Metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased expression of peroxisome proliferator-activated receptor gamma (PPARγ, Pparg) and reduced expression of genes involved in methionine metabolism in the liver. The nuclear receptor PPARγ is activated by fatty acids, and the knockout of Pparg in hepatocytes (Pparg ΔHep) reduced the negative effects of MASH on methionine metabolism. Here, we sought to determine whether hepatocyte Pparg is required for the transcriptional regulation of genes involved in hepatic methionine metabolism in conditions with altered fatty acid flux to the liver: fasting, refeeding, and high-fat diet (HFD)-induced obesity/steatosis. Fasting induced liver steatosis and increased the expression of key genes involved in the methionine metabolism in the liver, while 6h-refeeding reversed these effects and reduced the expression of phosphatidylethanolamine N-methyltransferase (Pemt) and cystathionine beta synthase (Cbs). Overall, fasting and refeeding did not alter hepatocyte Pparg expression nor Pparg ΔHep affected fasting and refeeding-mediated regulation of methionine metabolism gene expression. Diet-induced steatosis reduced hepatic Pemt expression in control (Pparg-intact) mice, and the thiazolidinedione (TZD)-mediated activation of PPARγ in diet-induced obese control (Pparg-intact) mice reduced the expression of betaine homocysteine S-methyltransferase (Bhmt) and Cbs. However, diet-induced steatosis increased hepatocyte Pparg expression, and Pparg ΔHep blocked the negative effects of HFD and TZD on hepatic methionine metabolism. The PPARγ-dependent reduction of hepatic Bhmt and Cbs expression was confirmed in mouse primary hepatocytes. Taken together, hepatocyte Pparg may serve as a negative regulator of hepatic methionine metabolism in diet-induced obese mice and these actions could contribute to promoting the onset of MASH.

Excess hepatic cholesterol contributes to the progression of metabolic liver diseases, including both metabolic dysfunction-associated steatotic liver disease (MASLD) and alcohol-associated liver disease (ALD), yet the mechanisms by which cholesterol disrupts hepatocellular metabolic homeostasis and promotes liver injury remain poorly understood. Here, we investigated whether disruption of hepatic NAD⁺ homeostasis contributes to cholesterol-induced hepatotoxicity and evaluated the underlying molecular mechanisms and therapeutic potential of restoring NAD+ metabolism. Using cholesterol-loading models in cultured hepatocytes and mice, we found that cholesterol markedly disrupted hepatic NAD+ homeostasis by suppressing nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage pathway. Pharmacological restoration of NAD+ homeostasis with nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), nicotinamide N-methyltransferase (NNMT) inhibitors, or preservation of protein PARylation through PARG inhibition effectively attenuated cholesterol-induced hepatocyte injury, restored PARP1 activity, and reactivated AMPK signaling both in vitro and in vivo. Mechanistically, cholesterol suppressed O-GlcNAc transferase (OGT) expression and protein O-GlcNAcylation, leading to SP1 inactivation, transcriptional repression of Nampt, disruption of NAD+ homeostasis, and subsequent inhibition of the PARP1-AMPK signaling axis. We further identified PARP1 as a positive regulator of AMPK via PARPylation, thereby linking impaired NAD+ homeostasis to defective adaptation to metabolic stress. In contrast to saturated fatty acid-induced lipotoxicity, cholesterol-induced hepatotoxicity occurred independently of SIRT1 and mTORC1 signaling, indicating a distinct pathogenic mechanism. Collectively, these findings identify the OGT-SP1-NAMPT-NAD+-PARP1-AMPK axis as a previously unrecognized mechanism underlying cholesterol-induced hepatotoxicity and demonstrate that pharmacological restoration of NAD+ homeostasis represents a promising therapeutic strategy for cholesterol-associated liver diseases.

Overactivation of hepatic de novo lipogenesis (DNL) contributes to fatty liver disease. Although glucose and fructose strongly promote DNL, diary-rich galactose is only weakly lipogenic. However, whether and how it regulates hepatic DNL remains unclear. In this study, we investigated whether low-dose galactose supplementation attenuates glucose- or fructose-induced DNL activation and protects against fatty liver diseases driven by DNL overactivation, such as alcohol-associated liver disease (ALD). In this study, we used integrated hepatocyte and mouse models to assess hepatic DNL and related signaling under high-glucose or high-fructose conditions, with or without low-dose galactose. Pharmacological and genetic interventions targeting the Leloir and hexosamine biosynthetic pathways (HBP) defined underlying mechanisms. For in vivo validation, male C57BL/6 mice were fed an isocaloric control or ethanol-containing diet for 4 wk. We found that glucose engages the HBP-mTORC1-SREBP-1c axis to stimulate hepatic DNL, whereas fructose acts predominantly through carbohydrate-responsive element-binding protein (ChREBP). Low-dose galactose selectively suppressed glucose-induced hepatic fat accumulation, concomitant with the inhibition of the HBP-mTORC1-SERBP-1c pathway. These effects required an intact Leloir pathway for galactose metabolism and were not observed with fructose. In alcohol-fed mice, hepatic HBP-mTORC1-SREBP-1c signaling was markedly upregulated, contributing to steatosis and liver injury. Replacing even a small fraction of dietary glucose with galactose normalized these alterations, attenuating hepatic lipid accumulation and injury without altering systemic glucose levels. In conclusion, glucose-induced hepatic lipogenesis involves the HBP-mTORC1-SREBP-1c pathway, which is also activated during chronic alcohol exposure. Low-dose galactose, obtainable from dairy sources, attenuates this pathway, thereby limiting excessive lipogenesis and protecting against early-stage ALD.NEW & NOTEWORTHY This study demonstrates that low-dose galactose, a dairy-derived monosaccharide, regulates hepatic de novo lipogenesis (DNL) by selectively inhibiting glucose-induced DNL activation. Mechanistically, low-dose galactose suppresses hexosamine biosynthetic pathway (HBP) flux, protein O-GlcNAcylation, and mTORC1 signaling, thereby inhibiting SREBP-1c activation in a Leloir pathway-dependent manner. Notably, galactose supplementation prevented early-stage alcohol-related liver disease by attenuating hepatic HBP-O-GlcNAcylation-SREBP-1c signaling.

Time-restricted feeding (TRF) is proposed as a relevant strategy to counteract obesity and metabolic disorders; however, the therapeutic efficacy of more pragmatic intermittent TRF (iTRF) regimens remains undefined. Herein, daily and intermittent TRF significantly improved whole-body physiology, promoted key hallmarks of energy restriction, and induced coordinated transcriptional and lipidomic remodeling in male mice with chronic metabolic dysfunction previously established by prolonged high-fat, high-cholesterol, high-fructose feeding. These effects were summarized using three composite scores (physiological/metabolic, energy restriction, and lipidic), which showed consistent associations with each other, suggesting convergence toward a TRF-associated metabolic state across biological scales. Lipidomic profiling identified a subset of hepatic lipids associated with systemic health, pointing to lipid remodeling, particularly at the endoplasmic reticulum, as a potential feature of the TRF response. Notably, TRF failed to histologically improve hepatic steatosis, ballooning, or inflammation, and its overall benefits were clearly inferior to dietary normalization to standard chow diet. Altogether, these data (i) demonstrate that intermittent TRF recapitulates the beneficial effects in advanced metabolic disease, (ii) provides translational support for flexible TRF strategies, and (iii) highlights the need to integrate TRF with additional therapeutic strategies.

Abstract Obesity and related metabolic disorders are often characterized by chronic adipose tissue inflammation, driving systemic insulin resistance and general metabolic dysfunction. Free Fatty Acid Receptor 2 (FFA2) has emerged as a potential modulator of adipocyte function, inflammation, and metabolism. To investigate the role of FFA2 expressed in the adipose tissue, we generated adipose‐specific FFA2 knockout mice (Adipoq‐F2‐KO) and assessed metabolic outcomes under standard laboratory chow and high‐fat, high‐sugar Western diet conditions, with and without dietary fiber supplementation. We found that adipose‐specific FFA2 deletion had minimal metabolic consequences under standard dietary conditions but significantly reduced body weight and adiposity when mice were fed a fiber (fructooligosaccharide)‐supplemented Western diet. Subsequent fecal analyses and transcriptomic profiling indicated impaired intestinal lipid absorption as the primary driver of reduced adiposity, suggesting disrupted adipose‐intestinal communication. Unexpectedly, the lighter Adipoq‐F2‐KO mice also exhibited heightened adipose inflammation, characterized by increased macrophage infiltration and pro‐inflammatory cytokine expression. Furthermore, in vitro loss‐of‐function experiments in adipocytes revealed that FFA2 knockdown impaired adipocyte maturation, lipid storage, and anti‐inflammatory signaling. Additional studies using intestinal epithelial cells exposed to adipocyte‐conditioned media implicated adipose‐derived signals in driving intestinal dysfunction. Collectively, our findings highlight adipose‐specific FFA2 as critical in regulating adipose tissue inflammation, lipid metabolism, and inter‐organ communication.

Mouse models of metabolic dysfunction-associated steatotic liver disease (MASLD) are valuable tools for identifying novel molecular mechanisms that drive progression from MASLD to metabolic dysfunction-associated steatohepatitis (MASH). However, generating a clinically relevant MASLD/MASH mouse model with obesity and peripheral metabolic dysfunction remains a challenge. In this study, we fed two different MASH-inducing diets to male mice with pre-existing high-fat (HF) diet-induced obesity. While a HF diet containing 40% Kcal from fat (mostly corn-oil shortening), 2% cholesterol, and 22% fructose reduced adiposity in these mice, a high-fat diet with 60% Kcal from fat (mostly lard), containing 2% cholesterol and supplemented with 10% fructose in the drinking water (HFC+Fr diet) promoted body weight and fat mass gain. Of note, 24 weeks of the HFC+Fr diet induced obesity, metabolic dysfunction, and liver steatosis in male and female mice, and promoted MASH with fibrosis in male mice. Furthermore, the HFC+Fr diet increased the expression of hepatocyte peroxisome proliferator-activated receptor γ (Pparg), but the knockout of Pparg in hepatocytes (PpargΔHep) reduced the development of MASH and fibrosis in male mice. In addition, the expression of key hepatic genes involved in methionine metabolism was downregulated by the HFC+Fr diet and upregulated by PpargΔHep only in male mice. Overall, the HFC+Fr diet is obesogenic and promotes MASLD in both male and female mice. However, the HFC+Fr diet promotes MASH in a sex- and hepatocyte Pparg-specific manner, which may be associated with downregulation of hepatic methionine metabolism.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the worst solid malignancies in regard to outcomes and metabolic dysfunction leading to cachexia. It is alarming that PDAC incidence rates continue to increase and warrant the need for innovative approaches to combat this disease. Due to its relatively slow progression (10-20 years), prevention strategies represent an effective means to improve outcomes. One of the risk factors for many cancers and for pancreatic cancer in particular is diet. Hence, our objective is to understand how a diet rich in omega 3 and omega 6 polyunsaturated fatty acids affects the progression of this disease. Methods: We investigated polyunsaturated fatty acid (PUFA) effects on disease progression employing both in vitro (PDAC cell lines) and in vivo (EL-Kras and KC mice) approaches. Also, we gathered data from the National Health and Nutrition Examination Survey (NHANES) and the National Cancer Institute (NCI) from 1999 to 2017 for a retrospective observational study. Results: The consumption of PUFAs in a patient population correlates with increased PDAC incidence, particularly when the omega 3 intake increases to a lesser extent than omega 6. Our data demonstrate dietary PUFAs can be incorporated into plasma membrane lipids affecting PI3K/AKT signaling and support the emergence of membrane-targeted therapies. Moreover, we show that the phospholipid composition of a lipid nanoparticle (LNP) can impact the cell membrane integrity and, ultimately, cell viability after administration of these LNPs. Conclusions: Cancer prevention is impactful particularly for those with very poor prognosis, including pancreatic cancer. Our results point to the importance of dietary intervention in this disease when detected early and the potential to improve the antiproliferative effect of drug efficacy when combined with these regimens in later stages of pancreatic cancer.

Nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome (MetS) have been linked to osteoporosis and osteoarthritis (OA), where the prevalence of all increase with age. Many individuals with NAFLD also exhibit MetS, a condition that is now termed metabolic dysfunction‐associated steatotic liver disease (MASLD). MASLD spans from simple hepatic steatosis to hepatocyte ballooning and inflammation, termed metabolic dysfunction‐associated steatohepatitis (MASH), which may occur with or without fibrosis. To delineate the contribution of liver injury to skeletal deterioration within the context of metabolic syndrome (MetS), we fed male mice a high-fat, cholesterol, and fructose (HFCF) diet that induces metabolic syndrome-associated steatohepatitis (MASH) and liver fibrosis, associated with moderate obesity but without profound insulin resistance. A nutrient-matched diet with high carbohydrates but low in fat, cholesterol, and fructose (LFCF), which induced steatosis in adult mice, served as the control. Micro‐CT analysis of the femur of HFCF-fed mice that developed MASH with fibrosis revealed significant cortical thinning (reduced bone area and thickness), decreased trabecular thickness, and lower bone mineral density compared to LFCF-fed mice, with no liver fibrosis. In the knee joint, MASH with fibrosis was associated with subchondral bone loss, medial cartilage erosion, and elevated chondrocyte expression of iNOS, NLRP3, and β‐galactosidase. Bulk RNA‐seq of knee tissue identified 152 differentially expressed genes: interferon‐related (Oas3, Nlrc5, Zbp1) and stress‐response (Slc6a4, Alox12, Cirbp, Trpc6, Tap1, Ubash3, Nrg1) pathways were upregulated, while extracellular matrix organization pathways were downregulated. Our findings reveal a mechanistic connection between the degree of liver injury and deterioration of bone and joint integrity, pointing to a shared pathophysiological axis in MASLD and OA. Clinically, this raises the prospect that a single therapeutic, designed to modulate inflammation, or hepatic lipid metabolism, could be repurposed or developed to concurrently treat both steatohepatitis and osteoarthritic degeneration.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic liver condition that often progresses to more advanced stages, such as metabolic dysfunction-associated steatohepatitis (MASH). MASH is characterized by inflammation and hepatocellular ballooning, in addition to hepatic steatosis. Despite the relatively high incidence of MASH in the population and its potential detrimental effects on human health, this liver disease is still not fully understood from a pathophysiological perspective. Deregulation of polyamine levels has been detected in various pathological conditions, including neurodegenerative diseases, inflammation, and cancer. However, the role of the polyamine pathway in chronic liver disorders such as MASLD has not been explored. In this study, we measured the expression of liver ornithine decarboxylase (ODC1), the rate-limiting enzyme responsible for the production of putrescine, and the hepatic levels of putrescine, in a preclinical model of MASH as well as in liver biopsies of patients with obesity undergoing bariatric surgery. Our findings reveal that expression of ODC1 and the levels of putrescine, but not spermidine nor spermine, are elevated in hepatic tissue of both diet-induced MASH mice and patients with biopsy-proven MASH compared with control mice and patients without MASH, respectively. Furthermore, we found that the levels of putrescine were positively associated with higher aspartate aminotransferase concentrations in serum and an increased SAF score (steatosis, activity, fibrosis). Additionally, in in vitro assays using human HepG2 cells, we demonstrate that elevated levels of putrescine exacerbate the cellular response to palmitic acid, leading to decreased cell viability and increased release of CK-18. Our results support an association between the expression of ODC1 and the progression of MASLD, which could have translational relevance in understanding the onset of this disease. © 2024 The Pathological Society of Great Britain and Ireland.

Supplementary Table 5: List of pathways from GO analyses in MALES. Significant pathways UP"over represented value" in red. Pathways DOWN "under reperesented value" in blue. In green pathways filtered for “lipid, fatty acid and sterol”Supplementary Table 6: List of pathways from GO analyses in FEMALES. Significant pathways UP"over-represented value" in red. Pathways DOWN "under-represented value" in blue. In orange pathways filtered for “lipid, fatty acid and sterol”.

Short-chain fatty acids (SCFAs) are key nutrients that play a diverse set of roles in physiological function, including regulating metabolic homeostasis. Generated through the fermentation of dietary fibers in the distal colon by the gut microbiome, SCFAs and their effects are partially mediated by their cognate receptors, including free fatty acid receptor 2 (FFA2). FFA2 is highly expressed in the intestinal epithelial cells, where its putative functions are controversial, with numerous in vivo studies relying on global knockout mouse models to characterize intestine-specific roles of the receptor. Here, we used the Villin-Cre mouse line to generate a novel, intestine-specific knockout mouse model for FFA2 (Vil-FFA2) to investigate receptor function within the intestine. Because dietary changes are known to affect the composition of the gut microbiome, and can thereby alter SCFA production, we performed an obesogenic challenge on male Vil-FFA2 mice and their littermate controls (FFA2-floxed, FFA2fl/fl) to identify physiological changes on a high-fat, high-sugar 'Western diet' (WD) compared to a low-fat control diet (CD). We found that the WD-fed Vil-FFA2 mice were transiently protected from the obesogenic effects of the WD and had lower fat mass and improved glucose homeostasis compared to the WD-fed FFA2fl/fl control group during the first half of the study. Additionally, major differences in respiratory exchange ratio and energy expenditure were observed in the WD-fed Vil-FFA2 mice, and food intake was found to be significantly reduced at multiple points in the study. Taken together, this study uncovers a novel role of intestinal FFA2 in mediating the development of obesity.

Growth hormone (GH) modifies liver gene transcription in a sexually dimorphic manner to meet liver metabolic demands related to sex; thus, GH dysregulation leads to sex-biased hepatic disease. We dissected the steps of the GH regulatory cascade modifying GH-dependent genes involved in metabolism, focusing on the male-predominant genes Lcn13, Asns, and Cyp7b1, and the female-predominant genes Hao2, Pgc1a, Hamp2, Cyp2a4, and Cyp2b9. We explored mRNA expression in 2 settings: (i) intact liver GH receptor (GHR) but altered GH and insulin-like growth factor 1 (IGF1) levels (NeuroDrd2KO, HiGH, aHepIGF1kd, and STAT5bCA mouse lines); and (ii) liver loss of GHR, with or without STAT5b reconstitution (aHepGHRkd, and aHepGHRkd + STAT5bCA). Lcn13 was downregulated in males in most models, while Asns and Cyp7b1 were decreased in males by low GH levels or action, or constant GH levels, but unexpectedly upregulated in both sexes by the loss of liver Igf1 or constitutive Stat5b expression. Hao, Cyp2a4, and Cyp2b9 were generally decreased in female mice with low GH levels or action (NeuroDrd2KO and/or aHepGHRkd mice) and increased in HiGH females, while in contrast, Pgc1a was increased in female NeuroDrd2KO but decreased in STAT5bCA and aHepIGF1kd females. Bioinformatic analysis of RNAseq from aHepGHRkd livers stressed the greater impact of GHR loss on wide gene expression in males and highlighted that GH modifies almost completely different gene signatures in each sex. Concordantly, we show that altering different steps of the GH cascade in the liver modified liver expression of Lcn13, Asns, Cyp7b1, Hao2, Hamp2, Pgc1a, Cyp2a4, and Cyp2b9 in a sex- and gene-specific manner.