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Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction- associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3- I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3- I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3- I148M and characterize changes induced by endogenous expression of the disease- causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3- I148M are not endoplasmic reticulum- resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3- I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3- I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild- type variant, PNPLA3- I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet--Golgi contact sites, which were also observed in I148M- expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3- I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3- I148M biology and identify the Golgi apparatus as a central hub of PNPLA3- I148M- driven cellular change. [ABSTRACT FROM AUTHOR]

Proinflammatory cytokine production by innate immune cells plays a crucial role in inflammatory diseases, but the molecular mechanisms controlling the inflammatory responses are poorly understood. Here, we show that TANK-binding kinase 1 (TBK1) serves as a vital regulator of proinflammatory macrophage function and protects against tissue inflammation. Myeloid cell-conditional Tbk1 knockout (MKO) mice spontaneously developed adipose hypertrophy and metabolic disorders at old ages, associated with increased adipose tissue M1 macrophage infiltration and proinflammatory cytokine expression. When fed with a high-fat diet, the Tbk1-MKO mice also displayed exacerbated hepatic inflammation and insulin resistance, developing symptoms of nonalcoholic steatohepatitis. Furthermore, myeloid cell-specific TBK1 ablation exacerbates inflammation in experimental colitis. Mechanistically, TBK1 functions in macrophages to suppress the NF-κB and MAP kinase signaling pathways and thus attenuate induction of proinflammatory cytokines, particularly IL-1ß. Ablation of IL-1 receptor 1 (IL-1R1) eliminates the inflammatory symptoms of Tbk1-MKO mice. These results establish TBK1 as a pivotal anti-inflammatory mediator that restricts inflammation in different disease models. [ABSTRACT FROM AUTHOR]

Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function.

Fatty liver disease (FLD) is a disorder in which accumulation of triglycerides (TGs) in the liver can lead to inflammation, fibrosis, and cirrhosis. Previously, we identified a variant (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) that is strongly associated with FLD, but the mechanistic basis for the association remains elusive. Although PNPLA3 has TG hydrolase activity in vitro, inactivation or overexpression of the WT protein in mice does not cause steatosis. In contrast, expression of two catalytically defective forms of PNPLA3 (I148M or S47A) in sucrose-fed mice causes accumulation of both PNPLA3 and TGs on hepatic lipid droplets (LDs). To determine if amassing PNPLA3 on LDs is a cause or consequence of steatosis, we engineered a synthetic isoform of PNPLA3 that uncouples protein accumulation from loss of enzymatic activity. Expression of a ubiquitylation-resistant form of PNPLA3 in mice caused accumulation of PNPLA3 on hepatic LDs and development of FLD. Lowering PNPLA3 levels by either shRNA knockdown or proteolysis-targeting chimera (PROTAC)-mediated degradation reduced liver TG content in mice overexpressing PNPLA3(148M). Taken together, our results show that the steatosis associated with PNPLA3(148M) is caused by accumulation of PNPLA3 on LDs. [ABSTRACT FROM AUTHOR]

Acetyl-CoA synthetase 2 (ACSS2) is a conserved nucleocytosolic enzyme that converts acetate to acetyl-CoA. Adult mice lacking ACSS2 appear phenotypically normal but exhibit reduced tumor burdens in mouse models of liver cancer. The normal physiological functions of this alternate pathway of acetyl-CoA synthesis remain unclear, however. Here, we reveal that mice lacking ACSS2 exhibit a significant reduction in body weight and hepatic steatosis in a diet-induced obesity model. ACSS2 deficiency reduces dietary lipid absorption by the intestine and also perturbs repartitioning and utilization of triglycerides from adipose tissue to the liver due to lowered expression of lipid transporters and fatty acid oxidation genes. In this manner, ACSS2 promotes the systemic storage or metabolism of fat according to the fed or fasted state through the selective regulation of genes involved in lipid metabolism. Thus, targeting ACSS2 may offer a therapeutic benefit for the treatment of fatty liver disease. [ABSTRACT FROM AUTHOR]

Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein-protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease. [ABSTRACT FROM AUTHOR]

Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function. [ABSTRACT FROM AUTHOR]

Endogenously formed reactive oxygen species continuously damage cellular constituents including DNA. These challenges, coupled with exogenous exposure to agents that generate reactive oxygen species, are both associated with normal aging processes and linked to cardiovascular disease, cancer, cataract formation, and fatty liver disease. Although not all of these diseases have been definitively shown to originate from mutations in nuclear DNA or mitochondrial DNA, repair of oxidized, saturated, and ring-fragmented bases via the base excision repair pathway is known to be critical for maintaining genomic stability. One enzyme that initiates base excision repair of ring-fragmented purines and some saturated pyrimidines is NEIL1, a mammalian homolog to Escherichia coliendonuclease VIII. To investigate the organismal consequences of a deficiency in NEIL1, a knockout mouse model was created. In the absence of exogenous oxidative stress, neil1 knockout (neil1-/-) and heterozygotic (neil1+/-) mice develop severe obesity, dyslipidemia, and fatty liver disease and also have a tendency to develop hyperinsulinemia. In humans, this combination of clinical manifestations, including hypertension, is known as the metabolic syndrome and is estimated to affect >40 million people in the United States. Additionally, mitochondrial DNA from neil1-/- mice show increased levels of steady-state DNA damage and deletions relative to wild-type controls. These data suggest an important role for NEIL1 in the prevention of the diseases associated with the metabolic syndrome. [ABSTRACT FROM AUTHOR]

Proinflammatory cytokine production by innate immune cells plays a crucial role in inflammatory diseases, but the molecular mechanisms controlling the inflammatory responses are poorly understood. Here, we show that TANK-binding kinase 1 (TBK1) serves as a vital regulator of proinflammatory macrophage function and protects against tissue inflammation. Myeloid cell-conditional Tbk1 knockout (MKO) mice spontaneously developed adipose hypertrophy and metabolic disorders at old ages, associated with increased adipose tissue M1 macrophage infiltration and proinflammatory cytokine expression. When fed with a high-fat diet, the Tbk1 -MKO mice also displayed exacerbated hepatic inflammation and insulin resistance, developing symptoms of nonalcoholic steatohepatitis. Furthermore, myeloid cell-specific TBK1 ablation exacerbates inflammation in experimental colitis. Mechanistically, TBK1 functions in macrophages to suppress the NF-κB and MAP kinase signaling pathways and thus attenuate induction of proinflammatory cytokines, particularly IL-1β. Ablation of IL-1 receptor 1 (IL-1R1) eliminates the inflammatory symptoms of Tbk1 -MKO mice. These results establish TBK1 as a pivotal anti-inflammatory mediator that restricts inflammation in different disease models., Competing Interests: Competing interest statement: D.J. is presently an employee of Ziopharm Oncology, Inc., and X.X. is presently an employee of AbbVie, although they participated in this research when they were employed in the University of Texas MD Anderson Cancer Center., (Copyright © 2022 the Author(s). Published by PNAS.)

Acetyl-CoA synthetase 2 (ACSS2) is a conserved nucleocytosolic enzyme that converts acetate to acetyl-CoA. Adult mice lacking ACSS2 appear phenotypically normal but exhibit reduced tumor burdens in mouse models of liver cancer. The normal physiological functions of this alternate pathway of acetyl-CoA synthesis remain unclear, however. Here, we reveal that mice lacking ACSS2 exhibit a significant reduction in body weight and hepatic steatosis in a diet-induced obesity model. ACSS2 deficiency reduces dietary lipid absorption by the intestine and also perturbs repartitioning and utilization of triglycerides from adipose tissue to the liver due to lowered expression of lipid transporters and fatty acid oxidation genes. In this manner, ACSS2 promotes the systemic storage or metabolism of fat according to the fed or fasted state through the selective regulation of genes involved in lipid metabolism. Thus, targeting ACSS2 may offer a therapeutic benefit for the treatment of fatty liver disease., Competing Interests: The authors declare no conflict of interest.