Funding InformationThis work was supported in part by a National Institutes of Health grant R01DK116746 to M.J.W.

Berberine Attenuates Hyperglycemia by Inhibiting the Hepatic Glucagon Pathway in Diabetic Mice.

Dysregulated glucagon drives hyperfunction in hepatic glucose output, which is the main cause of persistent hyperglycemia in type 2 diabetes. Berberine (Zhang et al., 2010) has been used as a hypoglycemic agent, yet the mechanism by which BBR inhibits hepatic gluconeogenesis remains incompletely understood. In this study, we treated diabetic mice with BBR, tested blood glucose levels, and then performed insulin, glucose lactate, and glucagon tolerance tests. Intracellular cAMP levels in hepatocytes were determined by ELISA, hepatic gluconeogenetic genes were assayed by RT-qPCR, and the phosphorylation of CREB, which is the transcriptional factor controlling the expression of gluconeogenetic genes, was detected by western blot. BBR reduced blood glucose levels, improved insulin and glucose tolerance, and suppressed lactate- and glucagon-induced hepatic gluconeogenesis in ob/ob and STZ-induced diabetic mice. Importantly, BBR blunted glucagon-induced glucose production and gluconeogenic gene expression in hepatocytes, presumably through reducing cAMP, which resulted in the phosphorylation of CREB. By utilizing a cAMP analogue, adenylate cyclase (AC), to activate cAMP synthetase, and an inhibitor of the cAMP degradative enzyme, phosphodiesterase (PDE), we revealed that BBR accelerates intracellular cAMP degradation. BBR reduces the intracellular cAMP level by activating PDE, thus blocking activation of downstream CREB and eventually downregulating gluconeogenic genes to restrain hepatic glucose production.

The effect of trans-palmitoleic acid on cell viability and sirtuin 1 gene expression in hepatocytes and the activity of peroxisome-proliferator-activated receptor-alpha.

Background:Accumulation of fatty acids in liver causes lipotoxicity which is followed by nonalcoholic fatty liver disease. The association between intakes of trans-fatty acids with metabolic diseases is still controversial. Accordingly, the objective of this study was to investigate the in vitro effects of trans-palmitoleic acid (tPA) and palmitic acid (PA) on lipid accumulation in hepatocytes, focusing on the gene expression of sirtuin 1 (SIRT1) as well as the transcriptional activity of peroxisome proliferator-activated receptor alpha (PPARα).Materials and Methods:In this experimental study, hepatocellular carcinoma (HepG2) cells were cultured and treated with various concentrations of tPA and PA (C16:0). The accumulation of triglyceride in the cells was measured by enzymatic method. Gene expression was evaluated by real-time polymerase chain reaction. The activity of PPARα was assessed by luciferase reporter assay after transfection of human embryonic kidney 293T cells by a vector containing the PPAR response element.Results:While concentration >1 mM for PA and cis-PA (cPA) reduced the viability of hepatocytes, tPA revealed an opposite effect and increased cell survival. Lipid accumulation in HepG2 cells after treatment with tPA was significantly lower than that in cells treated with PA. In addition, tPA at physiological concentration had no effect on the expression of SIRT1 while at high concentration significantly augmented its expression. There was a modest increase in PPARα activity at low concentration of tPA.Conclusion:tPA causes less lipid accumulation in hepatocytes with no detrimental effect on cell viability and might be beneficial for liver cells by the activation of SIRT1 and induction of PPARα activity.

Sirt1 regulates the expression of critical metabolic genes in chicken hepatocytes

Context Studies in mammals show that SIRT1 plays an important role in many biological processes including liver metabolism through histone and non-histone deacetylation. Little is known about the function of Sirt1 in the chicken. Aims The current study investigated the expression pattern of Sirt1 mRNA in the chicken and its functions in the chicken liver. Methods In this work, we used real-time quantitative polymerase chain reaction to quantify the expression levels of Sirt1 mRNA in major chicken organs and tissue types, siRNA to knock down Sirt1 expression in primary chicken hepatocytes, RNA sequencing to identify gene-expression changes induced by Sirt1 knockdown, and analysed the function of the differentially expressed genes (DEGs) through gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes ontology analysis. Key results In total, 86 DEGs were found between Sirt1 knockdown and control chicken hepatocytes, of which 63 genes were downregulated and 23 genes were upregulated by Sirt1 knockdown. The Kyoto Encyclopedia of Genes and Genomes analysis showed that 24 DEGs were involved in metabolism. Seven DEGs were involved in carbohydrate and lipid metabolism. Conclusions The present study showed that Sirt1 regulates the expression of genes involved in carbohydrate and lipid metabolism and many other biological processes in the chicken liver. Implications The results of the present study imply that Sirt1 has various functions in the chicken liver and that Sirt1 plays a potentially important role in hepatic carbohydrate and lipid metabolism in the chicken.

Hepatocyte gene expression and DNA methylation as ancestry-dependent mechanisms in African Americans

African Americans (AAs) are an admixed population with widely varying proportion of West African ancestry (WAA). Here we report the correlation of WAA to gene expression and DNA methylation in AA-derived hepatocytes, a cell type important in disease and drug response. We perform mediation analysis to test whether methylation is a mediator of the effect of ancestry on expression. GTEx samples and a second cohort are used as validation. One hundred and thirty-one genes are associated with WAA (FDR < 0.10), 28 of which replicate and represent 220 GWAS phenotypes. Among PharmGKB pharmacogenes, VDR, PTGIS, ALDH1A1, CYP2C19, and P2RY1 nominally associate with WAA (p < 0.05). We find 1037 WAA-associated, differentially methylated regions (FDR < 0.05), with hypomethylated genes enriched in drug-response pathways. In conclusion, WAA contributes to variability in hepatocyte expression and DNA methylation with identified genes previously implicated for diseases disproportionately affecting AAs, including cardiovascular (PTGIS, PLAT) and renal (APOL1) disease, and drug response (CYP2C19).

Pathogenic Oxidized Phospholipids Regulate Gene Expression, Cellular Metabolism, and Lipid Droplet Formation in Hepatocytes - Implications for Non-alcoholic Fatty Liver Disease

Pathogenic Oxidized Phospholipids Regulate Gene Expression, Cellular Metabolism, and Lipid Droplet Formation in Hepatocytes - Implications for Non-alcoholic Fatty Liver Disease

The Markedly Increased PAI1 in Obesity Induces a Compensatory Increase of Hepatocyte Tpa Expression By Activating a LRP1-CREB1 Pathway

Tissue-type plasminogen activator (tPA) initiates fibrinolysis, the primary mechanism that dissolves a thrombus. We have shown that in lean mice the hepatocytes maintain a basal level of circulating tPA that influences fibrinolysis in the occurrence of a vessel injury. As a serine protease, tPA is inhibited by the serpin plasminogen activator inhibitor 1 (PAI1). Both PAI1 and tPA which can be produced by hepatocytes, which is important for regulating energy metabolism and is sensitive to metabolic stress. In obesity, despite an increase in plasma tPA, blood tPA activity and fibrinolysis are reduced primarily due to a larger increase in PAI1. However, the source and regulatory mechanism of this phenomena are unknown. Palmitate treatment of human primary hepatocytes causes an increase in tPA mRNA/protein, reflecting the situation in the obese liver. This increase, however, is overcompensated by a larger increase in PAI1 mRNA, resulting in decreased net tPA activity in the cell culture medium. Silencing PAI1 mRNA with si-SERPINE1 in these cells increased tPA activity in the culture medium. Surprisingly, silencing PAI1 also prevented the increase of tPA mRNA induced by palmitate. Building on this observation, we further treated the cells with active recombinant PAI1 (rPAI1), which induced tPA expression. Next, we fed PAI1fl/flmice with a diet-induced obesity (DIO) diet for three months and silenced their hepatic PAI1 using AAV8-TBG-Cre, which inactivates ~95% floxed gene expression in hepatocytes but not in other cell types of the liver or other tissues. We found that while plasma tPA activity was increased in hepatocyte-PAI1 knockout mice, plasma tPA protein concentration was reduced. In other words, reducing hepatocyte PAI1 in obesity stopped the compensatory increase in liver tPA mRNA and protein. Thus, PAI1 induces tPA, which is likely a compensatory response. To explore the mechanism whereby rPAI1 induces tPA, we considered CREB1, as it is a transcription activator of tPA expression in endothelial cells and is expressed in hepatocytes. We found that CREB1 was activated (phosphorylated) by rPAI1 in human primary hepatocytes and that silencing CREB1 prevented the elevated tPA expression induced by rPAI1. Next, we silenced hepatic CREB1 in obese mice by treating DIO diet-fed CREB1fl/flmice with AAV8-TBG-Cre, which blocked the compensatory increase in tPA and thus worsened the impaired fibrinolysis in obesity. LDL receptor-related protein 1 (LRP1), the major cellular receptor of PAI1, can stimulate CREB1 transcription activity in neurons and adipocytes. As LRP1 is expressed on the surface of hepatocytes, we hypothesized that PAI1 activates CREB1 through LRP1-mediated signaling to increase tPA expression in obesity. Indeed, silencing LRP1 with si-LRP1 stopped the increase of tPA expression induced by rPAI1 in human primary hepatocytes. Interestingly, treating the cells with a mutant rPAI1 lacking the LRP1-interacting heparin-binding domain was unable to induce tPA expression. In summary, hepatocyte tPA mRNA/protein is increased in obesity, but this increase is ultimately overcompensated by a larger increase in PAI1, resulting in decreased plasma tPA activity and fibrinolysis. The markedly increased PAI1, through activating LRP1-CREB1 signaling pathway, drives an increase in hepatic tPA expression as a "compensatory" pathway in obesity. Preventing this compensatory pathway in obesity resulted in the worsening of fibrinolysis. Therapeutic boosting of this compensatory pathway may provide novel strategies to restore effective fibrinolysis in obesity. Disclosures No relevant conflicts of interest to declare.

FGF21 acts as a negative regulator of bile acid synthesis

Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid homeostasis in vivo; its most closely related subfamily member, FGF19, is known to be a critical negative regulator of bile acid synthesis. To delineate whether FGF21 also plays a functional role in bile acid metabolism, we evaluated the effects of short- and long-term exposure to native FGF21 and long-acting FGF21 analogs on hepatic signal transduction, gene expression and enterohepatic bile acid levels in primary hepatocytes and in rodent and monkey models. FGF21 acutely induced ERK phosphorylation and inhibited Cyp7A1 mRNA expression in primary hepatocytes and in different rodent models, although less potently than recombinant human FGF19. Long-term administration of FGF21 in mice fed a standard chow diet resulted in a 50-60% decrease in bile acid levels in the liver and small intestines and consequently a 60% reduction of bile acid pool size. In parallel, colonic and fecal bile acid was decreased, whereas fecal cholesterol and fatty acid excretions were elevated. The long-acting FGF21 analog showed superiority to recombinant human FGF21 and FGF19 in decreasing bile acid levels with long duration of effect action in mice. Long-term administration of the long-acting FGF21 analogs in obese cynomolgus monkeys suppressed plasma total bile acid and 7α-hydroxy-4-cholesten-3-one levels, a biomarker for bile acid synthesis. Collectively, these data reveal a previously unidentified role of FGF21 in bile acid metabolism as a negative regulator of bile acid synthesis.

CCR5 receptor antagonism inhibits hepatitis C virus (HCV) replication in vitro.

Background and aimThe hepatitis C virus (HCV) is a single-strand RNA virus that infects millions of people worldwide. Recent advances in therapy have led to viral cure using two- and three- drug combinations of direct acting inhibitors of viral replication. CCR5 is a chemokine receptor that is expressed on hepatocytes and represents a key co-receptor for HIV. We evaluated the effect of CCR5 blockade or knockdown on HCV replication in Huh7.5JFH1 cells.MethodsCells were exposed to varying concentrations of maraviroc (CCR5 inhibitor), cenicriviroc (CCR2/CCR5 inhibitor), sofosbuvir (nucleotide polymerase inhibitor), or raltegravir (HIV integrase inhibitor).ResultsHCV RNA was detected utilizing two qualitative strand-specific RT-PCR assays. HCV core antigen and NS3 protein was quantified in the supernatant and cell lysate, respectively. siRNA was utilized to knockdown CCR5 gene expression in hepatocytes. Alternatively, anti-CCR5 antibodies were employed to block the receptor. Supernatant levels of HCV RNA (expressed as fold change) were not reduced in the presence of raltegravir but were reduced 8.55-fold and 12.42-fold with cenicriviroc and maraviroc, respectively. Sofosbuvir resulted in a 16.20-fold change in HCV RNA levels. HCV core and NS3 protein production was also reduced in a dose-dependent manner. Two distinct anti-CCR5 antibodies also resulted in a significant reduction in HCV protein expression, as did siRNA knockdown of CCR5 gene expression.ConclusionsThese data provide evidence that CCR5 modulation could have a significant effect on HCV replication in an in vitro system. Further evaluation of the role of CCR5 inhibition in clinical settings may be warranted.

Comparison of metabolic and mitogenic response in vitro of the rapid-acting insulin lispro product SAR342434, and US- and EU-approved Humalog®

SAR342434 is a biosimilar of insulin lispro (Humalog® U-100). Batches of SAR342434 were compared with Humalog® batches of either EU or US origin in a panel of in vitro biological assays that included insulin binding to insulin receptor (IR) isoforms A (IR-A) and B (IR-B) and IR-A/IR-B autophosphorylation. A surface plasmon resonance biosensor-based assay was developed to characterize the kinetics of insulin binding to solubilized full-length IR-A or IR-B. Insulin-dependent metabolic activity assays included inhibition of lipolysis in in vitro differentiated human adipocytes, glucose uptake in L6-myocytes, and repression of glucose-6-phosphatase gene expression in human hepatocytes. Mitogenic activity assays included insulin binding to insulin-like growth factor-1 receptor (IGF1R), IGF1R autophosphorylation, and cell proliferation in MCF-7 cells. Weighted geometric means and their respective 95% confidence intervals (CI) were calculated for all 50% inhibitory or effective concentration values and kinetic binding constants for IR-A and IR-B. Statistical evaluation of the data demonstrated that the 90% CIs of the ratio of geometric means between SAR342434 and Humalog® EU or Humalog® US were within the predefined acceptance limits for each assay. Insulin lispro as SAR342434 solution demonstrated similarity to both US- and EU-approved Humalog® based on a side-by-side biological similarity assessment.

Schistosoma japonicum infection causes a reprogramming of glycolipid metabolism in the liver

BackgroundRecent investigations indicate that schistosome infection is closely associated with aberrant glycolipid metabolism. However, the actual glycolipid metabolism gene expression, as well as the possible pathways that regulate glycolipid metabolism in the schistosome-infected liver, has not been extensively explored.MethodsIn this study, we evaluated the dynamic expression of glycolipid metabolism-associated genes and proteins in the livers from mice infected with Schistosoma japonicum at the indicated time points using real-time PCR and immunofluorescence. Then, cultures of macrophages were treated with schistosome soluble egg antigen (SEA) to detect the expression levels of genes associated with glucose and lipid metabolism in order to identify macrophages metabolic characteristics in response to these antigens. Furthermore, SEA-stimulated macrophages were co-cultures with hepatocytes and detected the effects of macrophages on the gene expression of hepatocytes metabolism.ResultsThe expression of glycolysis-related genes (Ldha, Glut4, Pkm2, Glut1, Pfkfb3, Aldoc, HK2, Pfk) in the liver were upregulated but the gluconeogenesis gene (G6pc) was downregulated during S. japonicum infection. In addition, the mRNA levels of fatty acid (FA) oxidation-related genes (Ucp2, Atp5b, Pparg) in the liver were significantly upregulated; however, the FA synthesis genes (Fas, Acc, Scd1, Srebp1c) and lipid uptake gene (Cd36) were downregulated post-S. japonicum-infection. In consistence with these data, stimulation with SEA in vitro significantly enhanced the gene expression that involved in glycolysis and FA oxidation, but decreased genes related to gluconeogenesis, FA synthesis and lipid uptake in macrophages. The levels of phosphorylated AMPK, AKT and mTORC1 were increased in macrophages after SEA stimulation. Inhibition of phosphorylated AMPK, AKT and mTORC1 promoted SEA-treated macrophages to produce glucose. In addition, suppression of phosphorylated-AMPK, but not phosphorylated-AKT and phosphorylated-mTOR, induced the lipid accumulation in SEA-stimulated macrophages. Furthermore, SEA-treated macrophages significantly reduced the expression of Acc mRNA in hepatocytes in vitro.ConclusionsThese findings reveal S. japonicum infection induces dynamic changes in the expression levels of genes involved in catabolism (glucose uptake, glycolysis and fatty acid oxidation) and suppressing anabolism (glycogen synthesis) in the liver, which could occur via macrophages’ metabolic states, particularly those involved in the AMPK, AKT and mTORC1 pathways.

AMP-activated protein kinase signaling regulated expression of urea cycle enzymes in response to changes in dietary protein intake.

Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high-protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low-protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate-activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of carbamoylphosphate synthetase 1 and ornithine transcarbamylase mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with partial urea cycle disorders, which could be the basis of a new therapeutic approach.

LncRNA-H19 promotes hepatic lipogenesis by directly regulating miR-130a/PPARγ axis in non-alcoholic fatty liver disease.

Background: As one of the most common liver disorders worldwide, non-alcoholic fatty liver disease (NAFLD) begins with the abnormal accumulation of triglyceride (TG) in the liver. Long non-coding RNA-H19 was reported to modulate hepatic metabolic homeostasis in NAFLD. However, its molecular mechanism of NAFLD was not fully clear.Methods: In vitro and in vivo models of NAFLD were established by free fatty acid (FFA) treatment of hepatocytes and high-fat feeding mice, respectively. Hematoxylin and Eosin (H&E) and Oil-Red O staining detected liver tissue morphology and lipid accumulation. Immunohistochemistry (IHC) staining examined peroxisome proliferator-activated receptor γ (PPARγ) level in liver tissues. ELISA assay assessed TG secretion. Luciferase assay and RNA pull down were used to validate regulatory mechanism among H19, miR-130a and PPARγ. The gene expression in hepatocytes and liver tissues was detected by quantitative real-time PCR (qRT-PCR) and Western blotting.Results: H19 and PPARγ were up-regulated, while miR-130a was down-regulated in NAFLD mouse and cellular model. H&E and Oil-Red O staining indicated an increased lipid accumulation. Knockdown of H19 inhibited steatosis and TG secretion in FFA-induced hepatocytes. H19 could bind to miR-130a, and miR-130a could directly inhibit PPARγ expression. Meanwhile, miR-130a inhibited lipid accumulation by down-regulating NAFLD-related genes PPARγ, SREBP1, SCD1, ACC1 and FASN. Overexpression of miR-130a and PPARγ antagonist GW9662 inhibited lipogenesis and TG secretion, and PPARγ agonist GW1929 reversed this change induced by miR-130a up-regulation.Conclusion: Knockdown of H19 alleviated hepatic lipogenesis via directly regulating miR-130a/PPARγ axis, which is a novel mechanistic role of H19 in the regulation of NAFLD.

Guide to the Assessment of Mature Liver Gene Expression in Stem Cell-Derived Hepatocytes.

Differentiation of stem cells to hepatocyte-like cells (HLCs) holds great promise for basic research, drug and toxicological investigations, and clinical applications. There are currently no protocols for the production of HLCs from stem cells, such as embryonic stem cells or induced pluripotent stem cells, that produce fully mature hepatocytes with a wide range of mature hepatic functions. This report describes a standard method to assess the maturation of stem cell-derived HLCs with a moderately high-throughput format, by analysing liver gene expression by quantitative RT-qPCR. This method also provides a robust data set of the expression of 62 genes expressed in normal liver, generated from 17 fetal and 25 mature human livers, so that investigators can quickly and easily compare the expression of these genes in their stem cell-derived HLCs with the values obtained in authentic fetal and mature human liver. The simple methods described in this study will provide a quick and accurate assessment of the efficacy of a differentiation protocol and will help guide the optimization of differentiation conditions.

The survivin gene expression in neoplastic hepatocytes from chickens infected with Marek’s virus

P53 protein is one of the main proteins in apoptosis pathway. The role of survivin protein in inhibition of P53 in different human cancers has been proved. The expression of survivin can be a main marker in diagnosis and prognosis of different human cancer. Until now, the survivin gene expression in birds infected with Marek’s disease was not investigated. In this study, liver tissue samples of chickens infected with Marek’s disease virus (MDV) was collected. The identification of MDV was carried out with PCR and histopathologic examination. After identification of MDV, the pathogenicity of infected virus was investigated with specific primers targeted on 132 bp tandem repeat. After this, the survivin gene expression was examined in neoplastic liver samples by Real-Time PCR. The PCR amplification of tumor liver samples showed that all samples were infected with MDV. The result of histopathology and amplification of 132 bp tandem repeat in tumor samples showed that the chickens were infected to pathogenic MDV. Results showed that the survivin gene expression in neoplastic hepatocytes was significantly higher than normal hepatocytes. In conclusion, survivin gene expression can be utilized as a suitable biomarker in diagnosis of Marek’s disease in birds. It seems that viral meq oncogene in Marek’s disease can play role in induction of survivin expression in this disease that it is necessary to be proved.

Short communication: Effect of manipulating fatty acid profile on gluconeogenic gene expression in bovine primary hepatocytes

During the peripartum period, dairy cows experience both an increase in circulating fatty acid (FA) profile and a change in circulating FA profile, which have been shown to alter regulation of gluconeogenic genes. The objective was to quantify gene expression of key enzymes involved in gluconeogenesis and FA transport into the mitochondria in primary hepatocytes in response to exposure to an FA mixture mimicking what is circulating in a transition dairy cow with or without enrichment of C16:0, C18:0, and C18:1. Primary hepatocytes were isolated from 4 Holstein bull calves 3 d of age (± standard deviation 2 d) and cultured. Twenty-four hours after plating, treatments were applied to the cells for 24-h incubation. Treatments consisted of (1) control (1% BSA), (2) 0.75 mM FA cocktail (3% C14:0, 27% C16:0, 23% C18:0, 31% C18:1, 8% C18:2, and 8% C18:3 to mimic the FA profile of dairy cattle at calving), (3) 0.90 mM FA cocktail, (4) 0.75 mM FA cocktail + 0.15 mM C16:0, (5) 0.75 mM FA cocktail + 0.15 mM C18:0, and (6) 0.75 mM FA cocktail + 0.15 mM C18:1. After harvest in Trizol (Life Technologies, Carlsbad, CA), samples were stored at -80°C until RNA extraction, purification, and reverse transcription. Abundance of mRNA was measured using quantitative real-time PCR. Expression of genes of interest [carnitine palmitoyltransferase 1A, pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase] was calculated relative to the average abundance of 2 reference genes (ribosomal protein L32 and glyceraldehyde 3-phosphate dehydrogenase), which were the most stable out of 3 tested. Data were analyzed using PROC MIXED (SAS version 9.4; SAS Institute, Cary, NC) with the fixed effect of treatment and calf in the random statement. The addition of FA compared with the 1% BSA treatment increased the expression of carnitine palmitoyltransferase 1A and cytosolic PCK1. Enrichment with individual FA did not further regulate pyruvate carboxylase or PCK1 beyond that achieved by the basal profile. These results suggest that shifts in circulating FA profile within a biological range, without a difference in the total FA concentration, have minimal effects on transcriptional regulation of hepatic gluconeogenic genes in primary bovine hepatocytes.

Bone Morphogenetic Protein-8B Expression is Induced in Steatotic Hepatocytes and Promotes Hepatic Steatosis and Inflammation In Vitro

Non-alcoholic fatty liver disease (NAFLD) is considered to be the hepatic manifestation of the metabolic syndrome. The bone morphogenetic protein-8B (BMP8B) has been shown to be expressed in brown adipose tissues and the hypothalamus and to affect thermogenesis and susceptibility to diet-induced obesity. Here, we aimed to analyze BMP8B expression in NAFLD and to gain insight into BMP8B effects on pathophysiological steps of NAFLD progression. BMP8B mRNA and protein expression were dose-dependently induced in primary human hepatocytes in vitro upon incubation with fatty acids. Furthermore, hepatic BMP8B expression was significantly increased in a murine NAFLD model and in NAFLD patients compared with controls. Incubation with recombinant BMP8B further enhanced the fatty acid-induced cellular lipid accumulation as well as NFκB activation and pro-inflammatory gene expression in hepatocytes, while siRNA-mediated BMP8B depletion ameliorated these fatty acid-induced effects. Analysis of the expression of key factors of hepatocellular lipid transport and metabolisms indicated that BMP8B effects on fatty acid uptake as well as de novo lipogenesis contributed to hepatocellular accumulation of fatty acids leading to increased storage in the form of triglycerides and enhanced combustion by beta oxidation. In conclusion, our data indicate that BMP8B enhances different pathophysiological steps of NAFLD progression and suggest BMP8B as a promising prognostic marker and therapeutic target for NAFLD and, potentially, also for other chronic liver diseases.

SAT-183 Activating Transcription Factor 4 (ATF4) As A Possible Mediator Of Gluconeogenic And Lipogenic Actions Of Fat Mass And Obesity Associated (FTO) In Liver

Hepatic metabolism is critical for the maintenance of whole body glucose and lipid metabolism. Excessive hepatic glucose production and de novo lipogenesis contributes to fasting hyperglycemia in diabetes and hepatic steatosis, respectively. Common genetic variants of the fat mass and obesity associated (FTO) gene are strongly associated with obesity and type 2 diabetes. However, the role of FTO in the regulation of hepatic metabolism is not well understood. We have demonstrated that increased FTO level leads to an increase in gluconeogenic glucose-6-phosphatase (G6Pase) mRNA expression in hepatic cells. Conversely, reduced FTO level or inhibition of FTO activity caused a reduction of G6Pase mRNA expression. The transcription factor activating transcription factor 4 (ATF4) is a positive regulator of the expression of gluconeogenic and lipogenic genes in hepatocytes. These findings led to the hypothesis that increased hepatic FTO expression or activity leads to an increase expression of not only gluconeogenic genes but also lipogenic genes by activating ATF4, while reduced hepatic FTO expression or activity produces opposite effects. To address this hypothesis, we examined the effect of altered FTO expression on ATF4 and lipogenic stearoyl-CoA desaturase (SCD1) mRNA levels in alpha mouse liver 12 (AML12) immortalized hepatic cell line. We also treated AML12 cells with a small molecule FTO inhibitor rhein and measured the expression level of these genes. FTO overexpression resulted in increased ATF4 and SCD1 mRNA levels. FTO knockdown resulted in a reduction of SCD1 mRNA levels. Similarly, rhein treatment (1 μM) for 24 h reduced levels of ATF4 and SCD1 mRNA without causing cytotoxicity. These data support the hypothesis that FTO acts to shift hepatic metabolism toward anabolism, leading to increased gluconeogenesis and de novo lipogenesis and ATF4 is a possible mediator of this action. Our data also suggest that reduced activity of hepatic signaling pathways involving FTO and ATF4 may be effective in reversing hyperglycemic and hepatic steatosis partly by reducing gluconeogenesis and de novo lipogenesis in the liver. Sources of Research Support: Supported by University Research Grants Program (URGP) from University of Manitoba.

Spatial heterogeneity in the mammalian liver.

Hepatocytes operate in highly structured repeating anatomical units termed liver lobules. Blood flow along the lobule radial axis creates gradients of oxygen, nutrients and hormones, which, together with morphogenetic fields, give rise to a highly variable microenvironment. In line with this spatial variability, key liver functions are expressed non-uniformly across the lobules, a phenomenon termed zonation. Technologies based on single-cell transcriptomics have constructed a global spatial map of hepatocyte gene expression in mice revealing that ~50% of hepatocyte genes are expressed in a zonated manner. This broad spatial heterogeneity suggests that hepatocytes in different lobule zones might have not only different gene expression profiles but also distinct epigenetic features, regenerative capacities, susceptibilities to damage and other functional aspects. Here, we present genomic approaches for studying liver zonation, describe the principles of liver zonation and discuss the intrinsic and extrinsic factors that dictate zonation patterns. We also explore the challenges and solutions forobtaining zonation maps of liver non-parenchymal cells. These approaches facilitate globalcharacterization of liver function with high spatial resolution along physiological and pathological timescales.

Role of hepatocyte nuclear factor 4 alpha in cell proliferation and gemcitabine resistance in pancreatic adenocarcinoma

BackgroundHepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor that regulates the expression of numerous genes in hepatocytes and pancreatic β cells. HNF4α has been reported to affect cell proliferation and chemoresistance in several cancers. However, the role of HNF4α in pancreatic adenocarcinoma (PDAC) has not been studied extensively and remains unclear.MethodsBy utilizing immunohistochemical (IHC) staining, we measured the expression of HNF4α in PDAC tissues. By silencing HNF4α in PDAC cell lines, we assessed the impact of HNF4α on pancreatic cancer cell proliferation and gemcitabine sensitivity. We used CCK8 and colony formation assays to examine the effect of HNF4α on cell proliferation. A flow cytometry assay was used to assess cell apoptosis. The expression of gemcitabine-related genes was detected by quantitative real‑time PCR (qRT-PCR) and Western blotting. IHC was utilized to assess the correlation between HNF4α and human equilibrative nucleoside transporter 1 (hENT1) expression in PDAC patients. Chromatin immunoprecipitation (ChIP) and dual‑luciferase reporter assays were used to confirm that hENT1 is a target gene of HNF4α.ResultsIncreased HNF4α expression was detected in PDAC tissues; patients with higher HNF4α expression displayed worse prognosis. To elucidate the function of HNF4α, we examined its role in pancreatic cancer cell proliferation, apoptosis and gemcitabine resistance. In HNF4α-silenced Capan-1 and MiaPaCa-2 cells, we observed decreased cell proliferation and increased sensitivity to gemcitabine compared to those of controls. The mechanism of HNF4α in gemcitabine-related chemosensitivity was then explored. In response to HNF4α silencing, the expression levels of gemcitabine-related proteins, hENT1 and deoxycytidine kinase (dCK) were significantly increased. Additionally, hENT1 was negatively correlated with HNF4α in PDAC tissue samples. Moreover, we identified hENT1 as a downstream target of HNF4α.ConclusionHNF4α is a prognostic marker for overall survival, is required for pancreatic cancer cell proliferation and promotes resistance to gemcitabine by downregulating hENT1. Therefore, targeting HNF4α might reverse gemcitabine resistance and provide novel treatment strategies for PDAC.