Wild-type Hepatocytes Research Articles

Therapeutic liver cell transplantation to treat murine PKU.

For gene therapy of the liver, in vivo applications based on adeno-associated virus are the most advanced vectors despite limitations, including low efficacy and episomal loss, potential integration and safety issues, and high production costs. Alternative vectors and/or delivery routes are of high interest. The regenerative ability of the liver bears the potential for ex vivo therapy using liver cell transplantation for disease correction if provided with a selective advantage to expand and replace the existing cell mass. Here we present such treatment of a mouse model of human phenylketonuria (PKU). Primary hepatocytes from wild-type mice were gene modified in vitro (with a lentiviral vector) that carries a gene editing system (CRISPR) to inhibit Cypor. Cypor inactivation confers paracetamol (or acetaminophen) resistance to hepatocytes and thus a growth advantage to eliminate the pre-existing liver cells upon grafting (via the spleen) and exposure to repeated treatment with paracetamol. Grafting Cypor-inactivated wild-type hepatocytes into inbred young adult enu2 (PKU) mice, followed by selective expansion by paracetamol dosing, resulted in replacing up to 5% of cell mass, normalization of blood phenylalanine, and permanent correction of PKU. Hepatocyte transplantation offers thus an armamentarium of novel therapy options for genetic liver defects.

Intertwined roles for GDF-15, HMGB1, and MIG/CXCL9 in Pediatric Acute Liver Failure

IntroductionPediatric Acute Liver Failure (PALF) presents as a rapidly evolving, multifaceted, and devastating clinical syndrome whose precise etiology remains incompletely understood. Consequently, predicting outcomes—whether survival or mortality—and informing liver transplantation decisions in PALF remain challenging. We have previously implicated High-Mobility Group Box 1 (HMGB1) as a central mediator in PALF-associated dynamic inflammation networks that could be recapitulated in acetaminophen (APAP)-treated mouse hepatocytes (HC) in vitro. Here, we hypothesized that Growth/Differentiation Factor-15 (GDF-15) is involved along with HMGB1 in PALF.Methods28 and 23 inflammatory mediators including HMGB1 and GDF15 were measured in serum samples from PALF patients and cell supernatants from wild-type (C57BL/6) mouse hepatocytes (HC) and from cells from HC-specific HMGB1-null mice (HC-HMGB1−/−) exposed to APAP, respectively. Results were analyzed computationally to define statistically significant and potential causal relationships.ResultsCirculating GDF-15 was elevated significantly (P < 0.05) in PALF non-survivors as compared to survivors, and together with HMGB1 was identified as a central node in dynamic inflammatory networks in both PALF patients and mouse HC. This analysis also pointed to MIG/CXCL9 as a differential node linking HMGB1 and GDF-15 in survivors but not in non-survivors, and, when combined with in vitro studies, suggested that MIG suppresses GDF-15-induced inflammation.DiscussionThis study suggests GDF-15 as a novel PALF outcome biomarker, posits GDF-15 alongside HMGB1 as a central node within the intricate web of systemic inflammation dynamics in PALF, and infers a novel, negative regulatory role for MIG.

Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50–3000 ppm fluxapyroxad for 7 days and 250–3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250–3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1–100 μM fluxapyroxad or 500 μM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid β-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1–100 μM fluxapyroxad or 500 μM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1–16 μM fluxapyroxad or 500 μM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

Tezacaftor is a direct inhibitor of sphingolipid delta-4 desaturase enzyme (DEGS)

BackgroundWe recently demonstrated that 48 h exposure of primary human bronchial epithelial (hBE) cells, obtained from both CF (F508del homozygous) and non-CF subjects, to the triple drug combination Elexacaftor/Tezacaftor/Ivacaftor (ETI) results in a CFTR genotype-independent modulation of the de novo synthethic pathway of sphingolipids, with an accumulation of dihydroceramides (dHCer). Since dHCer are converted into ceramides (Cer) by the action of a delta-4 sphingolipid desaturase (DEGS) enzyme, we aimed to better understand this off-target effect of ETI (i.e., not related to CFTR rescue) MethodshBE cells, both F508del and wild-type, were cultured to create fully differentiated bronchial epithelia. We analyzed Cer and dHCer using an LC-MS based method previously developed by our lab. DEGS expression levels in differentiated hBE cells lysates were quantified by western blot analysis. ResultsWe demonstrated that 1) dHCer accumulate in hBE with time following prolonged ETI exposure, that 2) similar inhibition occurs in wild-type primary human hepatocytes and that 3) this does not result in an alteration of DEGS expression. We then proved that 4) ETI is a direct inhibitor of DEGS, that 5) Tezacaftor is the molecule responsible for this effect, that 6) the inhibition is concentration dependent. Finally, after repeated oral administration of ETI to naïve, non-CF, mice, we observed a slight accumulation of dHCer in the brain. ConclusionsWe believe that further investigations on Tezacaftor should be envisaged, particularly for the use of ETI during pregnancy, breastfeeding and in the early stages of development. DEGS dysfunction and dHCer accumulation causes impairment in the development of the nervous system, due to a derangement in myelin formation and maintenance.

Comparison of wild-type and high-risk PNPLA3 variants in a human biomimetic liver microphysiology system for metabolic dysfunction-associated steatotic liver disease precision therapy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a worldwide health epidemic with a global occurrence of approximately 30%. The pathogenesis of MASLD is a complex, multisystem disorder driven by multiple factors, including genetics, lifestyle, and the environment. Patient heterogeneity presents challenges in developing MASLD therapeutics, creating patient cohorts for clinical trials, and optimizing therapeutic strategies for specific patient cohorts. Implementing pre-clinical experimental models for drug development creates a significant challenge as simple in vitro systems and animal models do not fully recapitulate critical steps in the pathogenesis and the complexity of MASLD progression. To address this, we implemented a precision medicine strategy that couples the use of our liver acinus microphysiology system (LAMPS) constructed with patient-derived primary cells. We investigated the MASLD-associated genetic variant patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 (I148M variant) in primary hepatocytes as it is associated with MASLD progression. We constructed the LAMPS with genotyped wild-type and variant PNPLA3 hepatocytes, together with key non-parenchymal cells, and quantified the reproducibility of the model. We altered media components to mimic blood chemistries, including insulin, glucose, free fatty acids, and immune-activating molecules to reflect normal fasting (NF), early metabolic syndrome (EMS), and late metabolic syndrome (LMS) conditions. Finally, we investigated the response to treatment with resmetirom, an approved drug for metabolic syndrome-associated steatohepatitis (MASH), the progressive form of MASLD. This study, using primary cells, serves as a benchmark for studies using "patient biomimetic twins" constructed with patient induced pluripotent stem cell (iPSC)-derived liver cells using a panel of reproducible metrics. We observed increased steatosis, immune activation, stellate cell activation, and secretion of pro-fibrotic markers in the PNPLA3 GG variant compared to the wild-type CC LAMPS, consistent with the clinical characterization of this variant. We also observed greater resmetirom efficacy in the PNPLA3 wild-type CC LAMPS compared to the GG variant in multiple MASLD metrics, including steatosis, stellate cell activation, and the secretion of pro-fibrotic markers. In conclusion, our study demonstrates the capability of the LAMPS platform for the development of MASLD precision therapeutics, enrichment of patient cohorts for clinical trials, and optimization of therapeutic strategies for patient subgroups with different clinical traits and disease stages.

The mitochondrial calcium uniporter mediates mitochondrial Fe2+ uptake and hepatotoxicity after acetaminophen

Acetaminophen (APAP) overdose disrupts hepatocellular lysosomes, which release ferrous iron (Fe2+) that translocates into mitochondria putatively via the mitochondrial calcium uniporter (MCU) to induce oxidative/nitrative stress, the mitochondrial permeability transition (MPT), and hepatotoxicity. To investigate how MCU deficiency affects mitochondrial Fe2+ uptake and hepatotoxicity after APAP overdose, global MCU knockout (KO), hepatocyte specific (hs) MCU KO, and wildtype (WT) mice were treated with an overdose of APAP both in vivo and in vitro. Compared to strain-specific WT mice, serum ALT decreased by 88 and 56%, respectively, in global and hsMCU KO mice at 24 h after APAP (300 mg/kg). Hepatic necrosis also decreased by 84 and 56%. By contrast, when MCU was knocked out in Kupffer cells, ALT release and necrosis were unchanged after overdose APAP. Intravital multiphoton microscopy confirmed loss of viability and mitochondrial depolarization in pericentral hepatocytes of WT mice, which was decreased in MCU KO mice. CYP2E1 expression, hepatic APAP-protein adduct formation, and JNK activation revealed that APAP metabolism was equivalent between WT and MCU KO mice. In cultured hepatocytes after APAP, loss of cell viability decreased in hsMCU KO compared to WT hepatocytes. Using fructose plus glycine to prevent cell killing, mitochondrial Fe2+ increased progressively after APAP, as revealed with mitoferrofluor (MFF), a mitochondrial Fe2+ indicator. By contrast in hsMCU KO hepatocytes, mitochondrial Fe2+ uptake after APAP was suppressed. Rhod-2 measurements showed that Ca2+ did not increase in mitochondria after APAP in either WT or KO hepatocytes. In conclusion, MCU mediates uptake of Fe2+ into mitochondria after APAP and plays a central role in mitochondrial depolarization and cell death during APAP-induced hepatotoxicity.

Tau Loss of Function, by Deletion or Aggregation, Contributes to Peripheral Insulin Resistance.

Several epidemiological data revealed an association between Alzheimer's disease (AD) and type 2 diabetes. Researchers concentrated on brain insulin resistance with little emphasis on the link between systemic insulin resistance and AD, despite the fact that the incidence of type 2 diabetes is higher in AD patients and that impairment in insulin signaling is a risk factor for AD. The goal of this study is to determine the role of systemic insulin resistance in the pathogenesis of Alzheimer's disease by evaluating the consequences of tau loss-of-function on peripheral insulin sensitivity. Primary hepatocytes isolated from transgenic mouse models (Tau KO, P301 L) and wild type mice (C57BL/6) were evaluated for their insulin sensitivity using glucose uptake assays as well as biochemical analysis of insulin signaling markers. Our data show that tau deletion or loss of function promotes peripheral insulin resistance as seen in primary hepatocytes isolated from Tau KO and P301 L mice, respectively. Furthermore, exposure of wild-type primary hepatocytes to sub-toxic concentrations of tau oligomers results in a dose-dependent inhibition of glucose uptake, associated with downregulation of insulin signaling. Tau oligomers-induced inactivation of insulin signaling proteins was rescued by inhibition of p38 MAPK, suggesting the involvement of p38 MAPK. This is the first study testing tau role in peripheral insulin resistance at the cellular level using multiple transgenic mouse models. Moreover, this study suggests that tau should be functional for insulin sensitivity, therefore, any loss of function by deletion or aggregation would result in insulin resistance.

Ablation of Dual-Specificity Phosphatase 6 Protects against Nonalcoholic Fatty Liver Disease via Cytochrome P450 4A and Mitogen-Activated Protein Kinase

Dual-specificity phosphatase 6 (DUSP6) is a specific phosphatase for mitogen-activated protein kinase (MAPK). This study used a high-fat diet (HFD)-induced murine nonalcoholic fatty liver disease model to investigate the role of DUSP6 in this disease. Wild-type (WT) and Dusp6-haploinsufficiency mice developed severe obesity and liver pathology consistent with nonalcoholic fatty liver disease when exposed to HFD. In contrast, Dusp6-knockout (KO) mice completely eliminated these phenotypes. Furthermore, primary hepatocytes isolated from WT mice exposed to palmitic and oleic acids exhibited abundant intracellular lipid accumulation, whereas hepatocytes from Dusp6-KO mice showed minimal lipid accumulation. Transcriptome analysis revealed significant down-regulation of genes encoding cytochrome P450 4A (CYP4A), known to promote ω-hydroxylation of fatty acids and hepatic steatosis, in Dusp6-KO hepatocytes compared with that in WT hepatocytes. Diminished CYP4A expression was observed in the liver of Dusp6-KO mice compared with WT and Dusp6-haploinsufficiency mice. Knockdown of DUSP6 in HepG2, a human liver-lineage cell line, also promoted a reduction of lipid accumulation, down-regulation of CYP4A, and up-regulation of phosphorylated/activated MAPK. Furthermore, inhibition of MAPK activity promoted lipid accumulation in DUSP6-knockdown HepG2 cells without affecting CYP4A expression, indicating that CYP4A expression is independent of MAPK activation. These findings highlight the significant role of DUSP6 in HFD-induced steatohepatitis through two distinct pathways involving CYP4A and MAPK.

Hepatocyte FoxO1 Deficiency Protects From Liver Fibrosis via Reducing Inflammation and TGF-β1-mediated HSC Activation

The O-class of the forkhead transcription factor FoxO1 is a crucial factor mediating insulin→PI3K→Akt signaling and governs diverse cellular processes. However, the role of hepatocyte FoxO1 in liver fibrosis has not been well-established. In his study, we investigated the role of hepatocyte FoxO1 in liver fibrosis and uncovered the underlying mechanisms. Liver fibrosis was established by carbon tetrachloride (CCL4) administration and compared between liver-specific deletion of FoxO1 deletion (F1KO) and control (CNTR) mice. Using genetic and bioinformatic strategies invitro and invivo, the role of hepatic FoxO1 in liver fibrosis and associated mechanisms was established. Increased FoxO1 expression and FoxO1 signalingactivation were observed in CCL4-induced fibrosis. Hepatic FoxO1 deletion largely attenuated CCL4-induced liver injury and fibrosis compared with CNTR mice. F1KOmice showed ameliorated CCL4-induced hepatic inflammation and decreased TGF-β1 mRNA and protein levels compared with those of CNTR mice. In primary hepatocytes, FoxO1 deficiency reduced TGF-β1 expression andsecretion. Conditioned medium (CM) collected from wild-type hepatocytes treated with CCL4 activated humanHSC cell line (LX-2); such effect was attenuated byFoxO1 deletion in primary hepatocytes or neutralization of TGF-β1 in the CM using TGF-β1 antibody. Hepatic FoxO1overexpression in CNTR mice promoted CCL4-induced HSC activation; such effect was blocked in L-TGF-β1KO mice. Hepatic FoxO1 mediates CCL4-inducled liver fibrosis via upregulating hepatocyte TGF-β1 expression, stimulating hepatic inflammation and TGF-β1-mediated HSC activation. Hepatic FoxO1 may be a therapeutic target for prevention and treatment of liver fibrosis.

Hepatocyte ABCA1 deficiency is associated with reduced HDL sphingolipids.

ATP binding cassette transporter A1 (ABCA1) limits the formation of high density lipoproteins (HDL) as genetic loss of ABCA1 function causes virtual HDL deficiency in patients with Tangier disease. Mice with a hepatocyte-specific ABCA1 knockout (Abca1 HSKO) have 20% of wild type (WT) plasma HDL-cholesterol levels, suggesting a major contribution of hepatic ABCA1 to the HDL phenotype. Whether plasma sphingolipids are reduced in Tangier disease and to what extent hepatic ABCA1 contributes to plasma sphingolipid (SL) levels is unknown. Here, we report a drastic reduction of total SL levels in plasma of a Tangier patient with compound heterozygosity for mutations in ABCA1. Compared to mutation-free controls, heterozygous mutations in ABCA1 had no significant effect on total SLs in plasma; however, apoB-depleted plasma showed a reduction in total SL also in het carriers. Similarly, liver specific Abca1 KO mice (Abca1 HSKO) showed reduced total sphingolipids in plasma and liver. In parallel, apoM and sphingosine-1-phosphate (S1P) levels were reduced in plasma of Abca1 HSKO mice. Primary hepatocytes from Abca1 HSKO mice showed a modest, but significant reduction in total SLs concentration compared to WT hepatocytes, although SL de novo synthesis and secretion were slightly increased in Abca1 HSKO hepatocytes. We conclude that hepatic ABCA1 is a signficant contributor to maintaining total plasma pool of HDL sphingolipids, including sphingomyelins and S1P.

1596-P: Homozygous Mutations in MFN2 Aggravated Fat Deposition in Mouse Hepatocytes

Objective: Studies had found that homozygous mutations at p.R707W in the mitofusin-2 (MFN2) gene cause severe lipids metabolic abnormalities, and the mechanism has not been elucidated. In this study, we aim to explore the effect of the mutant protein on hepatocyte lipids deposition. Methods: Primary hepatocytes of wild-type (WT), heterozygous and homozygous mutation were isolated from 8 weeks old mice. Hepatocytes were treated with 600μmol/L free fat acid (FFA) of oleic acid/palmitic acid (2︰1) mixture for 12 hours to establish a NAFLD model in vitro. The lipid accumulation of hepatocytes was observed by Oil red O staining and the contents of triglyceride (TG). Results: Mutant mice carrying a novel p.R707W (CGA to TGG) mutation and silent mutation (p.D708=(GAT to GAC)) were identified (Fig. 1A). The results of Oil Red O staining and TG content measurement before FFA treating showed that more lipid was deposited in the homozygous hepatocyte than in WT cells (Fig. 1B, Fig. 1C) (*P<0.05), revealing that different genotypes of mice already have varied liver lipid contents at 8 weeks. After stimulation with FFA, TG content and lipidosis area in homozygous hepatocytes were significantly higher than those in WT (**P<0.01). And there was no significant difference between heterozygote and WT hepatocytes with or without FFA stimulation. Conclusion: Homozygous mutation at p.R707W in MFN2 aggravated lipid deposition in mouse. Disclosure Y.Yu: None. Y.Luo: None. X.Zhou: None. L.Ji: Other Relationship; Eli Lilly and Company, Novo Nordisk, Merck & Co., Inc., Bayer Inc., Sanofi-Aventis U.S., Roche Pharmaceuticals, MSD Life Science Foundation, AstraZeneca, Boehringer Ingelheim Inc., Abbott, Metronics. Funding China International Medical Foundation (2021-N-03)

Abstract 2559: Loss of AKT2 correlates with activation of β-catenin signaling in immortalized mouse hepatocytes

Abstract Background: Primary liver cancer is the second most common cause of cancer-related mortality. Abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/AKT and Wnt/β-catenin signaling pathways, which act as key regulators in cell proliferation and growth, is frequently observed in liver cancer. This study investigated the role of AKT2, the major AKT isoform expressed in the liver, on the activation of β-catenin signaling in immortalized mouse hepatocytes. Methods: Regulation of β-catenin signaling by AKT2 was examined in hepatocytes lacking the negative regulator of PI3K/AKT signaling, PTEN (phosphatase and tensin homolog deleted on chromosome 10). Immortalized hepatocytes were established from mice with liver specific deletions of Pten alone (PtenloxP/loxP; albumin [Alb]-Cre+) or in combination with Akt2 (PtenloxP/loxP; Akt2-/-; Alb-Cre+). Control mice were PtenloxP/loxP; Alb-Cre-. Immunoblotting was used to assess protein expression of AKT and β-catenin. Gene expression of β-catenin targets cyclin D1, CD44, and Jag1 was determined with quantitative PCR. Cell growth was evaluated with hemacytometer count. Results: Pharmacological inhibition of PI3K using LY294002 resulted in decreased phosphorylation levels of AKT (Ser473 and Thr308) and β-catenin (Ser552), suggesting crosstalk between the PI3K/AKT and β-catenin pathways. When compared to wild-type hepatocytes, Pten null and Pten;Akt2 double mutant (DM) expressed significantly higher protein levels of β-catenin (p≤0.0327), with no difference observed in Pten null vs. Pten;Akt2 double mutant groups (p>0.05). Phosphorylation of β-catenin at Ser552, a residue that has been previously shown to be phosphorylated by AKT, was also significantly enhanced in both Pten null and Pten;Akt2 double mutant groups, when compared to wild-type cells. Enhanced phosphorylation of β-catenin in these groups correlated with increased expression of β-catenin transcriptional targets CD44 and Jag1 (p≤0.0041), with no significant difference in mRNA levels of these targets observed in Pten null vs. DM group (p>0.05). Activation of the β-catenin signaling pathway in Pten null and DM groups correlated with enhanced cell growth and significantly increased mRNA levels of cyclin D1 (p<0.0001). Interestingly, when compared to the control group, DM group showed enhanced expression of Akt1 and total phosphorylated AKT. Conclusions: Pten loss alone or accompanied with Akt2 deletion resulted in enhanced activation of β-catenin signaling and cell proliferation. These changes correlated with increased expression of AKT1 and total phosphorylated AKT in the Pten;Akt2 DM group. These findings suggest the need to further evaluate the role of AKT isoforms in the PI3K/AKT/β-catenin signaling axis and a promise for evaluation of isoform-specific inhibitors targeted at AKT to block tumor growth. Citation Format: Ielyzaveta Slarve, Bangyan Stiles, Lina He. Loss of AKT2 correlates with activation of β-catenin signaling in immortalized mouse hepatocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2559.

MiR-194 Up-Regulates Cholesterol 7 Alpha-Hydroxylase Expression via β-Catenin Signaling and Aggravates Cholestatic Liver Diseases

miR-194 is abundantly expressed in hepatocytes, and its depletion increases hepatic resistance to acetaminophen-induced acute injuries. In this study, the biological role of miR-194 in cholestatic liver injury was investigated by using miR-194/miR-192 cluster liver-specific knockout (LKO) mice, in which no liver injuries or metabolic disorders were predisposed. Bile duct ligation (BDL) and 1-naphthyl isothiocyanate (ANIT) were applied to LKO and matched control wild-type (WT) mice to induce hepatic cholestasis. Periportal liver damage, mortality rate, and liver injury biomarkers in LKO mice were significantly less than in WT mice after BDL and ANIT injection. Intrahepatic bile acid level was significantly lower in the LKO liver within 48 hours of BDL- and ANIT-induced cholestasis compared with WT. Western blot analysis showed that β-catenin (CTNNB1) signaling and genes involved in cellular proliferation were activated in BDL- and ANIT-treated mice. The expression levels of cholesterol 7 alpha-hydroxylase (CYP7A1), pivotal in bile synthesis, and its upstream regulator hepatocyte nuclear factor 4α were reduced in primary LKO hepatocytes and liver tissues compared with WT. The knockdown of miR-194 using miRNA inhibitors reduced CYP7A1 expression in WT hepatocytes. In contrast, the knockdown of CTNNB1 and overexpression of miR-194, but not miR-192, in LKO hepatocytes and AML12 cells increased CYP7A1 expression. In conclusion, the results suggest that the loss of miR-194 ameliorates cholestatic liver injury and may suppress CYP7A1 expression via activation of CTNNB1 signaling.

Ganglioside GM3 prevents high fat diet-induced hepatosteatosis via attenuated insulin signaling pathway.

Gangliosides, sialic acid-containing glycosphingolipids, are widely involved in regulations of signal transductions to control cellular functions. It has been suggested that GM3, the simplest structure among gangliosides, is involved in insulin resistance, whereas it remains unclear whether insulin signaling diminished by GM3 actually aggravates the pathological conditions in metabolic disorders. Moreover, the functional roles of gangliosides in the regulation of insulin signaling have not yet been fully elucidated in liver or hepatocytes despite that it is one of the major insulin-sensitive organs. To understand physiological roles of GM3 in metabolic homeostasis in liver, we conducted a high fat diet (HFD) loading experiment using double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase, which lack all gangliosides except GM3, as well as wild-type (WT) mice. DKO mice were strikingly resistant to HFD-induced hepatosteatosis, and hepatic lipogenesis-related molecules including insulin signaling components were down-regulated in HFD-fed DKO. Furthermore, we established primary hepatocyte cultures from DKO and WT mice, and examined their responses to insulin in vitro. Following insulin stimulation, DKO hepatocytes expressing GM3 showed attenuated expression and/or activations in the downstream components compared with WT hepatocytes expressing GM2. While insulin stimulation induced lipogenic proteins in hepatocytes from both genotypes, their expression levels were lower in DKO than in WT hepatocytes after insulin treatment. All our findings suggest that the modified gangliosides, i.e., a shift to GM3 from GM2, might exert a suppressive effect on lipogenesis by attenuating insulin signaling at least in mouse hepatocytes, which might result in protection of HFD-induced hepatosteatosis.

Blockade of T-cell receptor with Ig and ITIM domains elicits potent antitumor immunity in naturally occurring HBV-related HCC in mice.

Chronic HBV infection is the leading cause of HCC and a serious health problem in China, East Asia, and North African countries. Effective treatment of HBV-related HCC is currently unavailable. This study evaluated the therapeutic potential of T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade in HBV-related HCC. A mouse model of spontaneous HBV-related HCC was generated by replacing wild-type hepatocytes with HBsAg + hepatocytes (namely HBs-HepR mice). The tumors in HBs-HepR mice were inflammation-associated HCC, similar to HBV-related HCC in patients, which was distinguished from other HCC mouse models, such as diethylnitrosamine-induced HCC, TGF-β-activated kinase 1 knockout-induced HCC, HCC in a stelic animal model, or NASH-induced HCC. HCC in HBs-HepR mice was characterized by an increased number of CD8 + T cells, whereas the production of IL-2, TNF-α, and interferon-gamma (IFN-γ) by intrahepatic CD8 + T cells was decreased. Increased expression of TIGIT on CD8 + T cells was responsible for functional exhaustion. The therapeutic effect of TIGIT blockade was investigated at the early and middle stages of HCC progression in HBs-HepR mice. TIGIT blockade reinvigorated intrahepatic CD8 + T cells with increased TNF-α and IFN-γ production and an increased number of CD8 + T cells in tumors, thereby slowing the development of HCC in HBs-HepR mice. Blocking PD-L1 did not show direct therapeutic effects or synergize with TIGIT blockade. Blockade of TIGIT alone enhanced the antitumor activity of CD8 + T cells during the progression of HBV-related HCC in a spontaneous HCC mouse model.

Tau Loss of Function, by Deletion or Aggregation, Contributes to Peripheral Insulin Resistance

AbstractBackgroundEpidemiological association between Alzheimer’s disease (AD) and type 2 diabetes is a critical health concern that has not been resolved yet at the molecular mechanistic level. While brains of AD patients and AD transgenic mouse models display impaired insulin signaling, there is no clear understanding of the role of peripheral insulin resistance in the pathogenesis of AD. Moreover, alterations in key insulin signaling proteins have been also implicated in AD, among which GSK‐3β, is also one of the important kinases for the phosphorylation of the microtubule‐associated protein tau (tau), one of the pathological hallmarks of AD. The goal of this study is to determine the role of systemic insulin resistance in the pathophysiology of AD by testing the hypothesis that functional tau is important for peripheral insulin sensitivity, and its deletion or loss‐of‐function promotes peripheral insulin resistance.MethodPrimary hepatocytes were isolated from transgenic mouse models (P301L, tau KO), and wild type mice (C57BL/6), and their insulin sensitivity was assessed by an insulin challenge, using biochemical analysis of insulin signaling markers, and glucose uptake assays. To confirm the involvement of tau, we treated wild type hepatocytes with tau aggregates and investigated their effect on insulin sensitivity. To investigate the involvement of p38 MAPK, a stress‐response protein, we pre‐treated wild type hepatocytes with a pharmacological inhibitor of p38 prior to tau aggregates treatment.ResultWe found that tau deletion or loss‐of‐function promotes peripheral insulin resistance as seen by the inhibition of insulin signaling proteins and the reduction of glucose uptake into primary hepatocytes isolated from tau KO and P301L mice, respectively. We also found that sub‐toxic exposure of wild‐type primary hepatocytes to tau aggregates resulted in inhibition of insulin signaling and glucose uptake, in a dose‐dependent manner. Our data also show that p38 MAPK may be involved in tau aggregates‐induced inhibition of insulin signaling.ConclusionThis is the first study testing the potential role of tau in peripheral insulin resistance at the cellular level using multiple mouse models. Our findings suggest that functional tau is required for insulin sensitivity, therefore, any loss‐of‐function by aggregation or deletion would result in insulin resistance.

Inhibition of polyploidization in Pten-deficient livers reduces steatosis.

The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver-specific deletion of Pten in mice results in the hyper-activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver-specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten-induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten-deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR-gamma signalling. However, we find no evidence that deregulated genes in Pten-deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten-deficient livers.

IPMK modulates hepatic glucose production and insulin signaling.

Hepatic glucose production (HGP) is crucial for the maintenance of normal glucose homeostasis. Although hepatic insulin resistance contributes to excessive glucose production, its mechanism is not well understood. Here, we show that inositol polyphosphate multikinase (IPMK), a key enzyme in inositol polyphosphate biosynthesis, plays a role in regulating hepatic insulin signaling and gluconeogenesis both in vitro and in vivo. IPMK-deficient hepatocytes exhibit decreased insulin-induced activation of Akt-FoxO1 signaling. The expression of messenger RNA levels of phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose 6-phosphatase (G6pc), key enzymes mediating gluconeogenesis, are increased in IPMK-deficient hepatocytes compared to wild type hepatocytes. Importantly, re-expressing IPMK restores insulin sensitivity and alleviates glucose production in IPMK-deficient hepatocytes. Moreover, hepatocyte-specific IPMK deletion exacerbates hyperglycemia and insulin sensitivity in mice fed a high-fat diet, accompanied by an increase in HGP during pyruvate tolerance test and reduction in Akt phosphorylation in IPMK deficient liver. Our results demonstrate that IPMK mediates insulin signaling and gluconeogenesis and may be potentially targeted for treatment of diabetes.

Gα12 overexpression in hepatocytes by ER stress exacerbates acute liver injury via ROCK1-mediated miR-15a and ALOX12 dysregulation.

Rationale: Liver injury must be further characterized to identify novel therapeutic approaches. Endoplasmic reticulum (ER) stress may cause hepatocyte death. Gα12 affects cell viability and its expression varies depending on physiological conditions. This study investigated whether hepatocyte-specific Gα12 overexpression affects acute liver injury, and if so, what the underlying mechanisms and treatment strategies are.Methods: All experiments were performed using human liver, hepatocytes, and toxicant injury models with Gna12 KO and/or hepatocyte-specific Gα12 overexpression. RNA-sequencing, immunoblotting, immunohistochemistry, reporter assays, and mutation assays were conducted.Results: Hepatic Gα12 was overexpressed in mice challenged with acetaminophen or other ER stress inducers or in patients with acute liver injury or fibrosis/cirrhosis. Several Gα12 and ER-associated pathways were identified using transcriptomic analysis. Acetaminophen intoxication was characterized by lipid peroxide-induced ferroptosis and was less severe in Gα12-deficient animals and cells. Conversely, Gα12 overexpression in wild-type or Gna12 KO hepatocytes increased hepatotoxicity, promoting lipid peroxidation, inflammation, and ferroptosis. IRE1α-dependent Xbp1 transactivated Gna12. Moreover, Gα12 overexpression enhanced the ability of acetaminophen to induce ALOX12, while downregulating GPX4. The level of miR-15a, herein identified as an ALOX12 inhibitor, was decreased. siRNA knockdown or pharmacological inhibition of ROCK1 prevented dysregulation of ALOX12 and GPX4, rescuing animals from toxicant-induced ferroptosis. These changes or correlations among the targets were confirmed in human liver specimens and datasets of livers exposed to other injurious medications.Conclusions: Gα12 overexpression by ER stress facilitates hepatocyte ferroptosis through ROCK1-mediated dysregulation of ALOX12, and miR-15a, supporting the concept that inhibition of Gα12 overexpression and/or ROCK1 axis may constitute a promising strategy for acute liver injury.

Methionine Metabolism in Aging Regulation

Aging is the major risk factor for many diseases but the mechanisms are poorly understood. The risk of developing hepatic steatosis increases with age and the health impact of this disease is negative and high. When challenged with high fat diets, long living Ames mice withstand the detrimental metabolic effects that occur in normal mice. We examined transcriptomic and epigenomic profiles of Ames and wild type hepatocytes in the presence or absence of fat to demonstrate that the epigenomic profile drives transcription factor and downstream gene expression resulting in susceptibility or resistance to fatty liver disease. We found that markers of steatosis are related to gene expression in wild type and Ames mice, and dwarf mice retain fewer lipid droplets compared to wild type mice. These studies will provide data to guide our understanding of mechanisms leading to hepatic disease and define factors that provide protection from age-related metabolic disorders.