Intracellular lipases can be broadly divided into two categories: neutral lipases and acid lipases. Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MAGL) are three key neutral lipases responsible for the hydrolysis of triacylglycerol (TAG) in lipid droplets (LDs). Although these three TAG intracellular lipase genes have been identified and characterized in multiple model species, their evolutionary history remains largely unknown. For the TAG intracellular lipase genes in Decapoda, there is also a large knowledge gap. Thus, in this study, we performed a genome-wide identification and investigation of TAG intracellular lipase genes in Decapoda and outgroups, analyzing their phylogenetics, structural features, conserved motifs, and expression patterns. In total, 22 ATGL genes, 23 HSL genes and 21 MAGL genes were identified in 17 selected species. HSL is the oldest and most conserved gene to exist in any species. Furthermore, RNA-seq analysis was conducted on two representative Decapod species, Chinese mitten crab (Eriocheir sinensis) and swimming crab (Portunus trituberculatus), which represent freshwater and marine environments, respectively. The analysis revealed a positive correlation between the expression levels of TAG intracellular lipase genes and the energy demand during different molting stages. Overall, the results of this study provide valuable insights into the evolutionary history of TAG intracellular lipase genes, which could enhance our understanding for the role of these genes during key physiological processes of Decapods.

PNPLA3(148M) (patatin-like phospholipase domain-containing protein 3) is the most impactful genetic risk factor for steatotic liver disease. A key unresolved issue is whether PNPLA3(148M) confers a loss- or gain-of-function. Here we test the hypothesis that PNPLA3 causes steatosis by sequestering ABHD5 (α/β hydrolase domain-containing protein 5), the cofactor of ATGL (adipose TG lipase), thus limiting mobilization of hepatic triglyceride (TG).

We quantified and compared the physical interactions between ABHD5 and PNPLA3/ATGL in cultured hepatocytes using NanoBiT complementation assays and immunocytochemistry. Recombinant proteins purified from human cells were used to compare TG hydrolytic activities of PNPLA3 and ATGL in the presence or absence of ABHD5. Adenoviruses and adeno-associated viruses were used to express PNPLA3 in liver-specific Atgl-/- mice and to express ABHD5 in livers of Pnpla3M/M mice, respectively.

ABHD5 interacted preferentially with PNPLA3 relative to ATGL in cultured hepatocytes. No differences were seen in the strength of the interactions between ABHD5 with PNPLA3(WT) and PNPLA3(148M). In contrast to prior findings, we found that PNPLA3, like ATGL, is activated by ABHD5 in in vitro assays using purified proteins. PNPLA3(148M)-associated inhibition of TG hydrolysis required that ATGL be expressed and that PNPLA3 be located on lipid droplets. Finally, overexpression of ABHD5 reversed the hepatic steatosis in Pnpla3M/M mice.

These findings support the premise that PNPLA3(148M) is a gain-of-function mutation that promotes hepatic steatosis by accumulating on lipid droplets and inhibiting ATGL-mediated lipolysis in an ABHD5-dependent manner. Our results predict that reducing, rather than increasing, PNPLA3 expression will be the best strategy to treat PNPLA3(148M)-associated steatotic liver disease.

Steatotic liver disease (SLD) is a common complex disorder associated with both environmental and genetic risk factors. PNPLA3(148M) is the most impactful genetic risk factor for SLD and yet its pathogenic mechanism remains controversial. Herein, we provide evidence that PNPLA3(148M) promotes triglyceride (TG) accumulation by sequestering ABHD5, thus limiting its availability to activate ATGL. Although the substitution of methionine for isoleucine reduces the TG hydrolase activity of PNPLA3, the loss of enzymatic function is not directly related to the steatotic effect of the variant. It is the resulting accumulation of PNPLA3 on LDs that confers a gain-of-function by interfering with ATGL-mediated TG hydrolysis. These findings have implications for the design of potential PNPLA3(148M)-based therapies. Reducing, rather than increasing, PNPLA3 levels is predicted to reverse steatosis in susceptible individuals.

Patatin-like phospholipase domain-containing 3 (PNPLA3) was the first gene identified through genome-wide association studies to be linked to hepatic fat accumulation. A missense variant, encoding the PNPLA3-148M allele, has since been shown to increase the risk for the full spectrum of steatotic liver disease (SLD), from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Despite extensive validation of this association and ongoing research into its pathogenic role, the precise mechanisms by which PNPLA3-148M contributes to the progression of SLD remain poorly understood. In this review, we evaluate preclinical in vitro and in vivo models used to investigate PNPLA3 and its involvement in SLD, with particular emphasis on metabolic dysfunction-associated steatotic liver disease. We assess the strengths and limitations of these models, as well as the challenges arising from species differences in PNPLA3 expression and function between human and murine systems.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is caused by metabolic triggers and genetic predisposition. Among the genetic MASLD risk variants identified today, the common PNPLA3 148M variant exerts the largest effect size of MASLD heritability. The PNPLA3 148M protein is causatively linked to the development of liver steatosis, inflammation and fibrosis in experimental studies and is therefore an appealing target for therapeutic approaches to treat this disease. Several PNPLA3 targeted approaches are currently being evaluated in clinical trials for the treatment of metabolic dysfunction-associated steatohepatitis (MASH), the most severe form of MASLD and promising proof of principle data with reduced liver fat content in homozygous PNPLA3 148M risk allele carriers has been reported from phase 1 trials following hepatic silencing of PNPLA3. Thus, targeting PNPLA3, the strongest genetic determinant of MASH may hold promise as the first precision medicine for the treatment of this disease. A histological endpoint-based phase 2b study has been initiated and several more are expected to be initiated to evaluate treatment effects on histological MASH and liver fibrosis in participants being homozygous for the PNPLA3 148M risk allele variant. The scope of this mini-review is to briefly describe the PNPLA3 148M genetics, function and preclinical experimental evidence with therapeutic approaches targeting PNPLA3 as well as to summarise the PNPLA3 based therapies currently in clinical development.

Adipose triglyceride lipase (ATGL) is an attractive therapeutic target in insulin resistance and metabolic dysfunction-associated steatotic liver disease (MASLD). This study investigated the effects of pharmacological ATGL inhibition on the development of metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis in mice.

Streptozotocin-injected male mice were fed a high-fat diet to induce MASH. Mice receiving the ATGL inhibitor atglistatin (ATGLi) were compared to controls using liver histology, lipidomics, metabolomics, 16s rRNA, and RNA sequencing. Human ileal organoids, HepG2 cells, and Caco2 cells treated with the human ATGL inhibitor NG-497, HepG2 ATGL knockdown cells, gel-shift, and luciferase assays were analysed for mechanistic insights. We validated the benefits of ATGLi on steatohepatitis and fibrosis in a low-methionine choline-deficient mouse model.

ATGLi improved serum liver enzymes, hepatic lipid content, and histological liver injury. Mechanistically, ATGLi attenuated PPARα signalling, favouring hydrophilic bile acid (BA) synthesis with increased Cyp7a1, Cyp27a1, Cyp2c70, and reduced Cyp8b1 expression. Additionally, reduced intestinal Cd36 and Abca1, along with increased Abcg5 expression, were consistent with reduced levels of hepatic triacylglycerol species containing polyunsaturated fatty acids, like linoleic acid, as well as reduced cholesterol levels in the liver and plasma. Similar changes in gene expression associated with PPARα signalling and intestinal lipid transport were observed in ileal organoids treated with NG-497. Furthermore, HepG2 ATGL knockdown cells revealed reduced expression of PPARα target genes and upregulation of genes involved in hydrophilic BA synthesis, consistent with reduced PPARα binding and luciferase activity in the presence of the ATGL inhibitors.

Inhibition of ATGL attenuates PPARα signalling, translating into hydrophilic BA composition, interfering with dietary lipid absorption, and improving metabolic disturbances. Validation with NG-497 opens a new therapeutic perspective for MASLD.

Despite the recent approval of drugs novel mechanistic insights and pathophysiology-oriented therapeutic options for MASLD (metabolic dysfunction-associated steatotic liver disease) are still urgently needed. Herein, we show that pharmacological inhibition of ATGL, the key enzyme in lipid hydrolysis, using atglistatin (ATGLi), improves MASH (metabolic dysfunction-associated steatohepatitis), fibrosis, and key features of metabolic dysfunction in mouse models of MASH and liver fibrosis. Mechanistically, we demonstrated that attenuation of PPARα signalling in the liver and gut favours hydrophilic bile acid composition, ultimately interfering with dietary lipid absorption. One of the drawbacks of ATGLi is its lack of efficacy against human ATGL, thus limiting its clinical applicability. Against this backdrop, we could show that ATGL inhibition using the human inhibitor NG-497 in human primary ileum-derived organoids, Caco2 cells, and HepG2 cells translated into therapeutic mechanisms similar to ATGLi. Collectively, these findings reveal a possible new avenue for MASLD treatment.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by increased hepatic steatosis with cardiometabolic disease and is a leading cause of advanced liver disease. We review here the genetic basis of MASLD. The genetic variants most consistently associated with hepatic steatosis implicate genes involved in lipoprotein input or output, glucose metabolism, adiposity/fat distribution, insulin resistance, or mitochondrial/ER biology. The distinct mechanisms by which these variants promote hepatic steatosis result in distinct effects on cardiometabolic disease that may be best suited to precision medicine. Recent work on gene-environment interactions has shown that genetic risk is not fixed and may be exacerbated or attenuated by modifiable (diet, exercise, alcohol intake) and nonmodifiable environmental risk factors. Some steatosis-associated variants, notably those in patatin-like phospholipase domain-containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2), are associated with risk of developing adverse liver-related outcomes and provide information beyond clinical risk stratification tools, especially in individuals at intermediate to high risk for disease. Future work to better characterize disease heterogeneity by combining genetics with clinical risk factors to holistically predict risk and develop therapies based on genetic risk is required.

Metabolic dysfunction-associated steatotic liver disease (MASLD) covers a range of liver conditions marked by the buildup of fat, spanning from simple fatty liver to more advanced stages like metabolic dysfunction-associated steatohepatitis and cirrhosis.

Our in-depth analysis of PNPLA3_WT and mutants (I148M (MT1) and C15S (MT2)) provides insights into their structure-function dynamics in lipid metabolism, especially lipid droplet hydrolysis and ABHD5 binding. Employing molecular docking, binding affinity, MD analysis, dissociation constant, and MM/GBSA analysis, we delineated distinct binding characteristics between wild-type and mutants.

Structural dynamics analysis revealed that unbound mutants exhibited higher flexibility, increased Rg and SASA values, and broader energy landscapes, indicating multiple inactive states. Mutations, especially in PNPLA3_MT1, reduced the exposure of the catalytic serine, potentially impairing enzymatic activity and LD hydrolysis efficiency. Altered interaction patterns and dynamics, particularly a shift in ABHD5 binding regions towards the C-terminal domain, underscore its role in LD metabolism. Energy dynamics analysis of the protein complexes revealed PNPLA3_WT exhibited multiple low-energy macrostates, whereas the mutants displayed narrower energy landscapes, suggesting a more stable functional state. PNPLA3_MT1 demonstrated the highest affinity towards ABHD5, highlighting the complex interplay between protein structure, dynamics, and lipid metabolism regulation.

PNPLA3_MT1 mutant exhibits the highest flexibility and significantly reduced catalytic serine accessibility, leading to impaired lipolysis. Contrarily, PNPLA3_WT maintains stable catalytic efficiency and effective LD hydrolysis, with PNPLA3_MT2 displaying intermediate behavior.

Our research provides valuable insights into the metabolic implications of PNPLA3 mutations, offering a path for potential therapeutic interventions in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD; formerly known as nonalcoholic fatty liver disease) is a chronic liver disease affecting over a billion individuals worldwide. MASLD can gradually develop into more severe liver pathologies, including metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and liver malignancy. Notably, although being a global health problem, there are very limited therapeutic options against MASLD and its related diseases. While a thyroid hormone receptor agonist (resmetirom) is recently approved for MASH treatment, other efforts to control these diseases remain unsatisfactory. Given the projected rise in MASLD and MASH incidence, it is urgent to develop novel and effective therapeutic strategies against these prevalent liver diseases. In this article, the pathogenic mechanisms of MASLD and MASH, including insulin resistance, dysregulated nuclear receptor signaling, and genetic risk factors (eg, patatin-like phospholipase domain-containing 3 and hydroxysteroid 17-β dehydrogenase-13), are introduced. Various therapeutic interventions against MASH are then explored, including approved medication (resmetirom), drugs that are currently in clinical trials (eg, glucagon-like peptide 1 receptor agonist, fibroblast growth factor 21 analog, and PPAR agonist), and those failed in previous trials (eg, obeticholic acid and stearoyl-CoA desaturase 1 antagonist). Moreover, given that the role of gut microbes in MASLD is increasingly acknowledged, alterations in the gut microbiota and microbial mechanisms in MASLD development are elucidated. Therapeutic approaches that target the gut microbiota (eg, dietary intervention and probiotics) against MASLD and related diseases are further explored. With better understanding of the multifaceted pathogenic mechanisms, the development of innovative therapeutics that target the root causes of MASLD and MASH is greatly facilitated. The possibility of alleviating MASH and achieving better patient outcomes is within reach. SIGNIFICANCE STATEMENT: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide, and it can progress to more severe pathologies, including steatohepatitis, cirrhosis, and liver cancer. Better understanding of the pathogenic mechanisms of these diseases has facilitated the development of innovative therapeutic strategies. Moreover, increasing evidence has illustrated the crucial role of gut microbiota in the pathogenesis of MASLD and related diseases. It may be clinically feasible to target gut microbes to alleviate MASLD in the future.

The liver acts as a central metabolic hub, integrating signals from the gastrointestinal tract and adipose tissue to regulate carbohydrate, lipid, and amino acid metabolism. Gut-derived metabolites, such as acetate and ethanol and non-esterified fatty acids from white adipose tissue (WAT), influence hepatic processes, which rely on mitochondrial function to maintain systemic energy balance. Metabolic dysregulation from obesity, insulin resistance, and type 2 diabetes disrupt these pathways, leading to metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH). This review explores the metabolic fluxes within the gut-adipose tissue-liver axis, focusing on the pivotal role of de novo lipogenesis (DNL), dietary substrates like glucose and fructose, and changes in mitochondrial function during MASLD progression. It highlights the contributions of white adipose tissue insulin resistance and impaired mitochondrial dynamics to hepatic lipid accumulation. Further understanding how the interplay between substrate flux from the gastro-intestinal tract integrates with adipose tissue and intersects with structural and functional alterations to liver mitochondria will be important to identify novel therapeutic targets and advance the treatment of MASLD and MASH.

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 days without any changes in total liver cholesterol content. Treating mice with antisense oligonucleotides (ASOs) against Coenzyme A synthase (Cosay) 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 COASY knockdown and bempedoic acid are novel therapeutic approaches to reduce liver lipid droplet cholesterol content and thereby prevent the development of MASH and liver fibrosis.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease linked to fibrosis. The role of specific lipid species in its pathogenesis remains debated. Using dietary, molecular, and genetic models, we found that mice on a choline-deficient, high-fat diet (CDAHFD) developed steatohepatitis and early fibrosis, marked by increased cholesterol in liver lipid droplets within five days. Targeting COASY with antisense oligonucleotides or treating with bempedoic acid or atorvastatin reduced lipid droplet cholesterol and prevented MASH. However, dietary cholesterol supplementation negated these effects. Human liver samples confirmed elevated lipid droplet cholesterol in MASH and fibrosis, especially in PNPLA3 I148M carriers. These findings highlight cholesterol reduction as a potential MASH therapy.

Non-alcoholic fatty liver disease (NAFLD) and its advanced form, non-alcoholic steatohepatitis (NASH), are the leading causes of chronic liver disease globally. They are driven by complex mechanisms where inflammation plays a pivotal role in disease progression. Current therapies, including lifestyle changes and pharmacological agents, are limited in efficacy, particularly in addressing the advanced stages of the disease. Emerging approaches targeting inflammation, metabolic dysfunction, and fibrosis offer promising new directions, though challenges such as treatment complexity and heterogeneity persist. This review concludes the main therapeutic targets and approaches to manage inflammation currently and emphasizes the critical need for future drug development and combination therapy for NAFLD/NASH management.

The storage and release of triacylglycerol (TAG) in lipid droplets (LDs) is regulated by dynamic protein interactions. α/β Hydrolase domain-containing protein 5 (ABHD5; also known as CGI-58) is a membrane/LD-bound protein that functions as a co-activator of patatin-like phospholipase domain-containing 2 (PNPLA2; also known as adipose triglyceride lipase) the rate-limiting enzyme for TAG hydrolysis. The dysregulation of TAG hydrolysis is involved in various metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). We previously demonstrated that ABHD5 interacted with PNPLA3, a closely related family member to PNPLA2. Importantly, a common missense variant in PNPLA3 (I148M) is the greatest genetic risk factor for MASLD. PNPLA3 148M functions to sequester ABHD5 and prevent coactivation of PNPLA2, which has implications for initiating MASLD; however, the exact mechanisms involved are not understood. Here, we demonstrate that LD targeting of both ABHD5 and PNPLA3 I148M is required for the interaction. Molecular modeling demonstrates important residues in the C terminus of PNPLA3 for LD binding and fluorescence cross-correlation spectroscopy demonstrates that PNPLA3 I148M has greater association with ABHD5 than WT PNPLA3. Moreover, the C terminus of PNPLA3 is sufficient for functional targeting of PNPLAs to LD and the interaction with ABHD5. In addition, ABHD5 is a general binding partner of LD-bound PNPLAs. Finally, PNPLA3 I148M targeting to LD is required to promote steatosis in vitro and in the liver. Overall results suggest that the interaction of PNPLA3 I148M with ABHD5 on LD is required to promote liver steatosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD), is the most common cause of liver disease, and its burden on health systems worldwide continues to rise at an alarming rate. MASLD is a complex disease in which the interactions between susceptible genes and the environment influence the disease phenotype and severity. Advances in human genetics over the past few decades have provided new opportunities to improve our understanding of the multiple pathways involved in the pathogenesis of MASLD. Notably, the PNPLA3, TM6SF2, GCKR, MBOAT7 and HSD17B13 single nucleotide polymorphisms have been demonstrated to be robustly associated with MASLD development and disease progression. These genetic variants play crucial roles in lipid droplet remodeling, secretion of hepatic very low-density lipoprotein and lipogenesis, and understanding the biology has brought new insights to this field. This review discusses the current body of knowledge regarding these genetic drivers and how they can lead to development of MASLD, the complex interplay with metabolic factors such as obesity, and how this information has translated clinically into the development of risk prediction models and possible treatment targets.

A common genetic variant (rs738409) encoding an isoleucine to methionine substitution at position 148 in the PNPLA3 protein is a determinant of hepatic steatosis, inflammation, fibrosis, cirrhosis, and liver-related mortality. AZD2693 is a liver-targeted antisense oligonucleotide against PNPLA3 mRNA. We evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics in single and multiple ascending dose studies.

AZD2693 was assessed in 3D cultures of homozygous PNPLA3 148M primary human hepatocytes and mice expressing human PNPLA3. The single ascending dose study investigated 2-110 mg doses in overweight/mildly obese but otherwise healthy volunteers. The multiple ascending dose study investigated three monthly doses (25 mg, 50 mg and 80 mg) in participants with MRI-proton density fat fraction (MRI-PDFF) ≥7%. Changes in liver fat content were assessed at baseline, weeks 8 and 12 by MRI-PDFF. PNPLA3 mRNA and protein knockdown levels were evaluated for the 80 mg dose.

AZD2693 potently reduced PNPLA3 expression in human hepatocytes and livers of mice. Clinically, AZD2693 was generally well tolerated (no adverse events leading to discontinuation or treatment-related serious adverse events). Half-life was 14-33 days across investigated doses. A least-square mean liver PNPLA3 mRNA knockdown of 89% and reduction of protein levels demonstrated target engagement. Changes in hepatic steatosis at week 12 were -7.6% and -12.2% (placebo-corrected least-square means) for the 25 mg and 50 mg doses, respectively. There was a dose-dependent increase of polyunsaturated fatty acids in serum triglycerides and decreases vs. placebo in high-sensitivity C-reactive protein and interleukin 6.

AZD2693 reduced liver PNPLA3 with an acceptable safety and tolerability profile. These findings support the continued development of AZD2693.

Clinical treatment options for metabolic dysfunction-associated steatohepatitis (MASH) are limited. The genetic risk factor with the largest effect size for progressing to poor liver-related outcomes in MASH is a single-nucleotide polymorphism in the gene PNPLA3 (p.I148M). In phase I single and multiple ascending dose studies, AZD2693, a liver-targeted antisense oligonucleotide, was well tolerated, reduced liver PNPLA3 mRNA and protein levels, and dose-dependently reduced liver fat content in homozygous PNPLA3 148M risk allele carriers. These data support continued development of AZD2693 as a potential precision medicine treatment for MASH. The phase IIb FORTUNA study is now ongoing.

NCT04142424, NCT04483947.

Many factors are associated with the development and progression of liver fat and fibrosis; however, genetics and the gut microbiota are representative factors. Moreover, recent studies have indicated a link between host genes and the gut microbiota. This study investigated the effect of patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 (C > G), which has been reported to be most involved in the onset and progression of fatty liver, on liver fat and fibrosis in a cohort study related to gut microbiota in a non-fatty liver population.

This cohort study included 214 participants from the health check-up project in 2018 and 2022 who had non-fatty liver with controlled attenuation parameter (CAP) values <248 dB/m by FibroScan and were non-drinkers. Changes in CAP values and liver stiffness measurement (LSM), liver-related items, and gut microbiota from 2018 to 2022 were investigated separately for PNPLA3 rs738409 CC, CG, and GG genotypes.

Baseline values showed no difference among the PNPLA3 rs738409 genotypes for any of the measurement items. From 2018 to 2022, the PNPLA3 rs738409 CG and GG genotype groups showed a significant increase in CAP and body mass index; no significant change was observed in the CC genotype group. LSM increased in all genotypes, but the rate of increase was highest in the GG genotype, followed by the CG and CC genotypes. Fasting blood glucose levels increased in all genotypes; however, HOMA-IR (Homeostasis Model Assessment of Insulin Resistance) increased significantly only in the GG genotype. HDL (high-density lipoprotein) and LDL (low-density lipoprotein) cholesterol levels significantly increased in all genotypes, whereas triglycerides did not show any significant changes in any genotype. As for the gut microbiota, the relative abundance of Feacalibacterium in the PNPLA3 rs738409 GG genotype decreased by 2% over 4 years, more than 2-fold compared to CC and GG genotypes. Blautia increased significantly in the CC group.

The results suggest that PNPLA3 G-allele carriers of non-fatty liver develop liver fat and fibrosis due to not only obesity and insulin resistance but also the deterioration of gut microbiota, which may require a relatively long course of time, even years.

The storage and release of triacylglycerol (TAG) in lipid droplets (LDs) is regulated by dynamic protein interactions. α/β hydrolase domain-containing protein 5 (ABHD5; also known as CGI-58) is a membrane/LD bound protein that functions as a co-activator of Patatin Like Phospholipase Domain Containing 2 (PNPLA2; also known as Adipose triglyceride lipase, ATGL) the rate-limiting enzyme for TAG hydrolysis. The dysregulation of TAG hydrolysis is involved in various metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). We previously demonstrated that ABHD5 interacted with PNPLA3, a closely related family member to PNPLA2. Importantly, a common missense variant in PNPLA3 (I148M) is the greatest genetic risk factor for MASLD. PNPLA3 148M functions to sequester ABHD5 and prevent co-activation of PNPLA2, which has implications for initiating MASLD; however, the exact mechanisms involved are not understood. Here we demonstrate that LD targeting of both ABHD5 and PNPLA3 I148M is required for the interaction. Molecular modeling demonstrates important resides in the C-terminus of PNPLA3 for LD binding and fluorescence cross-correlation spectroscopy demonstrates that PNPLA3 I148M greater associates with ABHD5 than WT PNPLA3. Moreover, the C-terminus of PNPLA3 is sufficient for functional targeting of PNPLAs to LD and the interaction with ABHD5. In addition, ABHD5 is a general binding partner of LD-bound PNPLAs. Finally, PNPLA3 I148M targeting to LD is required to promote steatosis in vitro and in the liver. Overall results suggest that PNPLA3 I148M is a gain of function mutation and that the interaction with ABHD5 on LD is required to promote liver steatosis.

Recent advances in the genetics of metabolic dysfunction-associated steatotic liver disease (MASLD) are gradually revealing the mechanisms underlying the heterogeneity of the disease and have shown promising results in patient stratification. Genetic characterization of the disease has been rapidly developed using genome-wide association studies, exome-wide association studies, phenome-wide association studies, and whole exome sequencing. These advances have been powered by the increase in computational power, the development of new analytical algorithms, including some based on artificial intelligence, and the recruitment of large and well-phenotyped cohorts. This review presents an update on genetic studies that emphasize new biological insights from next-generation sequencing approaches. Additionally, we discuss innovative methods for discovering new genetic loci for MASLD, including rare variants. To comprehensively manage MASLD, it is important to stratify risks. Therefore, we present an update on phenome-wide association study associations, including extreme phenotypes. Additionally, we discuss whether polygenic risk scores and targeted sequencing are ready for clinical use. With particular focus on precision medicine, we introduce concepts such as the interplay between genetics and the environment in modulating genetic risk with lifestyle or standard therapies. A special chapter is dedicated to gene-based therapeutics. The limitations of approved pharmacological approaches are discussed, and the potential of gene-related mechanisms in therapeutic development is reviewed, including the decision to perform genetic testing in patients with MASLD.

Exposure to ambient air pollutants has emerged as a risk for metabolic-dysfunction associated steatotic liver disease (MASLD).

We sought to examine associations between short-term (prior month) and long-term (prior year) ambient air pollution exposure with hepatic fat fraction (HFF) and liver stiffness in Latino youth with obesity. A secondary aim was to investigate effect modification by patatin-like phospholipase domain-containing protein 3 (PNPLA3) genotype and liver disease severity.

Data was analyzed from 113 Latino youth (age 11-19) with obesity in Southern California. Individual exposure to particulate matter with aerodynamic diameter ≤ 2.5μm (PM2.5), ≤ 10μm (PM10), nitrogen dioxide (NO2), 8-hour maximum ozone (8hrMax-O3), 24-hr O3, and redox-weighted oxidative capacity (Oxwt) were estimated using residential address histories and United States Environmental Protection Agency air quality observations. HFF and liver stiffness were measured using magnetic resonance imaging. Linear models were used to determine associations between short-term and long-term exposure to air pollutants with HFF and liver stiffness. Modification by PNPLA3 and liver disease severity was then examined.

Short-term exposure to 8hrMax-O3 was positively associated with HFF. Relationships between air pollution exposure and HFF were not impacted by PNPLA3 genotype or liver disease severity. Long-term exposure to 8hrMax-O3 and Oxwt were positively associated with liver stiffness. Associations between air pollution exposure and liver stiffness depended on PNPLA3 genotype, such that individuals with GG genotypes exhibited stronger, more positive relationships between short-term exposure to PM10, 8hrMax-O3, 24-hr O3, and Oxwt and liver stiffness than individuals with CC/CG genotypes. In addition, relationships between short-term exposure to NO2 and liver stiffness were stronger in those with severe liver disease.

Air pollution exposure may be a risk factor for liver disease among Latino youth with obesity, particularly in those with other preexisting risks for liver damage.

Membrane-associated ring-CH-type finger 6 (MARCH6), also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of March6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing in prevalence, impacting over a third of the global population. The advanced form of MASLD, Metabolic dysfunction-associated steatohepatitis (MASH), is on track to become the number one indication for liver transplant. FDA-approved pharmacological agents are limited for MASH, despite over 400 ongoing clinical trials, with only a single drug (resmetirom) currently on the market. This is likely due to the heterogeneous nature of disease pathophysiology, which involves interactions between highly individualized genetic and environmental factors. To apply precision medicine approaches that overcome interpersonal variability, in-depth insights into interactions between genetics, nutrition, and the gut microbiome are needed, given that each have emerged as dynamic contributors to MASLD and MASH pathogenesis. Here, we discuss the associations and molecular underpinnings of several of these factors individually and outline their interactions in the context of both patient-based studies and preclinical animal model systems. Finally, we highlight gaps in knowledge that will require further investigation to aid in successfully implementing precision medicine to prevent and alleviate MASLD and MASH.

Hepatic steatosis, the buildup of neutral lipids in lipid droplets (LDs), is commonly referred to as metabolic dysfunction-associated steatotic liver disease when alcohol or viral infections are not involved. Metabolic dysfunction-associated steatotic liver disease encompasses simple steatosis and the more severe metabolic dysfunction-associated steatohepatitis, characterized by inflammation, hepatocyte injury, and fibrosis. Previously viewed as inert markers of disease, LDs are now understood to play active roles in disease etiology and have significant nonpathological and pathological functions in cell signaling and function. These dynamic properties of LDs are tightly regulated by hundreds of proteins that coat the LD surface, controlling lipid metabolism, trafficking, and signaling. The following review highlights various facets of LD biology with the primary goal of discussing key mechanisms through which LDs promote the development of advanced liver diseases, including metabolic dysfunction-associated steatohepatitis.

Lipid droplets (LDs) are subcellular organelles that play an integral role in lipid metabolism by regulating the storage and release of fatty acids, which are essential for energy production and various cellular processes. Lipolysis and lipophagy are the two major LD degradation pathways that mediate the utilization of lipids stored in these organelles. Recent studies have further uncovered alternative pathways, including direct lysosomal LD degradation and LD exocytosis. Here, we highlight recent findings that dissect the molecular basis of these diverse LD degradation pathways. Then, we discuss speculations on the crosstalk among these pathways and the potential unconventional roles of LD degradation.

Lipid droplet (LD) is a metabolically active organelle, which changes dynamically with the metabolic state and energy requirements of cells. Proteins that either insert into the LD phospholipid monolayer or are present in the cytoplasm, playing a crucial role in lipid homeostasis and signaling regulation, are known as LD-associated proteins.

The keywords "lipid droplets" and "metabolic diseases" were used to obtain literature on LD metabolism and pathological mechanism. After searching databases including Scopus, OVID, Web of Science, and PubMed from 2013 to 2024 using terms like "lipid droplets", "lipid droplet-associated proteins", "fatty liver disease", "diabetes", "diabetic kidney disease", "obesity", "atherosclerosis", "hyperlipidemia", "natural drug monomers" and "natural compounds", the most common natural compounds were identified in about 954 articles. Eventually, a total of 91 studies of 10 natural compounds reporting in vitro or in vivo studies were refined and summarized.

The most frequently used natural compounds include Berberine, Mangostin, Capsaicin, Caffeine, Genistein, Epigallocatechin-3-gallate, Chlorogenic acid, Betaine, Ginsenoside, Resveratrol. These natural compounds interact with LD-associated proteins and help ameliorate abnormal LDs in various metabolic diseases.

Natural compounds involved in the regulation of LDs and LD-associated proteins hold promise for treating metabolic diseases. Further research into these interactions may lead to new therapeutic applications.

Lipolysis is the hydrolysis of triglycerides (TGs), commonly known as fats. Intracellular lipolysis of TG is associated with adipose triglyceride lipase (ATGL), which provides fatty acids during times of metabolic need. The aim of this study was to determine whether Coix lacryma-jobi L. var. ma-yuen Stapf (Coix) sprouts (CS) can alleviate obesity through lipolysis. Overall, we investigated the potential of CS under in vitro and in vivo conditions and confirmed the underlying mechanisms. Huh7 cells were exposed to free fatty acids (FFAs), and C57BL/6J mice were fed a 60% high-fat diet. When FFA were introduced into Huh7 cells, the intracellular TG levels increased within the Huh7 cells. However, CS treatment significantly reduced intracellular TG levels. Furthermore, CS decreased the expression of Pparγ and Srebp1c mRNA and downregulated the mutant Pnpla3 (I148M) mRNA. Notably, CS significantly upregulated ATGL expression. CS treatment at a dose of 200 mg/kg/day resulted in a significant and dose-dependent decrease in body weight gain and epididymal adipose tissue weight. Specifically, the group treated with CS (200 mg/kg/day) exhibited a significant modulation of serum lipid biomarkers. In addition, CS ameliorated histological alterations in both the liver and adipose tissues. In summary, CS efficiently inhibited lipid accumulation through the activation of the lipolytic enzyme ATGL coupled with the suppression of enzymes involved in TG synthesis. Consequently, CS show promise as a potential anti-obesity agent.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common paediatric liver disease. Latinos have high MASLD risk due to 50% prevalence of GG genotype of PNPLA3. Our primary aim was to evaluate associations between dietary carbohydrates/sugars and liver stiffness in Latino adolescents with obesity. Our secondary aim was to examine effect modification by (a) PNPLA3 genotype or (b) liver disease severity. Data were obtained from 114 Latino adolescents with obesity involved in two prior studies. No associations were seen between dietary carbohydrates/sugars and liver stiffness in the group as a whole. In subjects with GG genotype of PNPLA3, total sugar, fructose, sucrose, and glucose were associated with liver stiffness. Positive relationships between carbohydrate, total sugar, and sucrose and liver stiffness were stronger in those with MASLD and fibrosis compared to those with healthy livers and MASLD without fibrosis.

In this study, blackberry polysaccharide-selenium nanoparticles (BBP-24-3Se) were first prepared via Na2SeO3/Vc redox reaction, followed by coating with red blood cell membrane (RBC) to form core-shell structure polysaccharide-selenium nanoparticles (RBC@BBP-24-3Se). The particle size of BBP-24-3Se (167.1 nm) was increased to 239.8 nm (RBC@BBP-24-3Se) with an obvious core-shell structure after coating with RBC. FT-IR and XPS results indicated that the interaction between BBP-24-3 and SeNPs formed a new C-O···Se bond with valence state of Se0. Bioassays indicated that RBC coating markedly enhanced both the biocompatibility and bioabsorbability of RBC@BBP-24-3Se, and the absorption rate of RBC@BBP-24-3Se in HepG2 cells was 4.99 times higher than that of BBP-24-3Se at a concentration of 10 μg/mL. Compared with BBP-24-3Se, RBC@BBP-24-3Se possessed significantly heightened protective efficacy against oxidative damage and better regulation of glucose/lipid metabolism disorder induced by palmitic acid in HepG2 cells. Mechanistic studies demonstrated that RBC@BBP-24-3Se could effectively improve PI3K/AKT signaling pathway to promote glucose metabolism, inhibit the expression of lipid synthesis genes and up-regulate the expression of lipid-decomposing genes through AMPK signaling pathway to improve lipid metabolism. These results provided a theoretical basis for developing a new type of selenium supplement for the treatment of insulin resistance.

The I148M variant of PNPLA3 is closely associated with hepatic steatosis. Recent evidence indicates that the I148M mutant functions as an inhibitor of PNPLA2/ATGL-mediated lipolysis, leaving the role of wild-type PNPLA3 undefined. Despite showing a triglyceride hydrolase activity in vitro, PNPLA3 has yet to be established as a lipase in vivo. Here, we show that PNPLA3 preferentially hydrolyzes polyunsaturated triglycerides, mobilizing polyunsaturated fatty acids for phospholipid desaturation and enhancing hepatic secretion of triglyceride-rich lipoproteins. Under lipogenic conditions, mice with liver-specific knockout or acute knockdown of PNPLA3 exhibit aggravated liver steatosis and reduced plasma VLDL-triglyceride levels. Similarly, I148M-knockin mice show decreased hepatic triglyceride secretion during lipogenic stimulation. Our results highlight a specific context whereby the wild-type PNPLA3 facilitates the balance between hepatic triglyceride storage and secretion, and suggest the potential contribution of a loss-of-function by the I148M variant to the development of fatty liver disease in humans.

Fatty liver is characterized by the expansion of lipid droplets (LDs) and is associated with the development of many metabolic diseases. We assessed the morphology of hepatic LDs and performed quantitative proteomics in lean, glucose-tolerant mice compared with high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD- and HStD-fed mice had more and larger LDs than Chow-fed animals. We observed striking differences in liver LD proteomes of HFD- and HStD-fed mice compared with Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver LD proteome consisting of proteins common in HFD- and HStD-fed mice, as well as a proteome associated with glucose tolerance that included proteins shared in Chow and HStD but not HFD-fed mice. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase to perilipin 5 in the LD proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome.NEW & NOTEWORTHY This study identified a fatty liver lipid droplet proteome and one associated with glucose tolerance. Notably, glucose intolerance was linked with changes in the ratio of adipose triglyceride lipase to perilipin 5 that is indicative of dysregulation of neutral lipid homeostasis.

Our body stores energy mostly in form of fatty acids (FAs) in lipid droplets (LDs). From there the FAs can be mobilized and transferred to peroxisomes and mitochondria. This transfer is dependent on close opposition of LDs and mitochondria and peroxisomes and happens at membrane contact sites. However, the composition and the dynamics of these contact sites is not well understood, which is in part due to the dependence on the metabolic state of the cell and on the cell- and tissue-type. Here, we summarize the current knowledge on the contacts between lipid droplets and mitochondria both in mammals and in the yeast Saccharomyces cerevisiae, in which various contact sites are well studied. We discuss possible functions of the contact site and their implication in disease.

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.

The lipid droplet (LD) is a conserved organelle that exists in almost all organisms, ranging from bacteria to mammals. Dysfunctions in LDs are linked to a range of human metabolic syndromes. The formation of protein complexes on LDs is crucial for maintaining their function. Investigating how proteins interact on LDs is essential for understanding the role of LDs. We have developed an effective method to uncover protein-protein interactions and protein complexes specifically on LDs. In this method, we conduct co-immunoprecipitation (co-IP) experiments using LD proteins extracted directly from isolated LDs, rather than utilizing proteins from cell lysates. To elaborate, we begin by purifying LDs with high-quality and extracting LD-associated proteins. Subsequently, the co-IP experiment is performed on these LD-associated proteins directly, which would enhance the co-IP experiment specificity of LD-associated proteins. This method enables researchers to directly unveil protein complexes on LDs and gain deeper insights into the functional roles of proteins associated with LDs.

Genome-wide association studies have identified several genetic variants associated with nonalcoholic fatty liver disease. To emphasize metabolic abnormalities in fatty liver, metabolic (dysfunction)-associated fatty liver disease (MAFLD) has been introduced; thus, we aimed to investigate single-nucleotide polymorphisms related to MAFLD and its subtypes. A genome-wide association study was performed to identify genetic factors related to MAFLD. We used a Korean population-based sample of 2282 subjects with MAFLD and a control group of 4669. We replicated the results in a validation sample which included 639 patients with MAFLD and 1578 controls. Additionally, we categorized participants into three groups, no MAFLD, metabolic dysfunction (MD)-MAFLD, and overweight/obese-MAFLD. After adjusting for age, sex, and principal component scores, rs738409 [risk allele G] and rs3810622 [risk allele T], located in the PNPLA3 gene, showed significant associations with MAFLD (P-values, discovery set = 1.60 × 10-15 and 4.84 × 10-10; odds ratios, 1.365 and 1.284, validation set = 1.39 × 10-4, and 7.15 × 10-4, odds ratios, 1.299 and 1.264, respectively). An additional SNP rs59148799 [risk allele G] located in the GATAD2A gene showed a significant association with MAFLD (P-values, discovery set = 2.08 × 10-8 and validation set = 0.034, odds ratios, 1.387 and 1.250). rs738409 was significantly associated with MAFLD subtypes ([overweight/obese-MAFLD; odds ratio (95% confidence interval), P-values, 1.515 (1.351-1.700), 1.43 × 10-12 and MD-MAFLD: 1.300 (1.191-1.416), 2.90 × 10-9]. There was a significant relationship between rs3810622 and overweight/obese-MAFLD and MD-MAFLD [odds ratios (95% confidence interval), P-values, 1.418 (1.258, 1.600), 1.21 × 10-8 and 1.225 (1.122, 1.340), 7.06 × 10-6, respectively]; the statistical significance remained in the validation set. PNPLA3 was significantly associated with MAFLD and MAFLD subtypes in the Korean population. These results indicate that genetic factors play an important role in the pathogenesis of MAFLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the leading cause of chronic liver disease that affects over 30% of the world's population. For decades, the heterogeneity of non-alcoholic fatty liver disease (NAFLD) has impeded our understanding of the disease mechanism and the development of effective medications. However, a recent change in the nomenclature from NAFLD to MASLD emphasizes the critical role of systemic metabolic dysfunction in the pathophysiology of this disease and therefore promotes the progress in the pharmaceutical treatment of MASLD. In this review, we focus on the mechanism underlying the abnormality of hepatic lipid metabolism in patients with MASLD, and summarize the latest progress in the therapeutic medications of MASLD that target metabolic disorders.

A new nomenclature consensus has emerged for liver diseases that were previously known as non-alcoholic fatty liver disease (NAFLD) and metabolic dysfunction-associated fatty liver disease (MAFLD). They are now defined as metabolic dysfunction-associated steatotic liver disease (MASLD), which includes cardiometabolic criteria in adults. This condition, extensively studied in obese or overweight patients, constitutes around 30% of the population, with a steady increase worldwide. Lean patients account for approximately 10%-15% of the MASLD population. However, the pathogenesis is complex and is not well understood.

To systematically review the literature on the diagnosis, pathogenesis, characteristics, and prognosis in lean MASLD patients and provide an interpretation of these new criteria.

We conducted a comprehensive database search on PubMed and Google Scholar between January 2012 and September 2023, specifically focusing on lean NAFLD, MAFLD, or MASLD patients. We include original articles with patients aged 18 years or older, with a lean body mass index categorized according to the World Health Organization criteria, using a cutoff of 25 kg/m2 for the general population and 23 kg/m2 for the Asian population.

We include 85 studies in our analysis. Our findings revealed that, for lean NAFLD patients, the prevalence rate varied widely, ranging from 3.8% to 34.1%. The precise pathogenesis mechanism remained elusive, with associations found in genetic variants, epigenetic modifications, and adaptative metabolic response. Common risk factors included metabolic syndrome, hypertension, and type 2 diabetes mellitus, but their prevalence varied based on the comparison group involving lean patients. Regarding non-invasive tools, Fibrosis-4 index outperformed the NAFLD fibrosis score in lean patients. Lifestyle modifications aided in reducing hepatic steatosis and improving cardiometabolic profiles, with some medications showing efficacy to a lesser extent. However, lean NAFLD patients exhibited a worse prognosis compared to the obese or overweight counterpart.

MASLD is a complex disease comprising epigenetic, genetic, and metabolic factors in its pathogenesis. Results vary across populations, gender, and age. Limited data exists on clinical practice guidelines for lean patients. Future studies employing this new nomenclature can contribute to standardizing and generalizing results among lean patients with steatotic liver disease.

Lipid droplets are dynamic organelles that store neutral lipids, serve the metabolic needs of cells, and sequester lipids to prevent lipotoxicity and membrane damage. Here we review the current understanding of the mechanisms of lipid droplet biogenesis and turnover, the transfer of lipids and metabolites at membrane contact sites, and the role of lipid droplets in regulating fatty acid flux in lipotoxicity and cell death.

Patatin-like phospholipase domain containing proteins (PNPLAs) play diverse roles in lipid metabolism. In this review, we focus on the enzymatic properties and predicted 3D structures of PNPLA1-5. PNPLA2-4 exert both catabolic and anabolic functions. Whereas PNPLA1 is predominantly expressed in the epidermis and involved in sphingolipid biosynthesis, PNPLA2 and 4 are ubiquitously expressed and exhibit several enzymatic activities, including hydrolysis and transacylation of various (glycero-)lipid species. This review summarizes known biological roles for PNPLA-mediated hydrolysis and transacylation reactions and highlights open questions concerning their physiological function.

A missense variant in patatin-like phospholipase domain-containing protein 3 [PNPLA3(I148M)] is the most impactful genetic risk factor for fatty liver disease (FLD). We previously showed that PNPLA3 is ubiquitylated and subsequently degraded by proteasomes and autophagosomes and that the PNPLA3(148M) variant interferes with this process. To define the machinery responsible for PNPLA3 turnover, we used small interfering (si)RNAs to inactivate components of the ubiquitin proteasome system. Inactivation of bifunctional apoptosis regulator (BFAR), a membrane-bound E3 ubiquitin ligase, reproducibly increased PNPLA3 levels in two lines of cultured hepatocytes. Conversely, overexpression of BFAR decreased levels of endogenous PNPLA3 in HuH7 cells. BFAR and PNPLA3 co-immunoprecipitated when co-expressed in cells. BFAR promoted ubiquitylation of PNPLA3 in vitro in a reconstitution assay using purified, epitope-tagged recombinant proteins. To confirm that BFAR targets PNPLA3, we inactivated Bfar in mice. Levels of PNPLA3 protein were increased twofold in hepatic lipid droplets of Bfar-/- mice with no associated increase in PNPLA3 mRNA levels. Taken together these data are consistent with a model in which BFAR plays a role in the post-translational degradation of PNPLA3. The identification of BFAR provides a potential target to enhance PNPLA3 turnover and prevent FLD.

Subcutaneous fat deposition is an important index with which to evaluate meat-producing ducks, and affects their meat quality and feed conversion rate. Studying the differentially expressed genes in subcutaneous fat will help to comprehensively understand the potential mechanisms regulating fat deposition in ducks. In this study, 72 Nankou 1 Pekin Ducks and 72 Jingdian Pekin Ducks (half male and half female) at 42 days of age were selected for slaughter performance and transcriptome analysis. The results showed that the breast-muscle yield of Nankou 1 ducks was significantly higher than that of Jingdian ducks, but that the abdominal fat yield and subcutaneous fat yield were higher than that of Jingdian ducks. Thousands of DEGs, including many important genes involved in fat metabolism regulation, were detected by transcriptome. KEGG enrichment analysis showed that the DEGs were significantly enriched on pathways such as regulation of lipolysis in adipocytes, primary bile acid biosynthesis, and biosynthesis of unsaturated fatty acids. SCD, FGF7, LTBP1, PNPLA3, ADCY2, and ACOT8 were selected as candidate genes for regulating subcutaneous fat deposition. The results indicated that Nankou 1 had superior fat deposition ability compared to Jingdian ducks, and that the candidate genes regulated fat deposition by regulating fat synthesis and decomposition.

Cardiometabolic disease (CMD), which encompasses metabolic-associated fatty liver disease (MAFLD), chronic kidney disease (CKD) and cardiovascular disease (CVD), has been increasing considerably in the past 50 years. CMD is a complex disease that can be influenced by genetics and environmental factors such as diet. With the increased reliance on processed foods containing saturated fats, fructose and cholesterol, a mechanistic understanding of how these molecules cause metabolic disease is required. A major pathway by which excessive nutrients contribute to CMD is through oxidative stress. In this review, we discuss how oxidative stress can drive CMD and the role of aberrant nutrient metabolism and genetic risk factors and how they potentially interact to promote progression of MAFLD, CVD and CKD. This review will focus on genetic mutations that are known to alter nutrient metabolism. We discuss the major genetic risk factors for MAFLD, which include Patatin-like phospholipase domain-containing protein 3 (PNPLA3), Membrane Bound O-Acyltransferase Domain Containing 7 (MBOAT7) and Transmembrane 6 Superfamily Member 2 (TM6SF2). In addition, mutations that prevent nutrient uptake cause hypercholesterolemia that contributes to CVD. We also discuss the mechanisms by which MAFLD, CKD and CVD are mutually associated with one another. In addition, some of the genetic risk factors which are associated with MAFLD and CVD are also associated with CKD, while some genetic risk factors seem to dissociate one disease from the other. Through a better understanding of the causative effect of genetic mutations in CMD and how aberrant nutrient metabolism intersects with our genetics, novel therapies and precision approaches can be developed for treating CMD.

In the realm of oncology, the transformative impact of PROTAC (PROteolysis TAgeting Chimeras) technology has been particularly pronounced since its introduction in the 21st century. Initially conceived for cancer treatment, PROTACs have evolved beyond their primary scope, attracting increasing interest in addressing a diverse array of medical conditions. This expanded focus includes not only oncological disorders but also viral infections, bacterial ailments, immune dysregulation, neurodegenerative conditions, and metabolic disorders. This comprehensive review explores the broadening landscape of PROTAC application, highlighting ongoing developments and innovations aimed at deploying these molecules across a spectrum of diseases. Careful consideration of the design challenges associated with PROTACs reveals that, when appropriately addressed, these compounds present significant advantages over traditional therapeutic approaches, positioning them as promising alternatives. To evaluate the efficacy of PROTAC molecules, a diverse array of assays is employed, ranging from High-Throughput Imaging (HTI) assays to Cell Painting assays, CRBN engagement assays, Fluorescence Polarization assays, amplified luminescent proximity homogeneous assays, Timeresolved fluorescence energy transfer assays, and Isothermal Titration Calorimetry assays. These assessments collectively contribute to a nuanced understanding of PROTAC performance. Looking ahead, the trajectory of PROTAC technology suggests its potential recognition as a versatile therapeutic strategy for an expansive range of medical conditions. Ongoing progress in this field sets the stage for PROTACs to emerge as valuable tools in the multifaceted landscape of medical treatments.