HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen triggers an unfolded protein response (UPR) for stress adaptation, the failure of which induces cell apoptosis and tissue/organ damage. The molecular switches underlying how the UPR selects for stress adaptation over apoptosis remain unknown. Here, we discovered that accumulation of unfolded/misfolded proteins selectively induces N6-adenosine-methyltransferase-14 (METTL14) expression. METTL14 promotes C/EBP-homologous protein (CHOP) mRNA decay through its 3' UTR N6-methyladenosine (m6A) to inhibit its downstream pro-apoptotic target gene expression. UPR induces METTL14 expression by competing against the HRD1-ER-associated degradation (ERAD) machinery to block METTL14 ubiquitination and degradation. Therefore, mice with liver-specific METTL14 deletion are highly susceptible to both acute pharmacological and alpha-1 antitrypsin (AAT) deficiency-induced ER proteotoxic stress and liver injury. Further hepatic CHOP deletion protects METTL14 knockout mice from ER-stress-induced liver damage. Our study reveals a crosstalk between ER stress and mRNA m6A modification pathways, termed the ERm6A pathway, for ER stress adaptation to proteotoxicity.

In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.

Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.

CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.

Pacer Is a Mediator of mTORC1 and GSK3-TIP60 Signaling in Regulation of Autophagosome Maturation and Lipid Metabolism.

mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism.

Dietary fructose feeds hepatic lipogenesis via microbiota-derived acetate.

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods1, and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease2-4. Fructose intake triggers de novo lipogenesis in the liver4-6, in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates7. Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases8. However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota9, and this supplies lipogenic acetyl-CoA independently of ACLY10. Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA.

Oxygen-dependent histone lysine demethylase 4 restricts hepatitis B virus replication.

Mammalian cells have evolved strategies to regulate gene expression when oxygen is limited. Hypoxia-inducible factors (HIF) are the major transcriptional regulators of host gene expression. We previously reported that HIFs bind and activate hepatitis B virus (HBV) DNA transcription under low oxygen conditions; however, the global cellular response to low oxygen is mediated by a family of oxygenases that work in concert with HIFs. Recent studies have identified a role for chromatin modifiers in sensing cellular oxygen and orchestrating transcriptional responses, but their role in the HBV life cycle is as yet undefined. We demonstrated that histone lysine demethylase 4 (KDM4) can restrict HBV, and pharmacological or oxygen-mediated inhibition of the demethylase increases viral RNAs derived from both episomal and integrated copies of the viral genome. Sequencing studies demonstrated that KDM4 is a major regulator of the hepatic transcriptome, which defines hepatocellular permissivity to HBV infection. We propose a model where HBV exploits cellular oxygen sensors to replicate and persist in the liver. Understanding oxygen-dependent pathways that regulate HBV infection will facilitate the development of physiologically relevant cell-based models that support efficient HBV replication.

Fazirsiran for Liver Disease Associated with Alpha1-Antitrypsin Deficiency.

BACKGROUND: Alpha1-antitrypsin (AAT) deficiency results from carriage of a homozygous SERPINA1 "Z" mutation (proteinase inhibitor [PI] ZZ). The Z allele produces a mutant AAT protein called Z-AAT, which accumulates in hepatocytes and can lead to progressive liver disease and fibrosis. This open-label, phase 2 trial investigated the safety and efficacy of fazirsiran, an RNA interference therapeutic, in patients with liver disease associated with AAT deficiency. METHODS: We assigned adults with the PI ZZ genotype and liver fibrosis to receive fazirsiran at a dose of 200 mg (cohorts 1 [4 patients] and 2 [8 patients]) or 100 mg (cohort 1b [4 patients]) subcutaneously on day 1 and week 4 and then every 12 weeks. The primary end point was the change from baseline to week 24 (cohorts 1 and 1b) or week 48 (cohort 2) in liver Z-AAT concentrations, which were measured by means of liquid chromatography-mass spectrometry. RESULTS: All the patients had reduced accumulation of Z-AAT in the liver (median reduction, 83% at week 24 or 48). The nadir in serum was a reduction of approximately 90%, and treatment was also associated with a reduction in histologic globule burden (from a mean score of 7.4 [scores range from 0 to 9, with higher scores indicating a greater globule burden] at baseline to 2.3 at week 24 or 48). All cohorts had reductions in liver enzyme concentrations. Fibrosis regression was observed in 7 of 15 patients and fibrosis progression in 2 of 15 patients after 24 or 48 weeks. There were no adverse events leading to trial or drug discontinuation. Four serious adverse events (viral myocarditis, diverticulitis, dyspnea, and vestibular neuronitis) resolved. CONCLUSIONS: In this small trial, fazirsiran was associated with a strong reduction of Z-AAT concentrations in the serum and liver and concurrent improvements in liver enzyme concentrations. (Funded by Arrowhead Pharmaceuticals; AROAAT-2002 ClinicalTrials.gov number, NCT03946449.).

p38γ is essential for cell cycle progression and liver tumorigenesis.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.

VPS34 Acetylation Controls Its Lipid Kinase Activity and the Initiation of Canonical and Non-canonical Autophagy.

The class III phosphoinositide 3-kinase VPS34 plays a key role in the regulation of vesicular trafficking and macroautophagy. So far, we know little about the molecular mechanism of VPS34 activation besides its interaction with regulatory proteins to form complexes. Here, we report that VPS34 is specifically acetylated by the acetyltransferase p300, and p300-mediated acetylation represses VPS34 activity. Acetylation at K771 directly diminishes the affinity of VPS34 for its substrate PI, while acetylation at K29 hinders the VPS34-Beclin 1 core complex formation. Inactivation of p300 induces VPS34 deacetylation, PI3P production, and autophagy, even in AMPK-/-, TSC2-/-, or ULK1-/- cells. In fasting mice, liver autophagy correlates well with p300 inactivation/VPS34 deacetylation, which facilitates the clearance of lipid droplets in hepatocytes. Thus, p300-dependent VPS34 acetylation/deacetylation is the physiological key to VPS34 activation, which controls the initiation of canonical autophagy and of non-canonical autophagy in which the upstream kinases of VPS34 can be bypassed.

Calcium-dependent O-GlcNAc signaling drives liver autophagy in adaptation to starvation.

Starvation induces liver autophagy, which is thought to provide nutrients for use by other organs and thereby maintain whole-body homeostasis. Here we demonstrate that O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is required for glucagon-stimulated liver autophagy and metabolic adaptation to starvation. Genetic ablation of OGT in mouse livers reduces autophagic flux and the production of glucose and ketone bodies. Upon glucagon-induced calcium signaling, calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates OGT, which in turn promotes O-GlcNAc modification and activation of Ulk proteins by potentiating AMPK-dependent phosphorylation. These findings uncover a signaling cascade by which starvation promotes autophagy through OGT phosphorylation and establish the importance of O-GlcNAc signaling in coupling liver autophagy to nutrient homeostasis.

Activation of hepatic acetyl-CoA carboxylase by S-nitrosylation in response to diet.

Nitric oxide (NO), produced primarily by nitric oxide synthase enzymes, is known to influence energy metabolism by stimulating fat uptake and oxidation. The effects of NO on de novo lipogenesis (DNL), however, are less clear. Here we demonstrate that hepatic expression of endothelial nitric oxide synthase is reduced following prolonged administration of a hypercaloric high-fat diet. This results in marked reduction in the amount of S-nitrosylation of liver proteins including notably acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in DNL. We further show that ACC S-nitrosylation markedly increases enzymatic activity. Diminished endothelial nitric oxide synthase expression and ACC S-nitrosylation may thus represent a physiological adaptation to caloric excess by constraining lipogenesis. Our findings demonstrate that S-nitrosylation of liver proteins is subject to dietary control and suggest that DNL is coupled to dietary and metabolic conditions through ACC S-nitrosylation.

My lifelong dedication to bile acid research.

It is a great honor to be invited to write a reflections article on my scientific journey and lifelong bile acid research for the Journal of Biological Chemistry, in which I am proud to have published 24 articles. I have also published 21 articles in the Journal of Lipid Research, another journal of the American Society of Biochemistry and Molecular Biology. I begin my reflections from my early education in Taiwan, my coming to America for graduate study, and continue with my postdoctoral training in cytochrome P450 research, and my lifelong bile acid research career at Northeast Ohio Medical University. I have witnessed and helped in the transformation of this rural not so visible medical school to a well-funded leader in liver research. Writing this reflections article on my long and rewarding journey in bile acid research brings back many good memories. I am proud of my scientific contributions and attribute my academic success to hard work, perseverance, good mentoring, and networking. I hope these reflections of my academic career would help inspire young investigators to pursue an academic career in biochemistry and metabolic diseases.

Cyp26a1 supports postnatal retinoic acid homeostasis and glucoregulatory control.

Considerable evidence confirms the importance of Cyp26a1 to all-trans-retinoic acid (RA) homeostasis during embryogenesis. In contrast, despite its presence in postnatal liver as a potential major RA catabolizing enzyme and its acute sensitivity to induction by RA, some data suggested that Cyp26a1 contributes only marginally to endogenous RA homeostasis postnatally. We report reevaluation of a conditional Cyp26a1 knockdown in the postnatal mouse. The current results show that Cyp26a1 mRNA in WT mouse liver increases 16-fold upon refeeding after a fast, accompanied by an increased rate of RA elimination and a 41% decrease in the RA concentration. In contrast, Cyp26a1 mRNA in the refed homozygotic knockdown reached only 2% of its extent in WT during refeeding, accompanied by a slower rate of RA catabolism and no decrease in liver RA, relative to fasting. Refed homozygous knockdown mice also had decreased Akt1 and 2 phosphorylation and pyruvate dehydrogenase kinase 4 (Pdk4) mRNA and increased glucokinase (Gck) mRNA, glycogen phosphorylase (Pygl) phosphorylation, and serum glucose, relative to WT. Fasted homozygous knockdown mice had increased glucagon/insulin relative to WT. These data indicate that Cyp26a1 participates prominently in moderating the postnatal liver concentration of endogenous RA and contributes essentially to glucoregulatory control.

Oral arsenic administration to humanizedUDP-glucuronosyltransferase1 neonatal mice induces UGT1A1 through a dependence on Nrf2 and PXR.

Inorganic arsenic (iAs) is an environmental toxicant that can lead to severe health consequences, which can be exacerbated if exposure occurs early in development. Here, we evaluated the impact of oral iAs treatment on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) mice. We found that oral administration of iAs to neonatal hUGT1 mice that display severe neonatal hyperbilirubinemia leads to induction of intestinal UGT1A1 and a reduction in total serum bilirubin values. Oral iAs administration accelerates neonatal intestinal maturation, an event that is directly associated with UGT1A1 induction. As a reactive oxygen species producer, oral iAs treatment activated the Keap-Nrf2 pathway in the intestinal tract and liver. When Nrf2-deficient hUGT1 mice (hUGT1/Nrf2-/-) were treated with iAs, it was shown that activated Nrf2 contributed significantly toward intestinal maturation and UGT1A1 induction. However, hepatic UGT1A1 was not induced upon iAs exposure. We previously demonstrated that the nuclear receptor PXR represses liver UGT1A1 in neonatal hUGT1 mice. When PXR was deleted in hUGT1 mice (hUGT1/Pxr-/-), derepression of UGT1A1 was evident in both liver and intestinal tissue in neonates. Furthermore, when neonatal hUGT1/Pxr-/- mice were treated with iAs, UGT1A1 was superinduced in both tissues, confirming PXR release derepressed key regulatory elements on the gene that could be activated by iAs exposure. With iAs capable of generating reactive oxygen species in both liver and intestinal tissue, we conclude that PXR deficiency in neonatal hUGT1/Pxr-/- mice allows greater access of activated transcriptional modifiers such as Nrf2 leading to superinduction of UGT1A1.

Tissue-, Region-, and Gene-Specific Induction of Microsomal Epoxide Hydrolase Expression and Activity in the Mouse Intestine by Arsenic in Drinking Water.

This study aimed to characterize the effects of arsenic exposure on the expression of microsomal epoxide hydrolase (mEH or EPHX1) and soluble epoxide hydrolase (sEH or EPHX2) in the liver and small intestine. C57BL/6 mice were exposed to sodium arsenite in drinking water at various doses for up to 28 days. Intestinal, but not hepatic, mEH mRNA and protein expression was induced by arsenic at 25 ppm, in both males and females, whereas hepatic mEH expression was induced by arsenic at 50 or 100 ppm. The induction of mEH was gene specific, as the arsenic exposure did not induce sEH expression in either tissue. Within the small intestine, mEH expression was induced only in the proximal, but not the distal segments. The induction of intestinal mEH was accompanied by increases in microsomal enzymatic activities toward a model mEH substrate, cis-stilbene oxide, and an epoxide-containing drug, oprozomib, in vitro, and by increases in the levels of PR-176, the main hydrolysis metabolite of oprozomib, in the proximal small intestine of oprozomib-treated mice. These findings suggest that intestinal mEH, playing a major role in converting xenobiotic epoxides to less reactive diols, but not sEH, preferring endogenous epoxides as substrates, is relevant to the adverse effects of arsenic exposure, and that further studies of the interactions between drinking water arsenic exposure and the disposition or possible adverse effects of epoxide-containing drugs and other xenobiotic compounds in the intestine are warranted. SIGNIFICANCE STATEMENT: Consumption of arsenic-contaminated water has been associated with increased risks of various adverse health effects, such as diabetes, in humans. The small intestinal epithelial cells are the main site of absorption of ingested arsenic, but they are not well characterized for arsenic exposure-related changes. This study identified gene expression changes in the small intestine that may be mechanistically linked to the adverse effects of arsenic exposure and possible interactions between arsenic ingestion and the pharmacokinetics of epoxide-containing drugs in vivo.

Impact of flaxseed supplementation on lipid profile and liver enzymes in patients with non-alcoholic fatty liver disease: Systematic review and meta-analysis of randomized controlled trials.

Since the effects of flaxseed supplementation on lipid profile and liver enzymes are still controversial, a meta-analysis of randomized controlled trials was conducted in the present study to assess the effect of flaxseed supplementation on lipid profile and liver enzymes. The study was designed, conducted, and reported according to the guidelines of the 2020 preferred reporting items for systematic reviews and meta-analysis (PRISMA) statement. A systematic and comprehensive search was performed in several databases from inception up to January 10, 2024. The meta-analysis on the impact of flaxseed supplementation on lipid profile and liver enzymes indicates that the overall effect of flaxseed supplementation on triglycerides, combining different doses, revealed a significant reduction with a WMD of - 230.72 (-53.95, - 27.49) and a P-value of 0.010. High-density lipoprotein (HDL) demonstrated a positive effect, with an overall WMD of 1.82 (0.27, 3.38) and a P-value of 0.021, indicating an increase in HDL levels. The liver enzymes AST and ALT displayed reductions in their levels, with overall WMDs of - 21.18 (-2.95, 0.59) and - 24.83 (-8.74, - 20.91), respectively. Subgroup analysis based on dosage revealed more pronounced reductions in ALT levels for doses below 2000 mg/day. Findings from this study suggest that a flaxseed supplement might be beneficial to modulate the blood lipid profile and liver enzymes.

Relationship between changes in blood glucose, glycosylated hemoglobin [HbA1c], cholesterol, and triglyceride and changes in hepatic enzymes.

The main risk factors for non-alcoholic fatty liver disease (NAFLD) are strongly associated with obesity, diabetes, hyperlipidemia, and metabolic syndrome. The best clinical evaluation of the liver is done through studying changes in liver enzymes' activity, especially alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. Therefore, this study aimed to investigate the relationship between changes in factors such as blood glucose, cholesterol, glycosylated hemoglobin, and triglyceride and changes in hepatic enzymes in patients who visited Fajr Hospital in Tehran. Samples with SGPT levels > 40 U/L were selected and blood samples from the same individuals were collected in the next testing which was six months later. The changes in four factors of blood glucose, glycosylated hemoglobin, cholesterol, and triglyceride were calculated in these two consecutive visits, and finally, they were compared with changes in the hepatic enzymes and the relationship between them was evaluated by SPSS V. 23. Fifty-seven individuals with a mean age of 48 ± 15 years and SGPT > 40 U/L were included in the present study. Six samples were female (10.52 %) and 51 samples were male (89.48 %). The results showed that there was no significant relationship between blood glucose and glycosylated hemoglobin changes and hepatic enzymes. However, there was a significant relationship between cholesterol and triglyceride changes and hepatic enzymes of SGPT and SGOT (p ˂ 0.05). Based on the results of the current study, changes in FBS and HbA1c in two consecutive visits cannot be used to follow up on the treatment of fatty liver. However, changes in cholesterol and triglyceride can be used for monitoring the treatment in people with abnormal levels of hepatic enzymes.

The relationship between liver enzymes, prehypertension and hypertension in the Azar cohort population.

BACKGROUND: The incidence of hypertension (HTN) as a worldwide health problem is rising rapidly. Early identification and management of pre-HTN before HTN development can help reduce its related complications. We evaluated the relationship between liver enzymes levels and pre-HTN/HTN in the Azar cohort population. METHOD: This cross-sectional study was based on data from the large Azar cohort study and a total of 14,184 participants were included. Pre-HTN and HTN were defined based on the American Heart Association guideline. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT) levels were measured by Pars Azmoon kits. The relationship between pre-HTN/HTN and liver enzyme levels was evaluated by logistic regression. RESULTS: Of 14,184 participants, 5.7% and 39.6% had pre-HTN and HTN, respectively. In the adjusted model, AST levels of 19-23 IU/l were associated with an elevated risk of pre-HTN (OR [95% CI]: 1.24 [1.04-1.48]). A dose-response increase was seen in pre-HTN in relation to ALT, with the highest OR in the third tertile (1.34 [1.09-1.63]). The odds of pre-HTN also increased with GGT in the third tertile (1.25[1.03-1.52]). In addition, the odds of HTN increased with increased levels of AST, ALT, ALP, and GGT, such that the highest ORs were recorded in the third tertile (OR 1.22 [1.09-1.37], 1.51 [1.35-1.70], 1.19 [1.07-1.34], and 1.68 [1.49-1.89], respectively). Among these enzymes, GGT had the highest OR regarding HTN. CONCLUSION: This study indicates that AST, ALT, ALP and GGT levels were associated with pre-HTN (except for ALP) and HTN, independent of known risk factors. Hence, it may be possible to use liver enzymes to predict the incidence of pre-HTN and HTN, empowering primary care providers to make the necessary interventions promptly.