BACKGROUND:MASLD/MASH can occur in lean individuals; however, the environmental triggers and molecular mechanisms underlying lean MASH are unclear, and suitable animal models are lacking. RESULTS:Mice fed a Western diet with liquid fructose (WDF) develops obesity and MASH (obese MASH). In contrast, high salt supplementation of WDF (HSWDF) produced a lean MASH phenotype with reduced steatosis but induced significant inflammation and fibrosis (lean MASH). In WDF-induced obese MASH, we observed decreased urea cycle activity and flux, along with reduced eukaryotic translation initiation factor 5 A hypusination (EIF5AH) and mitochondrial biosynthesis. High salt supplementation of WDF unexpectedly ameliorated these alterations, enhanced hepatic fatty acid oxidation and reduced hepatosteatosis. However, single-cell sequencing revealed that dietary high salt was associated with pro-inflammatory responses in hepatic immune cell subpopulations. CONCLUSIONS:In summary, we have established a dietary mouse model of lean MASH that differs from obese-MASH in hepatic urea cycle, mitochondrial protein synthesis, and immune cell activation, providing new mechanistic insight into lean MASH.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an entity closely linked to obesity and/or insulin resistance, projected to affect about half of the global adult population by 2040. New therapeutic strategies that are both effective and affordable are needed to address this impending public health crisis. A narrative review was conducted to identify studies supporting the use of levothyroxine and thyroid hormone analogs in treating MASLD and metabolic dysfunction-associated steatohepatitis (MASH). Clinical studies have identified clear pathophysiological links between low (or low-normal) thyroid function and increased risk of hepatic steatosis. Of the two main thyroid hormone receptor (THR) isoforms, THRα and THRβ, the latter primarily mediates the metabolic effects of thyroid hormones in the liver, which could greatly benefit patients with MASLD. Recently, selective analogs of hepatic THRβ receptors have been developed to treat MASLD, with early clinical data demonstrating an effective profile. However, due to the increasing prevalence of MASLD in both developed and developing countries, the high cost of branded drugs may hinder their widespread use. Although the development of thyromimetics mainly aims to prevent activation of THRα in other tissues, there is evidence supporting the efficacy and safety of low-dose levothyroxine (LT4) therapy for treating MASLD. This review aims to systematically explore the potential of LT4 therapy as an alternative approach to managing MASLD.

Background: The liver and thyroid have complicated effects on each other's functions and metabolic homeostasis in the body. Both hypo- and hyperthyroidism influence hepatic carbohydrate and fat metabolism to regulate circulating glucose, cholesterol, and triglyceride levels. Hypothyroidism and "intrahepatic" hypothyroidism also contribute to the development of hypercholesterolemia and metabolic dysfunction-associated steatotic liver disease (MASLD). Likewise, hepatic dysfunction can modulate thyroid function by reducing thyroid hormone (TH) concentrations and their effects on peripheral tissues.Summary: In this review, we examine the impact of thyroid disorders and their treatment on hepatic physiology, metabolism, and pathology, as well as the influence of liver disease on thyroid function. We also describe the clinical and experimental evidence for THs playing significant roles in metabolic conditions such as metabolic syndrome, hyperlipidemia, and MASLD. Additionally, we summarize the current literature on the use of thyromimetics for the treatment of metabolic diseases.Conclusions: Recognizing the effects of the thyroid and THs on hepatic metabolism and fuel utilization, and the liver's role in modulating systemic TH action, is important for optimal clinical management of patients with thyroid and/or liver diseases. New emerging concepts on TH actions in the liver and the efficacy of thyromimetics for the treatment of MASLD have reshaped our understanding of the thyroid-liver relationship and the roles of THs in the pathogenesis and treatment of metabolic diseases.

Glycine plays a central role in human metabolism, and an adequate supply is required for synthesizing glutathione (GSH), eliminating excess metabolites as acylglycine via the glycine conjugation detoxification pathway, and maintaining 1-carbon cycle activity. However, glycine is deficient in individuals with severe obesity, which may compromise these pathways and metabolic health. This exploratory study examines whether dietary glycine supplementation could correct glycine deficiency and impairments in glycine-dependent metabolic pathways. 19 participants with severe obesity (BMI 38.3 ± 5.3 kg/m2) were treated with dietary glycine (100 mg/kg/day) for two weeks. We found that treatment significantly increased the plasma concentration of glycine and enhanced the urinary excretion of isobutyrylglycine, tigylglycine, isovalerylglycine, and hexanoylglycine. There were no changes in body weight but significant reductions in plasma triglyceride and aminotransferases. The glutamate-serine-glycine index, an indirect marker of metabolic dysfunction-associated liver disease (MASLD), also improved. Treatment did not affect GSH but raised the plasma concentrations of serine, homocysteine, cysteine, and folate, which are 1-carbon cycle metabolites. We conclude that dietary glycine supplementation reversed obesity-associated glycine deficiency and enhanced the glycine conjugation detoxification reaction, 1-carbon cycle flux, and potentially the severity of MASLD. Glycine supplementation should be further investigated as a novel treatment for MASLD.Clinical trials registry number NCT04658134 ( https://tinyurl.com/7rthbwjb ).

Abstract Disclosure: M. Tripathi: None. S. Sakthivel: None. A. Suzuki: None. B.K. Singh: None. P.M. Yen: None. Introduction: NAFLD (nonalcoholic fatty liver disease) and its more severe form, NASH (nonalcoholic steatohepatitis) are sexually dimorphic conditions that exhibit higher prevalence in men than women during reproductive ages but have similar prevalence after menopause. Reproductive age women are protected from hepatic fibrosis relative to men, although they lose this protection after menopause. These sex differences in NASH have been attributed, at least partly, to different circulating estrogen levels. However, the underlying mechanism(s) is not fully understood. We thus conducted animal experiments to investigate: 1) sex differences in the histologic severity of NASH, necroinflammatory and fibrogenic activities, and other relevant pathways in young and old male and female mice and 2) the effects of estradiol administration on these parameters in old male and female mice to evaluate the therapeutic potential of estradiol in NASH. Methodology: Young/reproductive age (12-weeks old) and old/post-reproductive age female and male (48∼50-weeks weeks old) C57BL/6J mice were fed control chow diet (NCD) or high-fat methionine/choline-deficient (MCD) diet for six weeks to induce NASH. The old mice were administered 200nM 17β-estradiol (physiological) in drinking water. We analyzed serum and hepatic NASH parameters, ER stress, and lysosome-autophagy defects. Results and Conclusion: Among reproductive age mice with NASH, females developed less severe histologic inflammation and fibrosis and decreased gene expression of Il1b, Il6, Tgfb, Col1a1, Ccl2, and Ccl5, and lower hepatic collagen than males. Females with NASH also had less inhibition of hepatic autophagy and lysosomal protein expression than males with NASH. Among old mice, post-reproductive age females fed NCD or MCD exhibited more severe hepatic inflammation and fibrosis and had higher gene expression of inflammatory and fibrogenic markers with comparable autophagic inhibition and lysosomal defects as similar age males. Physiological doses of estradiol significantly reduced hepatic gene expression of inflammatory and fibrogenic markers, ER stress and lysosomal-autophagic defects in post-reproductive age females. In conclusion, similar to clinical NASH, we observed marked sex and age-related differences in the severity of NASH. Furthermore, physiological doses of estradiol treatment improved the hepatic histology, lysosome-autophagy, inflammation, and fibrosis in post-reproductive age female mice with NASH and suggests that estradiol supplementation may decrease or reverse NASH progression in post-menopausal women with NASH. Presentation: 6/2/2024

OBJECTIVES:Stroke is common following left ventricular assist device (LVAD) implantation, although comprehensive data on perioperative strokes in this uncommon population is lacking. The current study aim was to characterize the presentation, features, and outcomes of perioperative cerebrovascular ischemia post-LVAD implantation at the authors' institution. DESIGN:Single-center retrospective cohort. SETTING:St. Paul's Hospital, Vancouver, British Columbia, Canada. PARTICIPANTS:Adult patients who received an LVAD between January 1, 2008, and August 31, 2021, were included, and those who died intraoperatively or underwent a concurrent cardiac surgical procedure were excluded. INTERVENTIONS:Data on demographics, comorbidities, stroke risk factors and characteristics, management, and outcomes (transplant, explant, death with LVAD in situ) were extracted. MEASUREMENTS AND MAIN RESULTS:After exclusions, 172 adult patients who underwent LVAD implantation during the study period were included and analyzed. The rate of perioperative stroke was 12.8% (22/172). Of these, 72.7% (16/22) had a stroke occur within 7 days of surgery, and 86.4% (19/22) had a primarily ischemic (v hemorrhagic) event. A total of 68.2% (15/22) were intubated, sedated, or recently extubated at symptom onset, complicating diagnosis. All were managed supportively or palliated without specific stroke intervention. Patients who experienced a perioperative stroke had a significantly lower cumulative incidence of survival to cardiac transplantation and a significantly higher cumulative incidence of dying with their device in situ. CONCLUSIONS:LVAD patients carry a high risk of perioperative stroke. They experience delayed recognition and diagnosis, limited intervention, and poor outcomes. Frequent neurological assessment and a low threshold for neuroimaging are prudent.

Abstract Disclosure: M. Tripathi: None. K. Gauthier: None. R. Sandireddy: None. S. Park: None. V. Giguere: None. P.K. Chow: None. S. Ghosh: None. D.P. McDonnell: None. P.M. Yen: None. B. Singh: None. The regulation of global and specific protein synthesis during the progression of metabolic dysfunction-associated steatohepatitis (MASH) is poorly understood. Accordingly, we performed a comprehensive analyses of protein translation by employing label-free quantitative proteomics, puromycin-labeling, and polysome profiling, and found a reduction in protein synthesis during lipotoxic stress in vitro and in liver tissues from MASH mouse models. We also found that expression of estrogen receptor-related receptor-α(ERRa/Esrra) and ribosomal Rplp1 were significantly downregulated at protein levels. Additionally, Esrra recruitment was decreased on the Rplp1 promoter, and led to diminished Rplp1 mRNA and protein expression. Taken together, these events reduced overall translation activity and the specific translation of lysosomal (e.g.,Lamp2, Ctsd) and autophagy (e.g., Sqstm1, Map1lc3b) proteins during MASH. Moreover, Esrra-Rplp1-mediated translation of these lysosomal and autophagy proteins and autophagy was compromised in MASH patients and liver-specific Esrra-deficient mice. Remarkably, alternate day fasting induced hepatic Esrra expression and reactivated Esrra-Rplp1 signaling in mice with MASH, leading to the re-expression of autophagy and lysosomal proteins to restore autophagy and reduce lipotoxicity, inflammation, and fibrosis. Thus, the Esrra-Rplp1-mediated translation of lysosomal and autophagy proteins critical for preventing lipotoxicity, inflammation, and fibrosis was suppressed during MASH. Reactivation of this pathway by intermittent fasting was able to reverse MASH. Our findings showed that Esrra not only regulated the transcription of genes involved in lipid metabolism, but also the specific translation of autophagy and lysosomal proteins via induction of Rplp1; thus, providing a molecular explanation for the beneficial hepatic effects of alternate day fasting in MASH. Presentation: 6/2/2024

Thyroid hormones (THs) are key hormones that regulate development and metabolism in mammals. In man, the major target tissues for TH action are the brain, liver, muscle, heart, and adipose tissue. Defects in TH synthesis, transport, metabolism, and nuclear action have been associated with genetic and endocrine diseases in man. Over the past few years, there has been renewed interest in TH action and the therapeutic potential of THs and thyromimetics to treat several metabolic disorders such as hypercholesterolemia, dyslipidaemia, non-alcoholic fatty liver disease (NAFLD), and TH transporter defects. Recent advances in the development of tissue and TH receptor isoform-targeted thyromimetics have kindled new hope for translating our fundamental understanding of TH action into an effective therapy. This review provides a concise overview of the historical development of our understanding of TH action, its physiological and pathophysiological effects on metabolism, and future therapeutic applications to treat metabolic dysfunction. Sinha and Yen provide a comprehensive overview of thyroid hormones and their role in development and in regulation of whole-body metabolic homeostasis

Aim: Hepatic homocysteine (Hcy) accumulation promotes inflammation and fibrosis in experimental nonalcoholic fatty liver disease (NAFLD), while vitamin B12 and folate reduce hepatic Hcy and protect animals from nonalcoholic steatohepatitis. This suggests clinical implications for preventing/treating patients with NAFLD. Given the known sex-specific regulation of one-carbon metabolism (OCM), the response to various OCM cofactors may vary by sex and reproductive status. We aimed to strategize an effective Hcy-lowering treatment in broader NAFLD patients while discerning disparities in treatment responses. Methods: We analyzed existing hepatic microarray data relevant to Hcy metabolism with clinical and histologic data from patients with NAFLD (N = 82), while considering potential age/sex disparities. Additionally, we performed computer simulation analyses using a mathematical model of OCM to predict hepatic Hcy-lowering effects of OCM cofactors by sex. Results: Of 82 patients with NAFLD, 98% had at least one metabolic feature [i.e., metabolic dysfunction-associated steatotic liver disease (MASLD)]. Lower hepatic gene expressions of cystathionine-beta synthase (CBS ) and phosphatidyl- ethanolamine N-methyltransferase (PEMT ) were associated with more severe fibrosis in NAFLD, while sub-analysis suggested possible variations by age and sex. The simulation analysis demonstrated sex differences in the Hcy-lowering effects of the OCM cofactors (vitamins B6 and B12, folate, and betaine), with the combination of these cofactors consistently showing the maximum Hcy-lowering effect in both sexes. Conclusion: We theorize that the combination of OCM cofactors would maximize Hcy-lowering effects in the broader MASLD population. Our findings also underscore the importance of considering sex and age in designing future studies on homocysteine metabolism.

OBJECTIVE:Currently, little is known about the mechanism(s) regulating global and specific protein translation during metabolic dysfunction-associated steatohepatitis (MASH; previously known as non-alcoholic steatohepatitis, NASH). METHODS:Unbiased label-free quantitative proteome, puromycin-labelling and polysome profiling were used to understand protein translation activity in vitro and in vivo. RESULTS:We observed a global decrease in protein translation during lipotoxicity in human primary hepatocytes, mouse hepatic AML12 cells, and livers from a dietary mouse model of MASH. Interestingly, proteomic analysis showed that Rplp1, which regulates ribosome and translation pathways, was one of the most downregulated proteins. Moreover, decreased Esrra expression and binding to the Rplp1 promoter, diminished Rplp1 gene expression during lipotoxicity. This, in turn, reduced global protein translation and Esrra/Rplp1-dependent translation of lysosome (Lamp2, Ctsd) and autophagy (sqstm1, Map1lc3b) proteins. Of note, Esrra did not increase its binding to these gene promoters or their gene transcription, confirming its regulation of their translation during lipotoxicity. Notably, hepatic Esrra-Rplp1-dependent translation of lysosomal and autophagy proteins also was impaired in MASH patients and liver-specific Esrra knockout mice. Remarkably, alternate day fasting induced Esrra-Rplp1-dependent expression of lysosomal proteins, restored autophagy, and reduced lipotoxicity, inflammation, and fibrosis in hepatic cell culture and in vivo models of MASH. CONCLUSIONS:Esrra regulation of Rplp1-mediated translation of lysosome/autolysosome proteins was downregulated during MASH. Alternate day fasting activated this novel pathway and improved MASH, suggesting that Esrra and Rplp1 may serve as therapeutic targets for MASH. Our findings also provided the first example of a nuclear hormone receptor, Esrra, to not only regulate transcription but also protein translation, via induction of Rplp1.

Thyroid hormones (triiodothyronine and thyroxine) are pivotal for metabolic balance in the liver and entire body. Dysregulation of the hypothalamus–pituitary–thyroid axis can contribute to hepatic metabolic disturbances, affecting lipid metabolism, glucose regulation and protein synthesis. In addition, reductions in circulating and intrahepatic thyroid hormone concentrations increase the risk of metabolic dysfunction-associated steatotic liver disease by inducing lipotoxicity, inflammation and fibrosis. Amelioration of hepatic metabolic disease by thyroid hormones in preclinical and clinical studies has spurred the development of thyromimetics that target THRB (the predominant thyroid hormone receptor isoform in the liver) and/or the liver itself to provide more selective activation of hepatic thyroid hormone-regulated metabolic pathways while reducing thyrotoxic side effects in tissues that predominantly express THRA such as the heart and bone. Resmetirom, a liver and THRB-selective thyromimetic, recently became the first FDA-approved drug for metabolic dysfunction-associated steatohepatitis (MASH). Thus, a better understanding of the metabolic actions of thyroid hormones and thyromimetics in the liver is timely and clinically relevant. Here, we describe the roles of thyroid hormones in normal liver function and pathogenesis of MASH, as well as some potential clinical issues that might arise when treating patients with MASH with thyroid hormone supplementation or thyromimetics. Thyroid hormones, triiodothyronine and thyroxine, are vital for metabolic homeostasis, and regulate essential hepatic functions. This Review summarizes the current knowledge of the role of thyroid hormones in the liver and examines the development of thyromimetics for liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) originates from a homeostatic imbalance in hepatic lipid metabolism. Increased fat deposition in the liver of people suffering from MASLD predisposes them to develop further metabolic derangements, including diabetes mellitus, metabolic dysfunction-associated steatohepatitis (MASH), and other end-stage liver diseases. Unfortunately, only limited pharmacological therapies exist for MASLD to date. Autophagy, a cellular catabolic process, has emerged as a primary mechanism of lipid metabolism in mammalian hepatocytes. Furthermore, preclinical studies with autophagy modulators have shown promising results in resolving MASLD and mitigating its progress into deleterious liver pathologies. In this review, we discuss our current understanding of autophagy-mediated hepatic lipid metabolism, its therapeutic modulation for MASLD treatment, and current limitations and scope for clinical translation.

Non-alcoholic steatohepatitis (NASH) is a condition characterized by inflammation and hepatic injury/fibrosis caused by the accumulation of ectopic fats in the liver. Recent advances in lipidomics have allowed the identification and characterization of lipid species and have revealed signature patterns of various diseases. Here, we describe a lipidomics workflow to assess the lipid profiles of liver homogenates taken from a NASH mouse model. The protocol described below was used to extract and analyze the metabolites from the livers of mice with NASH by liquid chromatography-mass spectrometry (LC-MS); however, it can be applied to other tissue homogenate samples. Using this method, over 1,000 species of lipids from five classes can be analyzed in a single run on the LC-MS. Also, partial elucidation of the identity of neutral lipid (triacylglycerides and diacylglycerides) aliphatic chains can be performed with this simple LC-MS setup. Key features Over 1,000 lipid species (sphingolipids, cholesteryl esters, neutral lipids, phospholipids, fatty acids) are analyzed in one run. Analysis of liver lipids in non-alcoholic steatohepatitis (NASH) mouse model. Normal-phase chromatography coupled to a triple quadrupole mass spectrometer.

LINC00116 encodes a microprotein first identified as Mitoregulin (MTLN), where it was reported to localize to the inner membrane of mitochondria to regulate fatty acid oxidation and oxidative phosphorylation. These initial discoveries were followed by reports with differing findings about its molecular functions and submitochondrial localization. To clarify the apparent discrepancies, we constructed multiple orthogonal methods of determining the localization of MTLN, including split GFP-based reporters that enable efficient and reliable topology analyses for microproteins. These methods unequivocally demonstrate MTLN primarily localizes to the outer membrane of mitochondria, where it interacts with enzymes of fatty acid metabolism including CPT1B and CYB5B. Loss of MTLN causes the accumulation of very long-chain fatty acids (VLCFAs), especially docosahexaenoic acid (DHA). Intriguingly, loss of MTLN protects mice against western diet/fructose-induced insulin-resistance, suggests a protective effect of VLCFAs in this context. MTLN thus serves as an attractive target to control the catabolism of VLCFAs.

Nonalcoholic steatohepatitis (NASH) is considered a pivotal stage in nonalcoholic fatty liver disease (NAFLD) progression and increases the risk of end-stage liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The etiology of NASH is multifactorial and identifying reliable molecular players has proven difficult. Presently, there are no approved drugs for NASH treatment, which has become a leading cause of liver transplants worldwide. Here, using public human transcriptomic NAFLD dataset, we uncover Cystic fibrosis transmembrane conductance receptor (CFTR) as a differentially expressed gene in the livers of human NASH patients. Similarly, murine Cftr expression was also found to be upregulated in two mouse models of diet-induced NASH. Furthermore, the pharmacological inhibition of CFTR significantly reduced NASH progression in mice and its overexpression aggravated lipotoxicity in human hepatic cells. These results, thus, underscore the involvement of murine Cftr in the pathogenesis of NASH and raise the intriguing possibility of its pharmacological inhibition in human NASH.