Liver RNA Research Articles

Modulation of AAV9 Galactose Binding Yields Novel Gene Therapy Vectors and Predicts Cross-Species Differences in Glycan Avidity.

Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extra-hepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect in vivo AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a six-fold reduction in liver RNA expression and a ten-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extra-hepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies.

Examining effects of a novel estrogenic perfluoro-alcohol, 1H,1H,8H,8H-Perfluorooctane-1,8-diol (FC8-diol), using the fathead minnow EcoToxChip.

In a previous in vivo study, adult male fathead minnows (Pimephales promelas) were exposed via water for 4 days to 1H,1H,8H,8H-perfluorooctane-1,8-diol (FC8-diol). The present study expands on the evaluation of molecular responses to this perfluoro-alcohol by analyzing 26 male fathead minnow liver RNA samples from that study (five from each test concentration: 0, 0.018, 0.051, 0.171, and 0.463 mg FC8-diol/L) using fathead minnow EcoToxChips Ver. 1.0. EcoToxChips are a quantitative polymerase chain reaction array that allows for simultaneous measurement of >375 species-specific genes of toxicological interest. Data were analyzed with the online tool EcoToxXplorer. Among the genes analyzed, 62 and 96 were significantly up- and downregulated, respectively, by one or more FC8-diol treatments. Gene expression results from the previous study were validated, showing an upregulation of vitellogenin mRNA (vtg) and downregulation of insulin-like growth factor 1 mRNA (igf1). Additional genes related to estrogen receptor activation including esr2a (estrogen receptor 2a) and esrrb (estrogen related receptor beta) were also affected, providing further confirmation of the estrogenic nature of FC8-diol. Furthermore, genes involved in biological pathways related to lipid and carbohydrate metabolism, innate immune response, endocrine reproduction, and endocrine thyroid were significantly affected. These results both add confidence in the use of the EcoToxChip tool for inferring chemical mode(s) of action and provide further insights into the possible biological effects of FC8-diol. Environ Toxicol Chem 2024;00:1-9. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Characterizing MetALD in a Nonhuman Primate Model using High Fat and Sucrose Diet and Chronic Binge Alcohol Administration

Mechanisms leading to metabolic and alcohol associated liver disease (MetALD) are not clearly delineated. This study examined the differential regulation of markers of hepatic mitochondrial function in nonhuman primates fed a high fat/high sucrose (HFSD) and administered chronic binge alcohol (CBA) or vehicle as compared to control-fed animals. We hypothesized that markers of hepatic dysregulation due to HFSD will be further altered by the combination of CBA/HFSD. Eight rhesus macaques were fed HFSD (27% sucrose and 42% fat, by kcal) with n=4 receiving vehicle and n=4 receiving 13-14g/kg/wk of ethanol through a gastric catheter. Rhesus macaques fed standard primate chow were used as reference controls (n=4-6). Liver RNA was isolated for determining expression of genes regulating mitochondrial biogenesis and function using RT-qPCR. Results were analyzed using one-way ANOVA and post-hoc pairwise multiple comparisons were performed using Tukey’s method, with alpha set to 0.05. Expression of inflammatory genes was significantly altered across the groups (p<0.05). HFSD increased tumor necrosis factor alpha (p=0.01), interleukin 1-beta (p=0.04), cluster of differentiation 38 (p=0.01), and transforming growth factor beta (p=0.01) expression compared to control. Both HFSD and CBA/HFSD increased interleukin-10 compared to control (p=0.02, p=0.03). Gene expression of mitochondrial function and oxidative stress regulators were significantly altered across the groups (p<0.05). CBA/HFSD decreased expression of peroxisome proliferator-activated receptor-gamma coactivator-1 beta (p=0.04) and nuclear respiratory factor 2 (p=0.04) compared to control. Both HFSD and CBA/HFSD decreased peroxisome proliferator-activated receptor alpha expression compared to control (p=0.01, 0.01). HFSD decreased superoxide dismutase 2 (SOD2) expression compared to control (p=0.01) and increased uncoupling protein 2 (p=0.01). SOD2 expression was increased only in CBA/HFSD compared to HFSD (p=0.01). These results reveal marked HFSD-mediated increases in markers of inflammation and CBA/HFSD-mediated changes in markers of mitochondrial function regulation and oxidative stress regulation. These findings suggest a greater impact of calorie-dense diets than CBA on hepatic inflammation and a combined impact of CBA and HFSD on mitochondrial regulation. Ongoing studies will examine oxidative phosphorylation complex protein activities and expression to elucidate and integrate HFSD or CBA mediated changes in hepatic bioenergetic capacity. Supported by F30AA030910, P60AA009803, and T32AA007577. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Therapeutic inhibition of monocyte recruitment prevents checkpoint inhibitor-induced hepatitis

BackgroundCheckpoint inhibitor-induced hepatitis (CPI-hepatitis) is an emerging problem with the widening use of CPIs in cancer immunotherapy. Here, we developed a mouse model to characterize the mechanism of CPI-hepatitis and...

VCD-induced menopause mouse model reveals reprogramming of hepatic metabolism

ObjectiveMenopause adversely impacts systemic energy metabolism and increases the risk of metabolic disease(s) including hepatic steatosis, but the mechanisms are largely unknown. Dosing female mice with vinyl cyclohexene dioxide (VCD) selectively causes follicular atresia in ovaries, leading to a murine menopause-like phenotype. MethodsIn this study, we treated female C57BL6/J mice with VCD (160 mg/kg i.p. for 20 consecutive days followed by verification of the lack of estrous cycling) to investigate changes in body composition, energy expenditure (EE), hepatic mitochondrial function, and hepatic steatosis across different dietary conditions. ResultsVCD treatment induced ovarian follicular loss and increased follicle-stimulating hormone (FSH) levels in female mice, mimicking a menopause-like phenotype. VCD treatment did not affect body composition, or EE in mice on a low-fat diet (LFD) or in response to a short-term (1-week) high-fat, high sucrose diet (HFHS). However, the transition to a HFHS lowered cage activity in VCD mice. A chronic HFHS diet (16 weeks) significantly increased weight gain, fat mass, and hepatic steatosis in VCD-treated mice compared to HFHS-fed controls. In the liver, VCD mice showed suppressed hepatic mitochondrial respiration on LFD, while chronic HFHS resulted in compensatory increases in hepatic mitochondrial respiration. Also, liver RNA sequencing revealed that VCD promoted global upregulation of hepatic lipid/cholesterol synthesis pathways. ConclusionOur findings suggest that the VCD-induced menopause model compromises hepatic mitochondrial function and lipid/cholesterol homeostasis that sets the stage for HFHS diet-induced steatosis while also increasing susceptibility to obesity.

Dietary intervention reverses molecular markers of hepatocellular senescence in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH.

Hepatocellular senescence may be a causal factor in the development and progression of non-alcoholic steatohepatitis (NASH). The most effective currently available treatment for NASH is lifestyle intervention, including dietary modification. This study aimed to evaluate the effects of dietary intervention on hallmarks of NASH and molecular signatures of hepatocellular senescence in the Gubra-Amylin NASH (GAN) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. GAN DIO-NASH mice with liver biopsy-confirmed NASH and fibrosis received dietary intervention by switching to chow feeding (chow reversal) for 8, 16 or 24 weeks. Untreated GAN DIO-NASH mice and chow-fed C57BL/6J mice served as controls. Pre-to-post liver biopsy histology was performed for within-subject evaluation of NAFLD Activity Score and fibrosis stage. Terminal endpoints included blood/liver biochemistry, quantitative liver histology, mitochondrial respiration and RNA sequencing. Chow-reversal promoted substantial benefits on metabolic outcomes and liver histology, as demonstrated by robust weight loss, complete resolution of hepatomegaly, hypercholesterolemia, elevated transaminase levels and hepatic steatosis in addition to attenuation of inflammatory markers. Notably, all DIO-NASH mice demonstrated ≥ 2 point significant improvement in NAFLD Activity Score following dietary intervention. While not improving fibrosis stage, chow-reversal reduced quantitative fibrosis markers (PSR, collagen 1a1, α-SMA), concurrent with improved liver mitochondrial respiration, complete reversal of p21 overexpression, lowered γ-H2AX levels and widespread suppression of gene expression markers of hepatocellular senescence. Dietary intervention (chow reversal) substantially improves metabolic, biochemical and histological hallmarks of NASH and fibrosis in GAN DIO-NASH mice. These benefits were reflected by progressive clearance of senescent hepatocellular cells, making the model suitable for profiling potential senotherapeutics in preclinical drug discovery for NASH.

Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

Acanthopanax senticosus ameliorates steatohepatitis through HNF4 alpha pathway activation in mice

Non-alcoholic fatty liver disease is a common liver disease worldwide, and is associated with dysregulation of lipid metabolism, leading to inflammation and fibrosis. Acanthopanax senticosus Harms (ASH) is widely used in traditional medicine as an adaptogen food. We examined the effect of ASH on steatohepatitis using a high-fat diet mouse model. Mice were fed a choline-deficient, l-amino acid-defined, high-fat diet with ASH extract (ASHE). After 6 weeks, liver RNA transcriptome sequencing (RNA-Seq) was performed, followed by Ingenuity Pathway Analysis (IPA). Our findings revealed that mice fed a high-fat diet with 5% ASHE exhibited significantly reduced liver steatosis. These mice also demonstrated alleviated inflammation and reduced fibrosis in the liver. IPA of RNA-Seq indicated that hepatocyte nuclear factor 4 alpha (HNF4 alpha), a transcription factor, was the activated upstream regulator (P-value 0.00155, z score = 2.413) in the liver of ASHE-fed mice. Adenosine triphosphate binding cassette transporter 8 and carboxylesterase 2, downstream targets of HNF4 alpha pathway, were upregulated. Finally, ASHE-treated HepG2 cells exposed to palmitate exhibited significantly decreased lipid droplet contents. Our study provides that ASHE can activate HNF4 alpha pathway and promote fat secretion from hepatocytes, thereby serving as a prophylactic treatment for steatohepatitis in mice.

219 Identification of Functional Single Nucleotide Polymorphisms Responsible for Alternative Splicing Affecting Gastrointestinal Nematodes Resistance in Grazing Sheep

Abstract Sheep infected with gastrointestinal nematodes (GIN) can show weight loss, anemia, diarrhea, decrease appetite, and hypoproteinemia, which can sometimes lead to death. These signs of infection decrease productivity and cause economic losses. Economic losses can also be associated with treatment without efficacy due to GIN resistance to most anthelmintic classes. Combined approaches need to be used to reduce the reliance on anthelmintic as the only control method to GIN. The aim of this study is the identification of potential functional single nucleotide polymorphisms (SNPs) responsible for alternate splicing in Rideau X Dorset crossbred sheep with different immune profiles using RNA-Sequencing (RNA-Seq). Animals were exposed to GIN while grazing, after that those with the greatest and least gastrointestinal tract parasite burdens were selected for liver RNA extracting. These animals can be separated into groups of high (H; n = 5) and medium (M; n = 6) innate immune responses. In addition, liver RNA was extracted for GIN-unexposed control lambs (U; n = 4). RNA-Seq analysis was performed using the CLC genomics workbench software, version 20.0.4 (CLC Bio, Aarhus, Denmark). Quality control analyses, including guanine-cytosine (GC) content, ambiguous base content, Phred score, base coverage, nucleotide contributions, and over-represented sequences parameters, were performed on fastq files. Sequence reads were aligned to the annotated Oar_rambouillet_v.1 (release 109) ovine reference genome. After filtering steps, mRNA isoforms with multiple (³ 2) expressed mRNA isoforms with at least one mRNA isoform differently expressed (DE) between H- and M-innate immune response and GIN-unexposed control groups (FDR < 0.05 and |FC| > 2) were keep. A total of 1, 75, and 58 DE mRNA isoforms were identified for M versus H, U versus M, and U versus H comparisons, respectively. Gene ontology (GO) analysis including the biological process, molecular function, and cellular component GO categories and metabolic pathways analysis were performed using STRING-db software. Enriched biological processes and metabolic pathways related to the immune system, as well as cholesterol and other sterol metabolism (e.g., sterol biosynthesis, steroid metabolism, cholesterol biosynthesis, cholesterol metabolism, ascorbate and aldarate metabolism, drug metabolism, and terpenoid biosynthesis) were identified (FDR < 0.05). Next, the list of DE mRNA isoforms identified will be deeply studied by performing SNP discovery analysis to validate if specific functional SNPs are responsible for the alternative splicing affecting GIN resistance in grazing sheep. The complete results will help to better understand the biological mechanisms that may be regulating the immune response to GIN. Candidate functional SNPs have the potential to be included in a commercial SNP panel for selection of more resistant animals to improve GIN resistance in sheep flocks.

Combination of an ACLY inhibitor with a GLP-1R agonist exerts additive benefits on nonalcoholic steatohepatitis and hepatic fibrosis in mice

Increased liver de novo lipogenesis (DNL) is a hallmark of nonalcoholic steatohepatitis (NASH). A key enzyme controlling DNL upregulated in NASH is ATP citrate lyase (ACLY). In mice, inhibition of ACLY reduces liver steatosis, ballooning, and fibrosis and inhibits activation of hepatic stellate cells. Glucagon-like peptide-1 receptor (GLP-1R) agonists lower body mass, insulin resistance, and steatosis without improving fibrosis. Here, we find that combining an inhibitor of liver ACLY, bempedoic acid, and the GLP-1R agonist liraglutide reduces liver steatosis, hepatocellular ballooning, and hepatic fibrosis in a mouse model of NASH. Liver RNA analyses revealed additive downregulation of pathways that are predictive of NASH resolution, reductions in the expression of prognostically significant genes compared with clinical NASH samples, and a predicted gene signature profile that supports fibrosis resolution. These findings support further investigation of this combinatorial therapy to treat obesity, insulin resistance, hypercholesterolemia, steatohepatitis, and fibrosis in people with NASH.

Multi‑omics‑based analysis of the regulatory mechanism of gypenosides on bile acids in hypercholesterolemic mice.

Gynostemma pentaphyllum is a traditional medicine used by ethnic minorities in southwest China and gypenosides are currently recognized as essential components of the pharmacological substances of Gynostemma pentaphyllum, which are effective in regulating metabolic syndrome, especially in improving hepatic metabolic disorders. The present study randomly divided C57BL/6J male mice into the normal diet control group (ND), high-fat diet modeling group (HFD) and gypenosides group (GP). Liquid chromatography-mass spectrometry (UPLC-MS) was applied to quantify bile acids in the liver, bile and serum of mice in ND, HFD and GP groups. Liver proteins were extracted for trypsin hydrolysis and analyzed quantitatively using UPLC-MS + MS/MS (timsTOF Pro 2). Total mouse liver RNA was extracted from ND, HFD and GP groups respectively, cDNA sequencing libraries constructed and sequenced using BGISEQ-500 sequencing platform. The expression of key genes Fxr, Shp, Cyp7a1, Cyp8b1, and Abab11 was detected by RT-qPCR. The results showed that gypenosides accelerated free bile acid synthesis by promoting the expression of bile acid synthase CYP7A1 and CYP8B1 genes and proteins and accelerating the secretion of conjugated bile acids from the liver to the bile ducts. GP inhibited the bile acid transporters solute carrier organic anion transporter family member (SLCO) 1A1 and SLCO1A4, reducing the reabsorption of free bile acids and accelerating the excretion of free bile acids from the blood to the kidneys. It also promoted the metabolic enzyme CYP3A11, which accelerated the metabolism and clearance of bile acids, thus maintaining the balance of the bile acid internal environment.

Liver transcriptomic and methylomic analyses identify transcriptional mitogen-activated protein kinase regulation in facultative hibernation of Syrian hamster.

Hibernation consists of alternating torpor-arousal phases, during which animals cope with repetitive hypothermia and ischaemia-reperfusion. Due to limited transcriptomic and methylomic information for facultative hibernators, we here conducted RNA and whole-genome bisulfide sequencing in liver of hibernating Syrian hamster (Mesocricetus auratus). Gene ontology analysis was performed on 844 differentially expressed genes and confirmed the shift in metabolic fuel utilization, inhibition of RNA transcription and cell cycle regulation as found in seasonal hibernators. Additionally, we showed a so far unreported suppression of mitogen-activated protein kinase (MAPK) and protein phosphatase 1 pathways during torpor. Notably, hibernating hamsters showed upregulation of MAPK inhibitors (dual-specificity phosphatases and sproutys) and reduced levels of MAPK-induced transcription factors (TFs). Promoter methylation was found to modulate the expression of genes targeted by these TFs. In conclusion, we document gene regulation between hibernation phases, which may aid the identification of pathways and targets to prevent organ damage in transplantation or ischaemia-reperfusion.

Urolithin C reveals anti-NAFLD potential via AMPK-ferroptosis axis and modulating gut microbiota.

The pharmacology of urolithin C (UroC) on non-alcoholic fatty liver disease (NAFLD) is largely undetermined. We sought to investigate the potential for NAFLD improvement by administration of UroC and the underlying mechanisms. We verified the therapeutic effect of UroC on choline-deficient amino acid-defined high fat diet (CDAHFD) induced NAFLD mice via evaluating NAFLD activity score (NAS), AST, ALT, hepatic phosphorylated AMPK, and 4-hydroxynonenal. Oleic acid-induced AML12 cell was appraised by oil red staining and western blotting to explore the effect and mechanism of UroC in vitro. Transcriptional regulation of UroC was explored by liver RNA sequencing, gut microbiota composition was explored by 16SrRNA sequencing, and colorectal tight junctional proteins were detected by western blotting and immunohistochemistry. The detrimental effects of CDAHFD included the increased liver index, AST, ALT, hepatic 4-hydroxynonenal, impaired intestinal mucosal barrier, and most importantly, pathological damage in liver. Oral administration of UroC largely protected against these harmful alterations. Remarkably, both RNA sequencing and western blotting results indicated an activation in hepatic AMPK signaling pathway which was thought to inhibit ferroptosis response to UroC in vivo, while no change were found in AMPK-ferroptosis axis response to UroC in oleic acid-induced AML12 cells, hinted an indispensable linkage between UroC and hepatic AMPK, presumably the gut-liver axis. Furthermore, UroC could neither alleviate lipid deposition nor inhibit ferroptosis in vitro. The 16SrRNA showed UroC partially counteracted the dysbiosis induced by CDAHFD. Specifically, UroC reversed the elevated proportion of Firmicutes/Bacteroidota and enhanced the level of Parabacteroides goldsteinii and Lactobacillus vaginalis, which played a beneficial role in metabolic disorders. Oral administration of Urolithin C protected against the detrimental impact of CDAHFD via regulating AMPK-ferroptosis axis, maintaining intestinal mucosal barrier and counteracting gut dysbiosis.

Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9+TREM2+ macrophages that express SPP1, GPNMB, FABP5, and CD63. In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-β1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-β1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-β1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-β1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

Effect of Administration of an Equal Dose of Selected Dietary Chemicals on Nrf2 Nuclear Translocation in the Mouse Liver.

Certain dietary chemicals influenced the expression of chemopreventive genes through the Nrf2-Keap1 pathway. However, the difference in Nrf2 activation potency of these chemicals is not well studied. This study is aimed at determining the difference in the potency of liver Nrf2 nuclear translocation induced by the administration of equal doses of selected dietary chemicals in mice. Male ICR white mice were administered 50 mg/kg of sulforaphane, quercetin, curcumin, butylated hydroxyanisole, and indole-3-carbinol for 14 days. On day 15, the animals were sacrificed, and their livers were isolated. Liver nuclear extracts were prepared, and Nrf2 nuclear translocation was detected through Western blotting. To determine the implication of the Nrf2 nuclear translocation on the expression levels of several Nrf2-regulated genes, liver RNA was extracted for qPCR assay. Equal doses of sulforaphane, quercetin, curcumin, butylated hydroxyanisole, and indole-3-carbinol significantly induced the nuclear translocation of Nrf2 with different intensities and subsequently increased the expression of Nrf2-regulated genes with an almost similar pattern as the Nrf2 nuclear translocation intensities (sulforaphane > butylated hydroxyanisole = indole-3-carbinol > curcumin > quercetin). In conclusion, sulforaphane is the most potent dietary chemical that induces the Nrf2 translocation into the nuclear fraction in the mouse liver.

The impact of hepatocyte-specific deletion of hypoxia-inducible factors on the development of polymicrobial sepsis with focus on GR and PPARα function.

Polymicrobial sepsis causes acute anorexia (loss of appetite), leading to lipolysis in white adipose tissue and proteolysis in muscle, and thus release of free fatty acids (FFAs), glycerol and gluconeogenic amino acids. Since hepatic peroxisome proliferator-activated receptor alpha (PPARα) and glucocorticoid receptor (GR) quickly lose function in sepsis, these metabolites accumulate (causing toxicity) and fail to yield energy-rich molecules such as ketone bodies (KBs) and glucose. The mechanism of PPARα and GR dysfunction is not known. We investigated the hypothesis that hypoxia and/or activation of hypoxia inducible factors (HIFs) might play a role in these issues with PPARα and GR. After cecal ligation and puncture (CLP) in mice, leading to lethal polymicrobial sepsis, bulk liver RNA sequencing illustrated the induction of the genes encoding HIF1α and HIF2α, and an enrichment of HIF-dependent gene signatures. Therefore, we generated hepatocyte-specific knock-out mice for HIF1α, HIF2α or both, and a new HRE-luciferase reporter mouse line. After CLP, these HRE-luciferase reporter mice show signals in several tissues, including the liver. Hydrodynamic injection of an HRE-luciferase reporter plasmid also led to (liver-specific) signals in hypoxia and CLP. Despite these encouraging data, however, hepatocyte-specific HIF1α and/or HIF2α knock-out mice suggest that survival after CLP was not dependent on the hepatocyte-specific presence of HIF proteins, which was supported by measuring blood levels of glucose, FFAs, and KBs. The HIF proteins were also irrelevant in the CLP-induced glucocorticoid resistance, but we found indications that the absence of HIF1α in hepatocytes causes less inactivation of PPARα transcriptional function. We conclude that HIF1α and HIF2α are activated in hepatocytes in sepsis, but their contribution to the mechanisms leading to lethality are minimal.

Maternal Western diet is associated with distinct preclinical pediatric NAFLD phenotypes in juvenile nonhuman primate offspring.

Pediatric NAFLD has distinct and variable pathology, yet causation remains unclear. We have shown that maternal Western-style diet (mWSD) compared with maternal chow diet (CD) consumption in nonhuman primates produces hepatic injury and steatosis in fetal offspring. Here, we define the role of mWSD and postweaning Western-style diet (pwWSD) exposures on molecular mechanisms linked to NAFLD development in a cohort of 3-year-old juvenile nonhuman primates offspring exposed to maternal CD or mWSD followed by CD or Western-style diet after weaning. We used histologic, transcriptomic, and metabolomic analyses to identify hepatic pathways regulating NAFLD. Offspring exposed to mWSD showed increased hepatic periportal collagen deposition but unchanged hepatic triglyceride levels and body weight. mWSD was associated with a downregulation of gene expression pathways underlying HNF4α activity and protein, and downregulation of antioxidant signaling, mitochondrial biogenesis, and PPAR signaling pathways. In offspring exposed to both mWSD and pwWSD, liver RNA profiles showed upregulation of pathways promoting fibrosis and endoplasmic reticulum stress and increased BiP protein expression with pwWSD. pwWSD increased acylcarnitines and decreased anti-inflammatory fatty acids, which was more pronounced when coupled with mWSD exposure. Further, mWSD shifted liver metabolites towards decreased purine catabolism in favor of synthesis, suggesting a mitochondrial DNA repair response. Our findings demonstrate that 3-year-old offspring exposed to mWSD but weaned to a CD have periportal collagen deposition, with transcriptional and metabolic pathways underlying hepatic oxidative stress, compromised mitochondrial lipid sensing, and decreased antioxidant response. Exposure to pwWSD worsens these phenotypes, triggers endoplasmic reticulum stress, and increases fibrosis. Overall, mWSD exposure is associated with altered expression of candidate genes and metabolites related to NAFLD that persist in juvenile offspring preceding clinical presentation of NAFLD.

Alterations in energy metabolism, total protein, uric and nucleic acids in African sharptooth catfish (Clarias gariepinus Burchell) exposed to crude oil and fractions

Water contamination by crude oil is a growing challenge and little is known about the probabilistic and non-probabilistic ecosystem and species consequences. Therefore, research aimed at understanding species survival strategy in crude oil-contaminated environments with focus on cellular metabolic alterations and dynamics is vital. This study assessed the alterations in lactate dehydrogenase (LDH), glucose (GLU), glucose-6-phosphate dehydrogenase (G-6-PDH), total protein (TP), uric and nucleic acids (UA, RNA, and DNA) in the liver, heart, kidney, blood supernatants, and muscle homogenates of African sharptooth catfish ([ASC] Clarias gariepinus) exposed to varying bonny-light crude oil concentrations to understand the underlying cause of their delayed development as well as potential health and wellbeing. Three concentrations (20, 50, and 100 mg/L) of diluted whole bonny-light crude oil (DWC), water-soluble (WSF), and water-insoluble (WIF) fractions of bonny-light crude oil were used to grow ASC for 9 weeks at room temperature. Biochemical assessments revealed significant (at p < 0.05) elevations in heart LDH (48.57 ± 4.67 to 3011.34 ± 4.67 U/L) and blood G-6-PDH activities (54.86 ± 0.00 to 128 ± 18.29 mU/mL), GLU (0.22 ± 0.01 to 0.77 ± 0.01 mg/dL), TP (5.15 ± 0.14 to 22.33 ± 0.21 g/L), UA (0.29 ± 0.05 to 10.05 ± 0.27 mg/dL), as well as liver DNA (0.38 ± 0.02 to 2.33 ± 0.09 μg/mL) and RNA (12.52 ± 0.05 to 30.44 ± 0.02 μg/mL) levels for laboratory-grown ASC in DWC, WSF, WIF, and oil-impacted Ubeji river collected ASC relative to the control. Due to greater levels of cellular metabolic alterations in oil-impacted Ubeji River collected ASC, it is evident that bonny-light contamination levels in the river is greater than 100 mg/L. In conclusion, bonny-light crude oil is toxic to ASC and induces stress response. The ecological changes caused by bonny-light crude oil contamination may ultimately affect niche functioning and the development of organs in ASC.

Transcriptional pathways linked to fetal and maternal hepatic dysfunction caused by gestational exposure to perfluorooctanoic acid (PFOA) or hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) in CD-1 mice

Per- and polyfluoroalkyl substances (PFAS) comprise a diverse class of chemicals used in industrial processes, consumer products, and fire-fighting foams which have become environmental pollutants of concern due to their persistence, ubiquity, and associations with adverse human health outcomes, including in pregnant persons and their offspring. Multiple PFAS are associated with adverse liver outcomes in adult humans and toxicological models, but effects on the developing liver are not fully described. Here we performed transcriptomic analyses in the mouse to investigate the molecular mechanisms of hepatic toxicity in the dam and its fetus after exposure to two different PFAS, perfluorooctanoic acid (PFOA) and its replacement, hexafluoropropylene oxide-dimer acid (HFPO-DA, known as GenX). Pregnant CD-1 mice were exposed via oral gavage from embryonic day (E) 1.5–17.5 to PFOA (0, 1, or 5 mg/kg-d) or GenX (0, 2, or 10 mg/kg-d). Maternal and fetal liver RNA was isolated (N = 5 per dose/group) and the transcriptome analyzed by Affymetrix Array. Differentially expressed genes (DEG) and differentially enriched pathways (DEP) were obtained. DEG patterns were similar in maternal liver for 5 mg/kg PFOA, 2 mg/kg GenX, and 10 mg/kg GenX (R2: 0.46–0.66). DEG patterns were similar across all 4 dose groups in fetal liver (R2: 0.59–0.81). There were more DEGs in fetal liver compared to maternal liver at the low doses for both PFOA (fetal = 69, maternal = 8) and GenX (fetal = 154, maternal = 93). Upregulated DEPs identified across all groups included Fatty Acid Metabolism, Peroxisome, Oxidative Phosphorylation, Adipogenesis, and Bile Acid Metabolism. Transcriptome-phenotype correlation analyses demonstrated > 1000 maternal liver DEGs were significantly correlated with maternal relative liver weight (R2 >0.92). These findings show shared biological pathways of liver toxicity for PFOA and GenX in maternal and fetal livers in CD-1 mice. The limited overlap in specific DEGs between the dam and fetus suggests the developing liver responds differently than the adult liver to these chemical stressors. This work helps define mechanisms of hepatic toxicity of two structurally unique PFAS and may help predict latent consequences of developmental exposure.

Pancreatic alpha cell glucagon–liver FGF21 axis regulates beta cell regeneration in a mouse model of type 2 diabetes

Aims/hypothesisGlucagon receptor (GCGR) antagonism ameliorates hyperglycaemia and promotes beta cell regeneration in mouse models of type 2 diabetes. However, the underlying mechanisms remain unclear. The present study aimed to investigate the mechanism of beta cell regeneration induced by GCGR antagonism in mice.MethodsThe db/db mice and high-fat diet (HFD)+streptozotocin (STZ)-induced mice with type 2 diabetes were treated with antagonistic GCGR monoclonal antibody (mAb), and the metabolic variables and islet cell quantification were evaluated. Plasma cytokine array and liver RNA sequencing data were used to screen possible mediators, including fibroblast growth factor 21 (FGF21). ELISA, quantitative RT-PCR and western blot were applied to verify FGF21 change. Blockage of FGF21 signalling by FGF21-neutralising antibody (nAb) was used to clarify whether FGF21 was involved in the effects of GCGR mAb on the expression of beta cell identity-related genes under plasma-conditional culture and hepatocyte co-culture conditions. FGF21 nAb-treated db/db mice, systemic Fgf21-knockout (Fgf21−/−) diabetic mice and hepatocyte-specific Fgf21-knockout (Fgf21Hep−/−) diabetic mice were used to reveal the involvement of FGF21 in beta cell regeneration. A BrdU tracing study was used to analyse beta cell proliferation in diabetic mice treated with GCGR mAb.ResultsGCGR mAb treatment improved blood glucose control, and increased islet number (db/db 1.6±0.1 vs 0.8±0.1 per mm2, p<0.001; HFD+STZ 1.2±0.1 vs 0.5±0.1 per mm2, p<0.01) and area (db/db 2.5±0.2 vs 1.2±0.2%, p<0.001; HFD+STZ 1.0±0.1 vs 0.3±0.1%, p<0.01) in diabetic mice. The plasma cytokine array and liver RNA sequencing data showed that FGF21 levels in plasma and liver were upregulated by GCGR antagonism. The GCGR mAb induced upregulation of plasma FGF21 levels (db/db 661.5±40.0 vs 466.2±55.7 pg/ml, p<0.05; HFD+STZ 877.0±106.8 vs 445.5±54.0 pg/ml, p<0.05) and the liver levels of Fgf21 mRNA (db/db 3.2±0.5 vs 1.8±0.1, p<0.05; HFD+STZ 2.0±0.3 vs 1.0±0.2, p<0.05) and protein (db/db 2.0±0.2 vs 1.4±0.1, p<0.05; HFD+STZ 1.6±0.1 vs 1.0±0.1, p<0.01). Exposure to plasma or hepatocytes from the GCGR mAb-treated mice upregulated the mRNA levels of characteristic genes associated with beta cell identity in cultured mouse islets and a beta cell line, and blockage of FGF21 activity by an FGF21 nAb diminished this upregulation. Notably, the effects of increased beta cell number induced by GCGR mAb were attenuated in FGF21 nAb-treated db/db mice, Fgf21−/− diabetic mice and Fgf21Hep−/− diabetic mice. Moreover, GCGR mAb treatment enhanced beta cell proliferation in the two groups of diabetic mice, and this effect was weakened in Fgf21−/− and Fgf21Hep−/− mice.Conclusions/interpretationOur findings demonstrate that liver-derived FGF21 is involved in the GCGR antagonism-induced beta cell regeneration in a mouse model of type 2 diabetes.Graphical abstract