Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.     

The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo

Both hemojuvelin (HJV) and bone morphogenic protein-6 (BMP6) are essential for hepcidin expression. Hepcidin is the key peptide hormone in iron homeostasis, and is secreted predominantly by hepatocytes. HJV expression is detected in hepatocytes, as well as in skeletal and heart muscle. HJV binds BMP6 and increases hepcidin expression presumably by acting as a BMP co-receptor. We characterized the role of hepatocyte HJV in the regulation of BMP6 and hepcidin expression. In HJV-null (Hjv^−/−) mice that have severe iron overload and marked suppression of hepcidin expression, we detected 4-fold higher hepatic BMP6 mRNA than in wild-type counterparts. These results indicate that Hjv^−/− mice do not lack BMP6. Furthermore, iron depletion in Hjv^−/− mice decreased hepatic BMP6 mRNA. Expression of HJV in hepatocytes of Hjv^−/− mice using an AAV2/8 vector, increased hepatic hepcidin mRNA by 65-fold and phosphorylated Smad1/5/8 in the liver by about 2.5-fold. However, no significant change in BMP6 mRNA was detected in either the liver or the small intestine of these animals. Our results revealed a close correlation of hepatic BMP6 mRNA expression with hepatic iron-loading. Together, our data indicate that the regulation of hepatic BMP6 expression by iron is independent of HJV, and that expression of HJV in hepatocytes plays an essential role in hepcidin expression by potentiating the BMP6-mediated signaling.     

Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP⁺) and AFP-nonproducing (AFP⁻) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP⁺ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP⁺ clones and likewise for adult hepatocytes and AFP⁻ clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, and ras-guanosine triphosphatase-activating protein, was observed in AFP⁺ clones, whereas the same proteins were not phosphorylated in AFP⁻ clones. We also observed that fetal hepatocytes and the AFP⁺ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP⁻ clones and, accordingly, that these AFP⁺ clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP⁺ clones was higher than that measured in cells of AFP⁻ clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP⁺ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.     

Thioesterase Superfamily Member 2 (Them2) and Phosphatidylcholine Transfer Protein (PC-TP) Interact To Promote Fatty Acid Oxidation and Control Glucose Utilization

Thioesterase superfamily member 2 (Them2) is a mitochondrion-associated long-chain fatty acyl coenzyme A (CoA) thioesterase that is highly expressed in the liver and oxidative tissues. Them2 activity in vitro is increased when it interacts with phosphatidylcholine transfer protein (PC-TP), a cytosolic lipid binding protein. Them2^−/− and Pctp^−/− mice exhibit enhanced hepatic insulin sensitivity and increased adaptive thermogenesis, and Them2^−/− mice are also resistant to diet-induced hepatic steatosis. Although we showed previously that a Them2–PC-TP complex suppresses insulin signaling, the enzymatic activity of Them2 suggests additional direct involvement in regulating hepatic nutrient homeostasis. Here we used cultured primary hepatocytes to elucidate biochemical and cellular mechanisms by which Them2 and PC-TP regulate lipid and glucose metabolism. Under conditions simulating fasting, Them2^−/− and Pctp^−/− hepatocytes each exhibited decreased rates of fatty acid oxidation and gluconeogenesis. In results indicative of Them2-dependent regulation by PC-TP, chemical inhibition of PC-TP failed to reproduce these changes in Them2^−/− hepatocytes. In contrast, rates of glucose oxidation and lipogenesis in the presence of high glucose concentrations were decreased only in Them2^−/− hepatocytes. These findings reveal a primary role for Them2 in promoting mitochondrial oxidation of fatty acids and glucose in the liver.     

Lipid Metabolism,Oxidative Stress and Cell Death Are Regulated by PKC Delta in a Dietary Model of Nonalcoholic Steatohepatitis

Steatosis, oxidative stress, and apoptosis underlie the development of nonalcoholic steatohepatitis (NASH). Protein kinase C delta (PKCδ) has been implicated in fatty liver disease and is activated in the methionine and choline-deficient (MCD) diet model of NASH, yet its pathophysiological importance towards steatohepatitis progression is uncertain. We therefore addressed the role of PKCδ in the development of steatosis, inflammation, oxidative stress, apoptosis, and fibrosis in an animal model of NASH. We fed PKCδ^−/− mice and wildtype littermates a control or MCD diet. PKCδ^−/− primary hepatocytes were used to evaluate the direct effects of fatty acids on hepatocyte lipid metabolism gene expression. A reduction in hepatic steatosis and triglyceride levels were observed between wildtype and PKCδ^−/− mice fed the MCD diet. The hepatic expression of key regulators of β-oxidation and plasma triglyceride metabolism was significantly reduced in PKCδ^−/− mice and changes in serum triglyceride were blocked in PKCδ^−/− mice. MCD diet-induced hepatic oxidative stress and hepatocyte apoptosis were reduced in PKCδ^−/− mice. MCD diet-induced NADPH oxidase activity and p47^phox membrane translocation were blunted and blocked, respectively, in PKCδ^−/− mice. Expression of pro-apoptotic genes and caspase 3 and 9 cleavage in the liver of MCD diet fed PKCδ^−/− mice were blunted and blocked, respectively. Surprisingly, no differences in MCD diet-induced fibrosis or pro-fibrotic gene expression were observed in 8 week MCD diet fed PKCδ^−/− mice. Our results suggest that PKCδ plays a role in key pathological features of fatty liver disease but not ultimately in fibrosis in the MCD diet model of NASH.     

PPARδ activation attenuates hepatic steatosis in Ldlr?/? mice by enhanced fat oxidation,reduced lipogenesis,and improved insulin sensitivity

PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr^−/− mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA β-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKβ1^−/− hepatocytes. However, FA oxidation was only partially reduced in AMPKβ1^−/− hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKβ1^−/− hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.     

Osteopontin deficiency aggravates hepatic injury induced by ischemia–reperfusion in mice

Osteopontin (OPN) is a multifunctional protein involved in hepatic steatosis, inflammation, fibrosis and cancer progression. However, its role in hepatic injury induced by ischemia–reperfusion (I–R) has not yet been investigated. We show here that hepatic warm ischemia for 45 min followed by reperfusion for 4 h induced the upregulation of the hepatic and systemic level of OPN in mice. Plasma aspartate aminotransferase and alanine aminotransferase levels were strongly increased in Opn^−/− mice compared with wild-type (Wt) mice after I–R, and histological analysis of the liver revealed a significantly higher incidence of necrosis of hepatocytes. In addition, the expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNFα), interleukin 6 (IL6) and interferon-γ were strongly upregulated in Opn^−/− mice versus Wt mice after I–R. One explanation for these responses could be the vulnerability of the OPN-deficient hepatocyte. Indeed, the downregulation of OPN in primary and AML12 hepatocytes decreased cell viability in the basal state and sensitized AML12 hepatocytes to cell death induced by oxygen–glucose deprivation and TNFα. Further, the downregulation of OPN in AML12 hepatocytes caused a strong decrease in the expression of anti-apoptotic Bcl2 and in the ATP level. The hepatic expression of Bcl2 also decreased in Opn^−/− mice versus Wt mice livers after I–R. Another explanation could be the regulation of the macrophage activity by OPN. In RAW macrophages, the downregulation of OPN enhanced iNOS expression in the basal state and sensitized macrophages to inflammatory signals, as evaluated by the upregulation of iNOS, TNFα and IL6 in response to lipopolysaccharide. In conclusion, OPN partially protects from hepatic injury and inflammation induced in this experimental model of liver I–R. This could be due to its ability to partially prevent death of hepatocytes and to limit the production of toxic iNOS-derived NO by macrophages.     

Hepatic Gluconeogenesis Is Enhanced by Phosphatidic Acid Which Remains Uninhibited by Insulin in Lipodystrophic Agpat2?/? Mice

In this study we examined the role of phosphatidic acid (PA) in hepatic glucose production (HGP) and development of hepatic insulin resistance in mice that lack 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2). Liver lysophosphatidic acid and PA levels were increased ∼2- and ∼5-fold, respectively, in male Agpat2^−/− mice compared with wild type mice. In the absence of AGPAT2, the liver can synthesize PAs by activating diacylglycerol kinase or phospholipase D, both of which were elevated in the livers of Agpat2^−/− mice. We found that PAs C16:0/18:1 and C18:1/20:4 enhanced HGP in primary WT hepatocytes, an effect that was further enhanced in primary hepatocytes from Agpat2^−/− mice. Lysophosphatidic acids C16:0 and C18:1 failed to increase HGP in primary hepatocytes. The activation of HGP was accompanied by an up-regulation of the key gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This activation was suppressed by insulin in the WT primary hepatocytes but not in the Agpat2^−/− primary hepatocytes. Thus, the lack of normal insulin signaling in Agpat2^−/− livers allows unrestricted PA-induced gluconeogenesis significantly contributing to the development of hyperglycemia in these mice.     

Evaluation of Antiviral Efficacy of Ribavirin,Arbidol, and T-705 (Favipiravir) in a Mouse Model for Crimean-Congo Hemorrhagic Fever

Background

Mice lacking the type I interferon receptor (IFNAR^−/− mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR^−/− mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus.

Methodology/Principal Findings

CCHF virus-infected IFNAR^−/− mice died 2–6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC₅₀ 0.6–2.8 µg/ml; IC₉₀ 1.2–4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR^−/− mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects.

Conclusions/Significance

Activated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin.     

Glycerol-3-phosphate Acyltransferase (GPAT)-1, but Not GPAT4, Incorporates Newly Synthesized Fatty Acids into Triacylglycerol and Diminishes Fatty Acid Oxidation

Four glycerol-3-phosphate acyltransferase (GPAT) isoforms, each encoded by a separate gene, catalyze the initial step in glycerolipid synthesis; in liver, the major isoforms are GPAT1 and GPAT4. To determine whether each of these hepatic isoforms performs a unique function in the metabolism of fatty acid, we measured the incorporation of de novo synthesized fatty acid or exogenous fatty acid into complex lipids in primary mouse hepatocytes from control, Gpat1^−/−, and Gpat4^−/− mice. Although hepatocytes from each genotype incorporated a similar amount of exogenous fatty acid into triacylglycerol (TAG), only control and Gpat4^−/− hepatocytes were able to incorporate de novo synthesized fatty acid into TAG. When compared with controls, Gpat1^−/− hepatocytes oxidized twice as much exogenous fatty acid. To confirm these findings and to assess hepatic β-oxidation metabolites, we measured acylcarnitines in liver from mice after a 24-h fast and after a 24-h fast followed by 48 h of refeeding with a high sucrose diet to promote lipogenesis. Confirming the in vitro findings, the hepatic content of long-chain acylcarnitine in fasted Gpat1^−/− mice was 3-fold higher than in controls. When compared with control and Gpat4^−/− mice, after the fasting-refeeding protocol, Gpat1^−/− hepatic TAG was depleted, and long-chain acylcarnitine content was 3.5-fold higher. Taken together, these data demonstrate that GPAT1, but not GPAT4, is required to incorporate de novo synthesized fatty acids into TAG and to divert them away from oxidation.     

Single Liver Lobe Repopulation with Wildtype Hepatocytes Using Regional Hepatic Irradiation Cures Jaundice in Gunn Rats

Background and Aims

Preparative hepatic irradiation (HIR), together with mitotic stimulation of hepatocytes, permits extensive hepatic repopulation by transplanted hepatocytes in rats and mice. However, whole liver HIR is associated with radiation-induced liver disease (RILD), which limits its potential therapeutic application. In clinical experience, restricting HIR to a fraction of the liver reduces the susceptibility to RILD. Here we test the hypothesis that repopulation of selected liver lobes by regional HIR should be sufficient to correct some inherited metabolic disorders.

Methods

Hepatocytes (10⁷) isolated from wildtype F344 rats or Wistar-RHA rats were engrafted into the livers of congeneic dipeptidylpeptidase IV deficient (DPPIV⁻) rats or uridinediphosphoglucuronateglucuronosyltransferase-1A1-deficient jaundiced Gunn rats respectively by intrasplenic injection 24 hr after HIR (50 Gy) targeted to the median lobe, or median plus left liver lobes. An adenovector expressing hepatocyte growth factor (10¹¹ particles) was injected intravenously 24 hr after transplantation.

Results

Three months after hepatocyte transplantation in DPPIV⁻ rats, 30–60% of the recipient hepatocytes were replaced by donor cells in the irradiated lobe, but not in the nonirradiated lobes. In Gunn rats receiving median lobe HIR, serum bilirubin declined from pretreatment levels of 5.17±0.78 mg/dl to 0.96±0.30 mg/dl in 8 weeks and remained at this level throughout the 16 week observation period. A similar effect was observed in the group, receiving median plus left lobe irradiation.

Conclusions

As little as 20% repopulation of 30% of the liver volume was sufficient to correct hyperbilirubinemia in Gunn rats, highlighting the potential of regiospecific HIR in hepatocyte transplantation-based therapy of inherited metabolic liver diseases.     

TNFR1 determines progression of chronic liver injury in the IKKγ/Nemo genetic model

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral and alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury, as well as cancer. Deletion of NF-κB essential modulator in hepatocytes (IKKγ/Nemo) causes spontaneous progression of TNF-mediated chronic hepatitis to hepatocellular carcinoma (HCC). Thus, we analyzed the role of death receptors including TNFR1 and TRAIL in the regulation of cell death and the progression of liver injury in IKKγ/Nemo-deleted livers. We crossed hepatocyte-specific IKKγ/Nemo knockout mice (Nemo^Δhepa) with constitutive TNFR1^−/− and TRAIL^−/− mice. Deletion of TNFR1, but not TRAIL, decreased apoptotic cell death, compensatory proliferation, liver fibrogenesis, infiltration of immune cells as well as pro-inflammatory cytokines, and indicators of tumor growth during the progression of chronic liver injury. These events were associated with diminished JNK activation. In contrast, deletion of TNFR1 in bone-marrow-derived cells promoted chronic liver injury. Our data demonstrate that TNF- and not TRAIL signaling determines the progression of IKKγ/Nemo-dependent chronic hepatitis. Additionally, we show that TNFR1 in hepatocytes and immune cells have different roles in chronic liver injury–a finding that has direct implications for treating chronic liver disease.     

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13^highCD133⁺ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.     

Integrin α1-null Mice Exhibit Improved Fatty Liver When Fed a High Fat Diet Despite Severe Hepatic Insulin Resistance

Hepatic insulin resistance is associated with increased collagen. Integrin α1β1 is a collagen-binding receptor expressed on hepatocytes. Here, we show that expression of the α1 subunit is increased in hepatocytes isolated from high fat (HF)-fed mice. To determine whether the integrin α1 subunit protects against impairments in hepatic glucose metabolism, we analyzed glucose tolerance and insulin sensitivity in HF-fed integrin α1-null (itga1^−/−) and wild-type (itga1^+/+) littermates. Using the insulin clamp, we found that insulin-stimulated hepatic glucose production was suppressed by ∼50% in HF-fed itga1^+/+ mice. In contrast, it was not suppressed in HF-fed itga1^−/− mice, indicating severe hepatic insulin resistance. This was associated with decreased hepatic insulin signaling in HF-fed itga1^−/− mice. Interestingly, hepatic triglyceride and diglyceride contents were normalized to chow-fed levels in HF-fed itga1^−/− mice. This indicates that hepatic steatosis is dissociated from insulin resistance in HF-fed itga1^−/− mice. The decrease in hepatic lipid accumulation in HF-fed itga1^−/− mice was associated with altered free fatty acid metabolism. These studies establish a role for integrin signaling in facilitating hepatic insulin action while promoting lipid accumulation in mice challenged with a HF diet.