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Objective: Alcohol-associated liver disease (ALD) is the leading cause of liver-related mortality worldwide. Current strategies to manage ALD focus largely on advanced stage disease, however, metabolic changes such as glucose intolerance are apparent at the earliest stage of alcoholic steatosis and increase the risk of disease progression. Ceramides impair insulin signaling and accumulate in ALD, and metabolic pathways involving ceramide synthase 6 (CerS6) are perturbed in ALD during hepatic steatosis. In this study, we aimed to investigate the role of CerS6 in ALD development and the relevance of CerS6 to human ALD., Methods: C57BL/6 WT and CerS6 KO mice of both sexes were fed either a Lieber-DeCarli control (CON) or 15% ethanol (EtOH) diet for six weeks. In vivo metabolic tests including glucose and insulin tolerance tests (GTT and ITT) and energy expenditure were performed. The mice were euthanized, and serum and liver lipids and liver histology were examined. For in vitro studies, CerS6 was deleted in human hepatocytes, VL17A and cells were incubated with EtOH and/or C 16:0 -ceramides. RNAseq analysis was performed in livers from mice and human patients with different stages of ALD and diseased controls., Results: After six weeks on an EtOH diet, CerS6 KO mice had reduced body weight, food intake, and %fat mass compared to WT mice. Energy expenditure increased in both male and female KO mice, however, was only statistically significant in male mice. In response to EtOH, WT mice developed mild hepatic steatosis, while steatosis was ameliorated in KO mice as determined by H&E and ORO staining. KO mice showed significantly decreased long-chain ceramide species, especially C 16:0 -ceramides, in the serum and liver tissues compared to WT mice. CerS6 deletion decreased serum TG and NEFA only in male not female mice. CerS6 deletion improved glucose tolerance and insulin resistance in EtOH-fed mice of both sexes. RNAseq analysis revealed that 74 genes are significantly upregulated and 66 genes are downregulated by CerS6 deletion in EtOH-fed male mice, with key network pathways including TG biosynthetic process, positive regulation of lipid localization, and fat cell differentiation. Similar to RNAseq results, absence of CerS6 significantly decreased mRNA expression of lipid droplet associated proteins in EtOH-fed mice. In vitro, EtOH stimulation significantly increased PLIN2 protein expression in VL17A cells while CerS6 deletion inhibited EtOH-mediated PLIN2 upregulation. C 16:0 -ceramide treatment significantly increased PLIN2 protein expression compared to CON. Notably, progression of ALD in humans was associated with increased hepatic CerS6 expression., Conclusions: Our findings demonstrate that CerS6 deletion improves glucose homeostasis in alcohol-fed mice and exhibits sex-based differences in the attenuation of EtOH-induced weight gain and hepatic steatosis. Additionally, we unveil that CerS6 plays a major role as a regulator of lipid droplet biogenesis in alcohol-induced intra-hepatic lipid droplet formation, identifying it as a putative target for early ALD management., Competing Interests: Declaration of Competing Interest RC receives research support from Intercept Pharmaceuticals and Merck, Inc., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The steatotic diseases of metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD), and chronic hepatitis C (HCV) account for the majority of liver disease prevalence, morbidity, and mortality worldwide. While these diseases have distinct pathogenic and clinical features, dysregulated lipid droplet (LD) organelle biology represents a convergence of pathogenesis in all three. With increasing understanding of hepatocyte LD biology, we now understand the roles of LD proteins involved in these diseases but also how genetics modulate LD biology to either exacerbate or protect against the phenotypes associated with steatotic liver diseases. Here, we review the history of the LD organelle and its biogenesis and catabolism. We also review how this organelle is critical not only for the steatotic phenotype of liver diseases but also for their advanced phenotypes. Finally, we summarize the latest attempts and challenges of leveraging LD biology for therapeutic gain in steatotic diseases. In conclusion, the study of dysregulated LD biology may lead to novel therapeutics for the prevention of disease progression in the highly prevalent steatotic liver diseases of MASLD, ALD, and HCV., Competing Interests: R.M.C. receives research support from Merck, Inc, and has consulted for Intercept Pharmaceuticals and Astrazeneca., (Thieme. All rights reserved.)

Alcohol's impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis, and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet (LD) accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and LD compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol's inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri- LD ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD., (Copyright © 2020 Correnti et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Perilipin 2 (Plin2) is a lipid droplet protein that has roles in both lipid and glucose homeostasis. An increase in Plin2 in liver is associated with the development of steatosis, glucose intolerance, and ceramide accumulation in alcoholic liver disease. We investigated the role of Plin2 on energy balance and glucose and lipid homeostasis in wildtype and Plin2 knockout (Plin2KO) mice chronically fed a Lieber-DeCarli liquid ethanol or control diet for six weeks., Methods: We performed in vivo measurements of energy intake and expenditure; body composition; and glucose tolerance. After sacrifice, liver was dissected for histology and lipid analysis., Results: We found that neither genotype nor diet had a significant effect on final weight, body composition, or energy intake between WT and Plin2KO mice fed alcohol or control diets. Additionally, alcohol feeding did not affect oxygen consumption or carbon dioxide production in Plin2KO mice. We performed glucose tolerance testing and observed that alcohol feeding failed to impair glucose tolerance in Plin2KO mice. Most notably, absence of Plin2 prevented hepatic steatosis and ceramide accumulation in alcohol-fed mice. These changes were related to downregulation of genes involved in lipogenesis and triglyceride synthesis., Conclusions: Plin2KO mice chronically fed alcohol are protected from hepatic steatosis, glucose intolerance, and hepatic ceramide accumulation, suggesting a critical pathogenic role of Plin2 in experimental alcoholic liver disease.

Background: Alcoholic liver disease (ALD) progresses from steatosis to inflammation, fibrosis, and cirrhosis. Although ALD has been associated with insulin resistance, it is unclear whether insulin resistance coincides with the development of steatosis., Methods: We studied the temporal relationship of steatosis and glucose homeostasis in mice fed a Lieber-DeCarli liquid control or ethanol (EtOH) diet for 2, 4, or 8 weeks. We studied the effects of alcohol consumption on energy balance, body composition, and hepatic lipids. Glucose tolerance test was performed, and insulin sensitivity was evaluated with hyperinsulinemic-euglycemic clamp., Results: EtOH-fed mice developed hepatic steatosis over time as compared with control-fed mice despite similar energy intake and expenditure, and gain in body weight and fat. EtOH-fed mice developed glucose intolerance as early as 2 weeks, while insulin resistance developed at 4 weeks. A hyperinsulinemic clamp study at 8 weeks revealed both hepatic and peripheral insulin resistance in EtOH-fed mice. Insulin resistance was associated with hepatic steatosis, increased ceramide levels, and Perilipin 2 expression., Conclusions: Chronic EtOH consumption leads to the development of hepatic steatosis, impaired glucose tolerance, and insulin resistance. These changes are independent of energy intake or expenditure, weight, whole body fat content, and inflammation. A better understanding of the processes linking EtOH-induced steatosis and abnormal glucose homeostasis may lead to novel therapies targeting the progression of ALD., (Copyright © 2013 by the Research Society on Alcoholism.)

Lipid droplets in the liver are coated with the perilipin family of proteins, notably adipocyte differentiation-related protein (ADRP) and tail-interacting protein of 47 kDa (TIP47). ADRP is increased in hepatic steatosis and is associated with hyperlipidemia, insulin resistance, and glucose intolerance. We have shown that reducing ADRP in the liver via antisense oligonucleotide (ASO) treatment attenuates steatosis and improves insulin sensitivity and glucose tolerance. We hypothesized that TIP47 has similar effects on hepatic lipid and glucose metabolism. We found that TIP47 mRNA and protein levels were increased in response to a high-fat diet (HFD) in C57BL/6J mice. TIP47 ASO treatment decreased liver TIP47 mRNA and protein levels without altering ADRP levels. Low-dose TIP47 ASO (15 mg/kg) and high-dose TIP47 ASO (50 mg/kg) decreased triglyceride content in the liver by 35% and 52%, respectively. Liver histology showed a drastic reduction in hepatic steatosis following TIP47 ASO treatment. The high dose of TIP47 ASO significantly blunted hepatic triglyceride secretion, improved glucose tolerance, and increased insulin sensitivity in liver, adipose tissue, and muscle. These findings show that TIP47 affects hepatic lipid and glucose metabolism and may be a target for the treatment of nonalcoholic fatty liver and related metabolic disorders.

Background and aims: There is significant overlap between non-alcoholic fatty liver disease (NAFLD) and alcohol-associated liver disease (ALD) with regards to risk factors and disease progression. However, the mechanism by which fatty liver disease arises from concomitant obesity and overconsumption of alcohol (syndrome of metabolic and alcohol-associated fatty liver disease; SMAFLD), is not fully understood. Methods: Male C57BL6/J mice were fed chow diet (Chow) or high-fructose, high-fat, high-cholesterol diet (FFC) for 4 weeks, then administered either saline or ethanol (EtOH, 5% in drinking water) for another 12 weeks. The EtOH treatment also consisted of a weekly 2.5 g EtOH/kg body weight gavage. Markers for lipid regulation, oxidative stress, inflammation, and fibrosis were measured by RT-qPCR, RNA-seq, Western blot, and metabolomics. Results: Combined FFC-EtOH induced more body weight gain, glucose intolerance, steatosis, and hepatomegaly compared to Chow, EtOH, or FFC. Glucose intolerance by FFC-EtOH was associated with decreased hepatic protein kinase B (AKT) protein expression and increased gluconeogenic gene expression. FFC-EtOH increased hepatic triglyceride and ceramide levels, plasma leptin levels, hepatic Perilipin 2 protein expression, and decreased lipolytic gene expression. FFC and FFC-EtOH also increased AMP-activated protein kinase (AMPK) activation. Finally, FFC-EtOH enriched the hepatic transcriptome for genes involved in immune response and lipid metabolism. Conclusions: In our model of early SMAFLD, we observed that the combination of an obesogenic diet and alcohol caused more weight gain, promoted glucose intolerance, and contributed to steatosis by dysregulating leptin/AMPK signaling. Our model demonstrates that the combination of an obesogenic diet with a chronic-binge pattern alcohol intake is worse than either insult alone. [ABSTRACT FROM AUTHOR]

Background & aims: Given ongoing challenges in non-invasive non-alcoholic liver disease (NAFLD) diagnosis, we sought to validate an ALT-based NAFLD phenotype using measures readily available in electronic health records (EHRs) and population-based studies by leveraging the clinical and genetic data in the Million Veteran Program (MVP), a multi-ethnic mega-biobank of US Veterans. Methods: MVP participants with alanine aminotransferases (ALT) >40 units/L for men and >30 units/L for women without other causes of liver disease were compared to controls with normal ALT. Genetic variants spanning eight NAFLD risk or ALT-associated loci (LYPLAL1, GCKR, HSD17B13, TRIB1, PPP1R3B, ERLIN1, TM6SF2, PNPLA3) were tested for NAFLD associations with sensitivity analyses adjusting for metabolic risk factors and alcohol consumption. A manual EHR review assessed performance characteristics of the NAFLD phenotype with imaging and biopsy data as gold standards. Genetic associations with advanced fibrosis were explored using FIB4, NAFLD Fibrosis Score and platelet counts. Results: Among 322,259 MVP participants, 19% met non-invasive criteria for NAFLD. Trans-ethnic meta-analysis replicated associations with previously reported genetic variants in all but LYPLAL1 and GCKR loci (P<6x10-3), without attenuation when adjusted for metabolic risk factors and alcohol consumption. At the previously reported LYPLAL1 locus, the established genetic variant did not appear to be associated with NAFLD, however the regional association plot showed a significant association with NAFLD 279kb downstream. In the EHR validation, the ALT-based NAFLD phenotype yielded a positive predictive value 0.89 and 0.84 for liver biopsy and abdominal imaging, respectively (inter-rater reliability (Cohen's kappa = 0.98)). HSD17B13 and PNPLA3 loci were associated with advanced fibrosis. Conclusions: We validate a simple, non-invasive ALT-based NAFLD phenotype using EHR data by leveraging previously established NAFLD risk-associated genetic polymorphisms. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) results in the accumulation of fat in the liver and can progress as an inflammatory disorder with considerable vascular endothelial dysfunction known as nonalcoholic steatohepatitis (NASH). Inflammatory signals can trigger the expression of vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells. VCAM-1 is a surface protein that induces adherence and extravasation of monocytes to blood vessels. In this issue of the JCI, Furuta et al. report on their sequencing of RNA transcripts from the livers of mice fed a NASH-inducing diet. VCAM-1 was upregulated in the whole liver as well as liver sinusoidal endothelial cells (LSECs). When the researchers incubated LSECs with palmitate, a toxic lipid, VCAM-1 was upregulated. Notably, inhibiting VCAM-1 in the NASH model reduced VCAM-1 expression, lessened infiltrating macrophages, and mitigated fibrosis. This study connects steatosis to endothelial dysfunction and inflammation and suggests that targeting VCAM-1 may address fibrosis in patients with NASH. [ABSTRACT FROM AUTHOR]

Background and Aim: Nonalcoholic fatty liver disease (NAFLD) is a common cause of hepatic steatosis in patients with inflammatory bowel disease (IBD). Both metabolic syndrome (MetS) and intestinal inflammation are implicated in NAFLD pathogenesis.Methods: We performed a retrospective cohort study of patients with IBD and NAFLD seen in our health system from January 1997 to December 2011 to examine associations between IBD severity and phenotype; MetS; and NAFLD fibrosis as estimated by the NAFLD Fibrosis Score (NFS).Results: A total of 84 patients were included in our analysis (24 UC, 60 CD). 23% of patients had MetS. IBD patients with MetS were significantly older at the time of IBD diagnosis (44 vs. 33, p = 0.005) and NAFLD diagnosis (55 vs. 47, p = 0.018). IBD patients with MetS had higher ALT (54 vs. 38 U/L, p = 0.02) and AST (52 vs. 35 U/L, p = 0.004). Comparing MetS patients to non-MetS IBD patients, there was no significant difference between IBD medication use (i.e., steroids, anti-TNFs, and immunomodulators) or NAFLD medication use, other than statins. Both UC and CD patients with concomitant MetS had significantly higher NFS scores than non-MetS patients: UC (-0.4 vs. -2.5, p = 0.02) and CD (-0.8 vs. -2.3, p = 0.03). IBD disease severity, disease location, or IBD medication use was associated with NAFLD severity.Conclusions: To our knowledge, this is the first study to demonstrate that NAFLD severity in both UC and CD IBD patients is associated with the presence of MetS but not with the severity of IBD. [ABSTRACT FROM AUTHOR]