Introduction and Objective: Tirzepatide, a dual GLP-1 and GIP receptor agonist, enhances glucose-stimulated insulin secretion, significantly reduces HbA1c, and promotes weight loss. Tirzepatide use leads to improvements in insulin sensitivity, both via weight loss-dependent and independent mechanisms. Pioglitazone, a PPARγ activator, enhances insulin sensitivity via distinct pathways. We hypothesized that combining Tirzepatide and pioglitazone would result in additive benefits for insulin action and adipose tissue remodeling. Methods: 32 male Sprague Dawley rats (6-8 weeks old) were fed high-fat diet for 12 weeks, then were treated with placebo, pioglitazone (10 mg/kg), Tirzepatide (100 nmol/kg), or their combination, for 8weeks. Body weight, fasting blood glucose (FBG), and calorie intake were monitored weekly. Oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were conducted at study end, and liver, adipose, muscle, and pancreatic tissues were collected. White adipose tissue single-nucleus RNASeq was performed to assess changes in the cellular makeup of the tissue. Results: Final body weights were 671.3 ± 26.2 g (placebo), 689.1 ± 23.4 g (pioglitazone), 532.3 ± 17.8 g(Tirzepatide), and 534.1 ± 18.6 g (combination). The combination group showed significant additive reductions in glucose area under the curve (AUC) compared to Tirzepatide alone (p-Value <0.05). Brown adipose tissue weight increased significantly with pioglitazone, alone or in combination, compared to Tirzepatide alone. Single-nucleus RNASeq analysis revealed a shift in adipocytic transcriptional profile from a more metabolically stressed to a metabolically healthy phenotype with the use of the drugs. Conclusion: Tirzepatide mitigated pioglitazone-induced weight gain, while their combination provided synergistic improvements in insulin sensitivity and adipocyte physiology. Disclosure M. Abu-Farha: None. D. Vatner: None. M. Qaddoumi: None. M. Abdul-Ghani: None. J. Golla: None. I. Al Khairi: None. A. Al Madhoun: None. Y. Al Sanae: None. F. Al-Mulla: None. N. Abukhalaf: None. J.A. Abubaker: None. Funding Kuwait Foundation for the Advancement of Sciences (KFAS). RA AM2023-029

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease globally. Disruptions in iron metabolism and mitochondrial oxidative function may cooperatively contribute to its pathogenesis. Ferredoxin reductase (FDXR), a mitochondrial flavoprotein, plays a critical role in mitochondrial respiratory supercomplex formation and iron-sulfur cluster biosynthesis-both essential for efficient oxidative metabolism. However, its role in MASLD remains unclear. Here, we knocked down hepatic Fdxr expression in the liver of C57BL/6 mice using N-acetyl galactosamineconjugated antisense oligonucleotides. [13C5]glutamine tracer infusions revealed that FDXR deficiency disrupted mitochondrial oxidative phosphorylation. In contrast, FDXR deficiency increased hepatic iron accumulation, reactive oxygen species, and lipid peroxidation. Mechanistically, FDXR deficiency disrupted iron-sulfur cluster assembly and reduced mitochondrial proteins such as succinate dehydrogenase complex iron-sulfur subunit B (SDHB), leading to mitochondrial dysfunction and steatosis. FDXR expression was upregulated in both human and murine MASLD livers, suggesting a compensatory protective response. Furthermore, hepatic overexpression of FDXR restored mitochondrial function, enhanced oxidative capacity, and ameliorated steatosis. These findings identify FDXR as a key regulator linking iron metabolism and mitochondrial integrity in MASLD and highlight its potential as a therapeutic target to prevent disease progression.

Increased de novo lipogenesis (DNL) contributes to hyperlipidemia, MASLD, and ASCVD in insulin-resistant subjects. However, multiple pathways support lipogenesis and few have sought to quantify the contributions of the discrete metabolic pathways that contribute to lipogenesis. In this study, antisense oligonucleotides (ASOs) targeting glucokinase (Gck), lactate dehydrogenase A (Ldha), and glutamic-pyruvic transaminase 2 (Gpt2) were utilized to restrict substrate flux from lipogenic precursors in C57BL6/J mice, comparing controls (CO) and chronic overnutrition (ON). In CO mice, ASO treatments did not significantly alter lipogenesis; however, there was a trend toward decreased hepatic triglyceride content and DNL, especially with the GPT2 ASO (TG = -46.8%; DNL = -53.7%). Expectedly, increased hepatic TG content and DNL (ON vs. CO: TG = +187.9%; DNL = +41.8%) were observed in mice with chronic overnutrition. Gas chromatography-mass spectrometry analyses demonstrated increased hepatic TCA cycle metabolites (ON vs. CO: fumarate +74.2%; malate +54.0%; and citrate +43.2) and decreased hepatic concentrations of multiple amino acids (ON vs. CO: Leu -41.7%; Ile -45.0%; Val -56.3%; Ser -22.6%). With ON, TG content and DNL were reduced by restricting lipogenic carbon entry from alanine (GPT2: TG = -45.5%; DNL = -48.1%), lactate (LDHA: TG = -25.8%; DNL = -33.1%), or glucose (GCK: TG = -59.2%; DNL = -69.2%). Amino acids appear to be a consistent carbon source for DNL in mice; however, carbon entry from all sources is required to maintain the significantly elevated rates of hepatic DNL in chronically overfed mice. These findings may inform the development of novel therapies and underscore the importance of peripheral substrate storage and oxidation in the prevention of dyslipidemia in the metabolic syndrome.

BACKGROUNDThis study examined the underlying cellular mechanisms associated with insulin resistance (IR) and metabolic disease risk within subcutaneous adipose tissue (SAT) in youth with obesity and IR compared with those without IR.METHODSThirteen adolescents who were insulin sensitive (IS) and 17 adolescents with IR and obesity underwent a 3-hour oral glucose tolerance test and MRI to measure abdominal fat distribution and liver fat content. Lipolysis was determined by glycerol turnover ([2H5]-glycerol infusion) and adipose triglyceride lipase (ATGL) phosphorylation (Western blot) from SAT samples biopsied prior to and 30-minutes following insulin infusion during a hyperinsulinemic-euglycemic clamp (HEC).RESULTSGlycerol turnover suppression during the HEC (first step) was lower in participants with IR compared with those with IS. Prior to insulin infusion, activated ATGL (reflected by the p-ATGL (Ser406)-to-ATGL ratio) was greater in participants with IR compared with those with IS and suppressed in response to a 30-minute insulin exposure in participants with IS, but not in those with IR. Lastly, greater ATGL inactivation is associated with greater glycerol suppression and lower liver fat.CONCLUSIONSInsulin-mediated inhibition of adipose tissue lipolysis via ATGL is dysregulated among adolescents with IR compared with those with IS, thereby serving as a vital mechanism linking glucose and insulin dysregulation and ectopic lipid storage within the liver.FUNDINGThis work was supported by funding from the NIH (R01-HD028016-25A1, T32- DK-007058, R01-DK124272, RO1-DK119968, R01MD015974, RO1-DK113984, P3-DK045735, RO1-DK133143, and RC2-DK120534) and the Robert E. Leet and Clara Guthrie Patterson Trust Mentored Research Award.

Metabolic dysfunction-associated steatohepatitis (MASH) represents a progressive form of steatotic liver disease which increases the risk for fibrosis and advanced liver disease. The accumulation of discrete species of bioactive lipids has been postulated to activate signaling pathways that promote inflammation and fibrosis. However, the key pathogenic lipid species is a matter of debate. We explored candidates using various dietary, molecular, and genetic models. Mice fed a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) developed steatohepatitis and manifested early markers of liver fibrosis associated with increased cholesterol content in liver lipid droplets within 5 d without any changes in total liver cholesterol content. Treating mice with antisense oligonucleotides against Coenzyme A synthase (Coasy) or treatment with bempedoic acid or atorvastatin decreased liver lipid droplet cholesterol content and prevented CDAHFD-induced MASH and the fibrotic response. All these salutary effects were abrogated with dietary cholesterol supplementation. Analysis of human liver samples demonstrated that cholesterol in liver lipid droplets was increased in humans with MASH and liver fibrosis and was higher in PNPLA3 I148M (variants rs738409) than in HSD17B13 variants (rs72613567). Together, these data identify cholesterol in liver lipid droplets as a critical mediator of MASH and demonstrate that Coenzyme A synthase knockdown and bempedoic acid are therapeutic approaches to reduce liver lipid droplet cholesterol content and thereby prevent the development of MASH and liver fibrosis.

Introduction and Objective: White adipose tissue (WAT) insulin resistance (IR) is essential to the pathogenesis of metabolic disease; yet defects in human WAT insulin signaling are not well characterized. This study elucidates alterations to WAT insulin signaling associated with human IR. Methods: Human WAT was obtained from three cohorts of patients with obesity: 1) in a bariatric surgery cohort (RESOLVE), RNASeq was performed on WAT collected before and after weight loss; 2) in another cohort (SODA), glucose or fructose-sweetened beverages were given before WAT collection and proteomics analyses were performed; 3) in an adolescent cohort, immunoblotting and qPCR assessed WAT biopsied before or during hyperinsulinemic euglycemic clamps. Results: Attenuation of insulin-stimulated AKT phosphorylation in IR vs. relatively insulin sensitive (IS) adolescents was not significant (IR vs IS: -37.6%, p>0.1). Expectedly, GLUT4 decreased in IR (Resolve: log PC = -1.54, p<0.000000001; SODA: R2=0.367, p<0.05; Adolescent: -60.3%, p<0.05). TUG, which traps insulin-responsive GLUT4, was increased in IR (Resolve: log PC +0.39, p<0.05; SODA: R2=0.277, p<0.05; Adolescent: +48.5%, p<0.01). Gene expression throughout the TC10 pathway that mediates insulin-stimulated TUG cleavage was changed pre- versus post- bariatric surgery in the RESOLVE study. A subset of TC10 pathway proteins also changed in the SODA and Adolescent studies. Conclusion: In three cohorts, IR is correlated with increased WAT TUG. As well, molecular regulation of TC10 pathway is altered, underscoring the importance of TUG regulation to WAT metabolic health. As one of the first descriptions of altered signaling by this pathway in human WAT, this data can drive future therapeutics for patients with metabolic disease. J.W. Strober: None. K.W. ter Horst: None. A. Slusher: None. J.A. Paulo: None. B. Gassaway: None. S. Shuken: None. S. Caprio: None. M. Serlie: None. J. Bogan: None. D.F. Vatner: None. National Institutes of Health (DK124272); National Institutes of Health (DK007058)

Metabolic dysfunction-associated steatohepatitis (MASH) is a frequent complication of type 2 diabetes. However, the molecular mechanisms responsible for the development of MASH are unknown. Loss of function mutations in the lipid droplet proteins Comparative Gene Identification-58 (CGI-58) and Cell Death-inducing DNA fragmentation factor-like effector B (CIDEB) promote and protect against MASH, respectively. We hypothesized that the discrete impact of these proteins on MASH is due to the alteration of lipid droplet morphology followed by cholesterol content. We tested these hypotheses using antisense oligonucleotides (ASO) to knock down the expression of CGI-58 or CideB in the liver of a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) mouse MASH model. CGI-58 ASO treatment increased plasma ALT, macrophage crown-like structures, and liver inflammation/fibrosis marker expression, which were associated with increased lipid droplet size and liver lipid droplet cholesterol content. Co-treatment with an ASO against glycerol-3-phosphate acyltransferase, mitochondrial (Gpam) decreased liver lipid droplet cholesterol and prevented CGI-58 ASO-induced MASH. In contrast, CideB ASO treatment in CDAHFD mice prevented liver inflammation and fibrosis and reduced liver lipid droplet size and lipid droplet cholesterol content. These protective effects of the CideB ASO were all abrogated with cholesterol supplementation to the diet. Conclusions: Knockdown of CGI-58 promotes liver inflammation and fibrosis by increasing liver lipid droplet size and lipid droplet cholesterol content, whereas knockdown of CideB protects against the development of liver inflammation and fibrosis by reducing liver lipid droplet size and cholesterol content. Furthermore, CIDEB and GPAM are potential therapeutic targets for MASH. Disclosure I. Sakuma: None. R.C. Gaspar: None. A. Nasiri: None. M. Kahn: None. J. Zheng: None. M. Guerra: None. D. Yimlamai: None. S. Murray: Employee; Ionis Pharmaceuticals. M. Perelis: Employee; Ionis Pharmaceuticals. W. Barnes: Employee; Ionis Pharmaceuticals. D.F. Vatner: None. K. Petersen: None. V. Samuel: None. G.I. Shulman: None.

Abstract Disclosure: I. Sakuma: None. M. Fujimoto: None. D.F. Vatner: None. K. Yokote: None. T. Tanaka: None. Context [1]23I-metaiodobenzylguanidine scintigraphy (MIBG) and [1]8F-fluoro-2-doxy-D-glucose positron emission tomography (FDG-PET) are helpful for the localization of pheochromocytoma. The norepinephrine transporter (NET) and the glucose transporter 1 (GLUT1) are thought to be critical for the uptake of these radioisotopes. NET expression is activated by Paired Like Homeobox (PHOX2) and glucocorticoid receptor (GR) in neuroblastoma cells. Around 70% of pheochromocytomas carry well-known mutations. Pheochromocytoma-associated genes are divided into two clusters: the pseudohypoxia-related cluster 1 and the kinase signaling-related cluster 2. Cluster 1 mutation are associated with noradrenergic pheochromocytomas with an elevated metastatic risk. Cluster 2 tumors demonstrate an adrenergic phenotype with a less aggressive course. How mutations of cluster 1 and cluster 2 impact the expression of NET/GLUT1 and on radiotracer uptake remains unclear. Hypothesis Cluster 1 and cluster 2 will have different effects on the expression of transcription factors associated with chromaffin cell differentiation with resultant differential effects on NET/GLUT1 expression. Methods 42 patients with pheochromocytoma were evaluated by MIBG and FDG-PET. Tumor gene expression was evaluated by real-time qPCR, and mutation analyses were performed by Sanger or next-generation sequencing. Results 88% of tumors were MIBG-avid, and 67% were FDG-avid. The expression of NET in MIBG-negative tumors was 70% lower than in MIBG-positive tumors. The expression of GLUT1 in FDG-PET-negative tumors was 75% lower than in FDG-PET-positive tumors. The expression of NET positively correlated with transcription factors involved in chromaffin cell maturation, including PHOX2A, PHOX2B, GATA binding protein 3 (GATA3), GR, catecholamine synthases, and insulinoma associated 1 (INSM1). The JASPAR database for promoter analysis revealed putative responsive regions of the transcription factors PHOX2A, PHOX2B, GATA3, GR, and INSM1 on the NET gene. Seven patients (16.7%) had mutations in cluster 1, and fourteen patients (33.3%) had mutations in cluster 2. Cluster 2 exhibited elevated NET, PHOX2A, PHOX2B, GATA3, GR, and INSM1 expression levels compared to cluster 1. Cluster 1 showed a higher expression level of GLUT1 and BMP4 that is associated with early chromaffin cell differentiation. Conclusions In pheochromocytomas, MIBG and FDG uptake correlated with the expression levels of NET and GLUT1, respectively. NET expression is associated with chromaffin cell differentiation transcription factor expression. We propose that pheochromocytomas that carry Cluster 1 mutations are more likely to be missed in MIBG due to low NET expression, while pheochromocytomas that carry Cluster 2 mutations are more likely to be missed on FDG-PET due to low GLUT1 expression with a more mature chromaffin cell phenotype. Presentation: 6/2/2024

Background In rodents, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the conversion of inactive 11-dehydrocorticosterone to the active hormone corticosterone. Dysregulation of intracellular glucocorticoid action is implicated in metabolic diseases. Assessing 11β-HSD1 enzyme levels in vivo may be key to understanding obesity pathophysiology. Objective We used a Zucker Fatty (ZF) rat model and [ 18 F]AS2471907 PET imaging to determine appropriate kinetic modeling methods and assess changes in 11β-HSD1 levels due to obesity in the liver, white and brown adipose tissue (WAT/BAT), and brain. Material and Methods To validate [ 18 F]AS2471907 PET in preclinical models, time-activity curves (TACs) were generated and kinetic modeling was performed with image-derived input functions (IDIFs) extracted from multiple locations. Quantitative estimates of radioligand binding were compared with ex vivo 11β-HSD1 protein expression. Validated quantitative PET kinetic modeling methods were then used to assess differences in 11β-HSD1 between lean and obese ZF rats. Metabolic disease status was confirmed with stable isotopes tracer studies of glucose and fatty acid metabolism. Results Obesity is associated with decreased brain 11β-HSD1 levels, measured by [ 18 F]AS2471907 PET, which correlated with measures of glucose and fatty acid metabolism. Conclusion We demonstrate that [ 18 F]AS2471907 PET can provide useful quantification of 11β-HSD1 levels in a rodent model of obesity.

Metabolic associated fatty liver disease (MAFLD) has become one of the most common causes of chronic liver disease and cirrhosis in the United States and around the world.1Wong R.J. Aguilar M. Cheung R. et al.Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States.Gastroenterology. 2015; 148: 547-555Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 2Le M.H. Le D.M. Baez T.C. et al.Global incidence of non-alcoholic fatty liver disease: a systematic review and meta-analysis of 63 studies and 1,201,807 persons.J Hepatol. 2023; 79: 287-295Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 3Zhai M. Liu Z. Long J. et al.The incidence trends of liver cirrhosis caused by nonalcoholic steatohepatitis via the GBD study 2017.Sci Rep. 2021; 11: 5195Crossref PubMed Scopus (18) Google Scholar It is a heterogeneous condition, and although some patients remain at simple steatosis, others progress to nonalcoholic steatohepatitis (NASH), to cirrhosis, and to hepatocellular carcinoma. There are few tools available to the clinician treating this common problem, and both pathophysiology and pharmacology studies of MAFLD are actively pursued in rodents and human beings. Hepatic triglyceride biosynthesis is a logical target for the prevention and treatment of steatohepatitis. Fatty acids can enter the fatty acyl CoA esterification pathway in both lean insulin-sensitive animals and obese insulin-resistant animals.4Vatner D.F. Majumdar S.K. Kumashiro N. et al.Insulin-independent regulation of hepatic triglyceride synthesis by fatty acids.Proc Natl Acad Sci U S A. 2015; 112: 1143-1148Crossref PubMed Scopus (163) Google Scholar Acyl-CoA:glycerol-sn-3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting enzyme of glycerolipid synthesis, combining a fatty acid from an acyl CoA with glycerol-3-phosphate to form lysophosphatidic acid. Deletion of mitochondrial GPAT1 can prevent both hepatic triglyceride accumulation and hepatic insulin resistance (a hallmark of hepatic metabolic dysfunction).5Neschen S. Morino K. Hammond L.E. et al.Prevention of hepatic steatosis and hepatic insulin resistance in mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase 1 knockout mice.Cell Metab. 2005; 2: 55-65Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar There is excellent evidence that mitochondrial GPAT1 variants also impact the predisposition to MAFLD in human beings. A gain-of-function mutation in GPAT1, p.Ile43Val GPAM, confers an increased risk of MAFLD.6Jamialahmadi O. Mancina R.M. Ciociola E. et al.Exome-wide association study on alanine aminotransferase identifies sequence variants in the GPAM and APOE associated with fatty liver disease.Gastroenterology. 2021; 160: 1634-1646.e7Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Subjects with this mutation have increased rates of hepatic steatosis, transaminitis, cirrhosis, and increased plasma cholesterol levels.7Sveinbjornsson G. Ulfarsson M.O. Thorolfsdottir R.B. et al.Multiomics study of nonalcoholic fatty liver disease.Nat Genet. 2022; 54: 1652-1663Crossref PubMed Scopus (29) Google Scholar On the other hand, a frameshift missense loss-of-function mutation, p.Thr189GlyfsTer5 GPAM, decreases the risk of MAFLD and is associated with reduced cholesterol levels.7Sveinbjornsson G. Ulfarsson M.O. Thorolfsdottir R.B. et al.Multiomics study of nonalcoholic fatty liver disease.Nat Genet. 2022; 54: 1652-1663Crossref PubMed Scopus (29) Google Scholar Ng et al8Ng S.W.K. Rouhani F.J. Brunner S.F. et al.Convergent somatic mutations in metabolism genes in chronic liver disease.Nature. 2021; 598: 473-478Crossref PubMed Scopus (51) Google Scholar observed somatic missense, nonsense, and frameshift mutations of GPAM in biopsy specimens of liver from patients with NAFLD and alcohol-related liver disease. These investigators hypothesized that attenuating the stress of hepatic steatosis could give a selective advantage to hepatic tissue with such GPAM mutations, underscoring the potential to protect hepatocytes against lipotoxicity by interfering with GPAT1 function. Much like more widely known genetic polymorphisms that alter the risk for MAFLD, such as those in patatin-like phospholipase domain containing 3 and hydroxysteroid 17-β dehydrogenase 13, this evidence suggests that GPAT1 may be a target for pharmacotherapy of MAFLD; however, as yet, no small-molecule inhibitors of GPAT1 or antisense oligonucleotide therapies targeting GPAM have progressed to clinical trials in patients.9Linden D. Romeo S. Therapeutic opportunities for the treatment of NASH with genetically validated targets.J Hepatol. 2023; 79: 1056-1064Abstract Full Text Full Text PDF Scopus (0) Google Scholar Gpam null mice are protected against both diet-induced and genetically driven hepatic steatosis5Neschen S. Morino K. Hammond L.E. et al.Prevention of hepatic steatosis and hepatic insulin resistance in mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase 1 knockout mice.Cell Metab. 2005; 2: 55-65Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar,10Hammond L.E. Gallagher P.A. Wang S. et al.Mitochondrial glycerol-3-phosphate acyltransferase-deficient mice have reduced weight and liver triacylglycerol content and altered glycerolipid fatty acid composition.Mol Cell Biol. 2002; 22: 8204-8214Crossref PubMed Scopus (164) Google Scholar,11Wendel A.A. Li L.O. Li Y. et al.Glycerol-3-phosphate acyltransferase 1 deficiency in ob/ob mice diminishes hepatic steatosis but does not protect against insulin resistance or obesity.Diabetes. 2010; 59: 1321-1329Crossref PubMed Scopus (46) Google Scholar and are protected against hepatic carcinogenic insults,12Ellis J.M. Paul D.S. Depetrillo M.A. et al.Mice deficient in glycerol-3-phosphate acyltransferase-1 have a reduced susceptibility to liver cancer.Toxicol Pathol. 2012; 40: 513-521Crossref PubMed Scopus (17) Google Scholar but there are no previous studies of the effect of Gpam deficiency in widely used MAFLD–NASH models. Smith et al in this issue use diet-induced MAFLD–NASH models to study the potential protective effect of disruption of mitochondrial GPAT1. Smith et al were able to reproduce the prior finding that Gpam deletion attenuates hepatic steatosis in both high-fat diet–fed mice and ob/ob mice. Two diets were used to induce a NASH-like phenotype: the choline-deficient, L-amino acid–defined, high-fat diet, and the Gubra–Amylin NASH high-saturated-fat, high-fructose, high-cholesterol diet. Compared with wild-type mice, Gpam null mice on the choline-deficient, L-amino acid–defined, high-fat diet showed reduced progression of hepatic stiffness by shear wave elastography in vivo, and reduced progression to NASH as reflected by reduced hepatic inflammation and fibrosis by both messenger RNA markers and by immunohistochemistry. In contrast, there was no significant difference between Gpam knockout mice and wild-type mice fed the Gubra–Amylin NASH diet, as reflected by in vivo shear wave elastography or by analysis of the livers at the end of the study. As may be predicted, lysophosphatidic acid levels were reduced drastically in all of Smith et al's Gpam null models, inclusive of the simple hepatic steatosis experiments and the NASH experiments. This is notable because lysophosphatidic acid has long been known to be an important bioactive lipid and an inflammatory mediator.13Moolenaar W.H. Development of our current understanding of bioactive lysophospholipids.Ann N Y Acad Sci. 2000; 905: 1-10Crossref PubMed Google Scholar, 14Lee H. Liao J.J. Graeler M. et al.Lysophospholipid regulation of mononuclear phagocytes.Biochim Biophys Acta. 2002; 1582: 175-177Crossref PubMed Scopus (52) Google Scholar, 15Goetzl E.J. Kong Y. Voice J.K. Cutting edge: differential constitutive expression of functional receptors for lysophosphatidic acid by human blood lymphocytes.J Immunol. 2000; 164: 4996-4999Crossref PubMed Google Scholar Recently, a brief course of exogenous lysophosphatidic acid was shown to cause liver inflammation in wild-type mice.16Fan G. Li Y. Zong Y. et al.GPAT3 regulates the synthesis of lipid intermediate LPA and exacerbates Kupffer cell inflammation mediated by the ERK signaling pathway.Cell Death Dis. 2023; 14: 208Crossref Scopus (0) Google Scholar Taken together, lysophosphatidic acid may be a key metabolite explaining the protective effect of loss-of-function GPAM alleles and the increased risk associated with the gain-of-function allele. In addition, it should be noted that metabolic diseases such as MAFLD tend to be heterogeneous, with variability in the severity and course of disease. The fact that lysophosphatidic acid was reduced by Gpam ablation in all models studied, but the progression to NASH was attenuated in only 1 of 2 NASH models, suggests that there may be some pathways to NASH that are mediated by lysophosphatidic acid, whereas others are mediated by divergent mechanisms. The findings presented in this article support the notion derived from human genome-wide association study data that mitochondrial GPAT1 inhibition may be a good candidate for MAFLD therapy. The heterogeneity seen in the genotype–NASH diet model interaction opens up new questions as much as the results provide answers. It will be exciting and important to see whether targeting the glycerolipid biosynthesis pathway or targeting lysophosphatidic acid directly will be of benefit to patients on the MAFLD spectrum, and which subgroups of MAFLD patients (as reflected by different animal models) might benefit from such interventions. GPAT1 Deficiency in Mice Modulates NASH Progression in a Model-Dependent MannerCellular and Molecular Gastroenterology and HepatologyPreviewNonalcoholic fatty liver disease (NAFLD), and its more severe form, nonalcoholic steatohepatitis (NASH), is the leading cause for liver failure and liver cancer. Although the etiology is likely multifactorial, genes involved in regulating lipid metabolism are enriched in human NAFLD genome-wide association studies (GWAS), pointing to dysregulated lipid metabolism as a major pathogenic factor. Glycerol-3-phosphate acyltransferase 1 (GPAT1), encoded by GPAM, converts acyl-CoAs and glycerol-3-phosphate into lysophosphatidic acid and has been shown to regulate lipid accumulation in the liver. Full-Text PDF Open Access

Hepatic insulin resistance (IR) is often said to be "pathway-selective" with preserved insulin stimulation of de novo lipogenesis (DNL) despite attenuated insulin signaling toward glucose metabolism. However, DNL has not been assessed in models of liver-specific IR. We studied mice with differential tissue-specific lipid-induced IR achieved by different durations of high-fat diet (HFD) feeding. Mice with isolated hepatic IR demonstrated markedly reduced DNL, with a rebound seen in mice with whole-body IR. Insr T1150A mice (protected against diacylglycerol-PKCε-induced hepatic IR) maintained normal DNL with HFD feeding. During hyperinsulinemic clamps, hepatic IR reduced DNL, but hyperglycemia augmented DNL in both resistant and sensitive animals. Regulation through SREBP1c did not consistently correlate with changes in DNL. These results demonstrate that hepatic IR is not pathway-selective, highlighting the primacy of lipogenic substrate in stimulation of DNL. Future therapeutics to reduce lipogenesis should target substrate drivers of DNL rather than targeting plasma insulin levels.

Metabolic dysfunction-associated steatohepatitis (MASH) is highly prevalent among patients with type 2 diabetes. Cholesterol is one of several candidates causing hepatic inflammation and fibrosis. We hypothesized that by interrupting the synthesis of cholesterol, we could prevent a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) C57BL/6J MASH mouse model. We found that cholesterol in liver lipid droplets accumulated on day 1, MASH-associated macrophage markers (Gpnmb and Trem2) mRNA expression and plasma ALT increased on day 2, macrophage crown-like-structures emerged on day 3, and fibrosis markers (Col1a1, αSMA) mRNA expression increased on day 7 in the CDAHFD-treated mice. Filipin staining demonstrated free cholesterol accumulation in liver lipid droplets. We next examined whether a N-acetylgalactosamine-modified antisense oligonucleotide (ASO) against coenzyme A synthase (Coasy), which catalyzes a rate controlling step in hepatic cholesterol synthesis, would reduce cholesterol in liver lipid droplets and inflammation/fibrosis in this model. Coasy ASO treatment prevented accumulation of cholesterol in liver lipid droplets (Control=1.02 ± 0.04 mg/g tissue; Coasy=0.68 ± 0.08, P<0.001) and decreased plasma ALT and liver inflammation/fibrosis markers expression. To examine whether these effects of the Coasy ASO were mediated by reductions in liver lipid droplet cholesterol we examined whether adding 2% cholesterol to the diet would prevent the Coasy ASO protective effect. Cholesterol treatment induced accumulation of cholesterol in liver lipid droplets (Coasy=0.68 ± 0.08 mg/g tissue; Coasy with cholesterol =1.40 ± 0.11, P<0.001) and abrogated the protective effects of the Coasy ASO on liver inflammation and fibrosis. Conclusions: These data demonstrate that cholesterol in liver lipid droplets is a key mediator of MASH and hepatic Coasy knockdown is a potential therapeutic approach. Disclosure I. Sakuma: None. R.C. Gaspar: None. A. Nasiri: None. M. Kahn: None. M. Guerra: None. D. Yimlamai: None. S. Murray: Employee; Ionis Pharmaceuticals. M. Perelis: Employee; Ionis Pharmaceuticals. W. Barnes: Employee; Ionis Pharmaceuticals. D.F. Vatner: None. K. Petersen: None. V. Samuel: None. G.I. Shulman: None.

PNPLA3 (Patatin like phospholipid domain containing protein 3) I148M is the most common human genetic mutation associated with hepatic steatosis. Alterations in white adipose tissue (WAT) function are often linked to hepatic lipid accumulation, but current understanding of the effect of PNPLA3I148M on WAT function is limited to the finding that this mutation does not appear to impact WAT insulin responsiveness. We evaluated WAT catecholamine responsiveness using the β3-adrenergic agonist CL 316243 (CL) in Pnpla3I148M mice. In vivo, the plasma NEFA response to CL was increased in mutant mice (Basal NEFA: WT 0.65 ± 0.07 mM, MUT 0.66 ± 0.07 mM, P = 0.5; CL stimulated NEFA: WT 1.3 ± 0.1 mM, MUT 1.6 ± 0.1 mM, P = 0.03). Ex vivo, CL-induced fatty acid release from gonadal WAT was increased in mutant mice (1.22-fold increased NEFA release, P = 0.03). High sucrose diet fed Pnpla3I148M mice are a known model of increased hepatic steatosis; beta adrenergic blockade with propranolol did not alter hepatic triglyceride content in WT mice as compared with vehicle, but markedly decreased hepatic triglyceride in Pnpla3I148M mice (WT: VEH 37 ± 5 mg/g; PRO 36 ± 6 mg/g. MUT: VEH 55 ± 6 mg/g; PRO 19 ± 4mg/g, P = 0.0002). Acipimox, an inhibitor of WAT lipolysis, similarly had no effect on WT hepatic steatosis and markedly decreased hepatic triglyceride in Pnpla3I148M mice (WT: VEH 46 ± 5 mg/g; ACI 49 ± 10 mg/g. MUT: VEH 78 ± 11 mg/g; ACI 40 ± 5mg/g, P = 0.006). Conclusions: WAT catecholamine responsiveness is increased in Pnpla3I148M mice contributing to hepatic steatosis in these mice. As human hepatic triglyceride biosynthesis is strongly weighted towards esterification of preformed fatty acids from extrahepatic sources, this biology may contribute to the increased susceptibility to fatty liver seen in patients with the PNPLA3I148M mutation. Disclosure J. Golla: None. R. Suh: None. L.M. Paolella: None. J.W. Strober: None. F. Zhang: None. W.M. Philbrick: None. D.F. Vatner: None. Funding National Institutes of Health (RO1DK124272;T32DK007058; P30DK045735)

AGPAT2 (1- acyl-sn- glycerol - 3- phosphate- acyltransferase-2)converts lysophosphatidic acid (LPA) into phosphatidic acid (PA), and mutations of the AGPAT2 gene cause the most common form of congenital generalized lipodystrophy which leads to steatohepatitis. The underlying mechanism by which AGPAT2 deficiency leads to lipodystrophy and steatohepatitis has not been elucidated. We addressed this question using an antisense oligonucleotide (ASO) to knockdown expression of Agpat2 in the liver and white adipose tissue (WAT) of adult male Sprague- Dawley rats. Agpat2 ASO treatment induced lipodystrophy and inflammation in WAT and the liver, which was associated with increased LPA content in both tissues, whereas PA content was unchanged. We found that a controlled- release mitochondrial protonophore (CRMP) prevented LPA accumulation and inflammation in WAT whereas an ASO against glycerol -3 -phosphate acyltransferase, mitochondrial (Gpam) prevented LPA content and inflammation in the liver in Agpat2 ASO- treated rats. In addition, we show that overnutrition, due to high sucrose feeding, resulted in increased hepatic LPA content and increased activated macrophage content which were both abrogated with Gpam ASO treatment. Taken together, these data identify LPA as a key mediator of liver and WAT inflammation and lipodystrophy due to AGPAT2 deficiency as well as liver inflammation due to overnutrition and identify LPA as a potential therapeutic target to ameliorate these conditions.

ObjectiveNonalcoholic fatty liver disease (NAFLD), the most common liver disease among youth with obesity, precedes more severe metabolic and liver diseases. However, the impact of the Sars-CoV-2 global pandemic on the prevalence and severity of NAFLD and the associated metabolic phenotype among youth with obesity is unknown. MethodsParticipants were recruited from the Yale Pediatric Obesity Clinic during the Sars-CoV-2 global pandemic (August 2020 to May 2022) and were compared with a frequency-matched control group of youth with obesity studied before the Sars-CoV-2 global pandemic (January 2017 to November 2019). Glucose metabolism differences were assessed during an extended 180-minute oral glucose tolerance test. Magnetic resonance imaging-derived proton density fat fraction (PDFF) was used to determine intrahepatic fat content in those with NAFLD (PDFF >= 5.5). ResultsNAFLD prevalence increased in participants prior to (36.2%) versus during the Sars-CoV-2 pandemic (60.9%), with higher PDFF values observed in participants with NAFLD (PDFF >= 5.5%) during versus before the pandemic. An increase in visceral adipose tissue and a hyperresponsiveness in insulin secretion during the oral glucose tolerance test were also observed. ConclusionsHepatic health differences were likely exacerbated by environmental and behavioral changes associated with the pandemic, which are critically important for clinicians to consider when engaging in patient care to help minimize the future risk for metabolic perturbations.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.