Phospholipase Domain-containing Research Articles

Lipid droplet targeting of ABHD5 and PNPLA3 I148M is required to promote liver steatosis.

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.

Investigation on the mechanism of hepatotoxicity of dictamnine on juvenile zebrafish by integrating metabolomics and transcriptomics

Dictamnine(DIC), as the key pharmacological component of the classical Chinese herbal medicine cortex dictamni, possesses multiple pharmacological activities such as anti-microbial, anti-allergic, anti-cancer, and anti-inflammatory activities, however it is also the main toxicant of cortex dictamni induced hepatic damage, yet the underlying molecular mechanisms causing hepatic damage are still largely unknown. With the purpose of explore possibilities hepatotoxicity of dictamnine in zebrafish and to identify the key regulators and metabolites involved in the biological process, we administered zebrafish to dictamnine at a sub-lethal dose (<LC10) for 24 h and performed biochemical index tests, pathological observations, metabolomics, and transcriptomics analyses. The results showed that the liver function indexes such as ALT and AST were affected after the exposure treatment with dictamnine, and the hepatic damage, lipid droplet formation, and increased apoptosis were observed in zebrafish by HE, oil red O(ORO), and Acridine orange hydrochloride(AO)staining. Transcriptome sequencing analysis showed that dictamnine exposure could generate 5696 down-regulated and 4936 up-regulated DEGs(Differential Expressed Genes); metabolomics analysis showed that 36 potential biomarkers were disturbed by dictamnine exposure treatment in juvenile zebrafish. Integration of metabolomics data and transcriptomics data showed that Patatin like phospholipase domain containing 3 Gene Patatin Like Phospholipase Domain Containing 3(PNPLA3), Lactase Gene(LCT), and Galactosidase Beta 1(GLB1) genes were involved in the regulation of 12 key potential biomarkers related to Galactose metabolism and Glycerophospholipid metabolism, such as LysoPC(16:0/0:0) and UDP-4-dehydro-6-deoxy-D-glucose, which in turn regulated pathways of Galactose metabolism and Glycerophospholipid metabolism and consequently induced hepatotoxicity. The comprehensive evaluation of the hepatotoxicity induced by dictamnine was realized from multiple levels, perspectives and indexes by the integrated evaluation method of zebrafish modeling, which supported the applicability of zebrafish in the evaluation of hepatotoxicity of traditional Chinese medicine, and supplied the scientific basis for elucidating the molecular mechanism of the hepatotoxicity induced by dictamnine, as well as guided the development of the toxicity-reducing therapies by dictamnine in the future.

Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) as a Non-Invasive Marker in Diagnosis of NASH

Abstract Background Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of progressive liver disease characterized by increased hepatic triglyceride content (HTGC) in the absence of excess alcohol consumption. NAFLD includes simple steatosis, non-alcoholic steatohepatitis, fibrosis and ultimately cirrhosis, and is strongly associated with features of the metabolic syndrome (obesity, insulin resistance/type 2 diabetes mellitus and dyslipidemia). Objective To study the role of PNPLA3 as non-invasive biomarker in diagnosis of NASH. Patients and Methods Single Centre randomized case control clinical study This study was conducted for patients attended Bariatric surgery unit at Ain-Shams University Hospital. Literature review designed and data collection started in October 2019 up to March 2021. This study performed on 50 Egyptian patients, proven NAFLD by histology, patients were biopsied during a bariatric Surgery at Ain-Shams University Hospital after written consent were obtained, and 25 healthy control Subjects were selected randomly after meted the criteria. Results The G risk allele shows higher level of ALT and statistically significant (p &amp;lt; 0.028*) but not significant with AST (p &amp;lt; 0.243), significant with triglycerides (p &amp;lt; 0.013*), with total cholesterol (p &amp;lt; 0.010*) and BMI (p &amp;lt; 0.016*) independent of the others parameter compared to wild type C allele. To greater number of G risk allele more higher scoring of NAS score (p &amp;lt; 0.002*) and increasing likelihood of advance fibrosis stage (p &amp;lt; 0.046*). By dominant analysis 54.77% of the steatohepatitis patients carrying the risk allele G while steatosis group only 22.42% carrying the risk allele the risk G allele had 4.19 fold more risk of having sheatohepatitis (NASH) than the wild type C allele among the studied NAFLD patients group (odds ratio 4.19, CI 95% 1.762-9.962, p &amp;lt; 0.001*). Homozygous and heterozygous Risk allele GG, CG had 4.37 fold risk of developing NAFLD than the homozygous wild type CC (CI 1.491-12.826, p &amp;lt; 0.005*) and by dominant analysis the risk G allele had 3.45 fold more risk of having NAFLD than the wild type C allele (CI 1.408-8.474, P-value &amp;lt;0.003*) in Egyptian Conclusion We demonstrated for the first time risk of PNPLA3 rs738409 polymorphism in Egyptian, by dominant analysis the risk G allele had 3.45 fold more risk of having NAFLD than the wild type C allele in Egyptian, the risk G allele had 4.19 fold more risk of having sheatohepatitis (NASH) than the wild type C allele among the studied NAFLD patients group.

432-P: Role of Patatin-Like Phospholipase Domain–Containing 3 Gene for Kidney Dysfunction in Diabetes Endotypes

Recent studies identified distinct endotypes of diabetes with differences in metabolic features and in risk for diabetes-related comorbidities. Of note, persons allocated to the severe insulin resistant diabetes (SIRD) endotype, who show increased prevalence of nonalcoholic fatty liver disease (NAFLD) and chronic kidney disease (CKD), are more frequently carriers of the G-allele in the single-nucleotide polymorphism (SNP) rs738409 in the patatin-like phospholipase domain containing 3 (PNPLA3) gene. This SNP associates with increased risk of NAFLD yet the association between the presence of the PNPLA3 SNP and CKD remains controversial. The present study examined whether this SNP differently associates with CKD in endotypes of recent-onset diabetes. Participants with newly diagnosed diabetes (n=707) from the prospective German Diabetes Study underwent k-means clustering, genotyping, magnetic resonance spectroscopy to determine hepatocellular lipid content (HCL) and laboratory analyses to calculate the glomerular filtration rates (eGFR). SIRD had the lowest eGFR and highest HCL compared to severe insulin deficient, moderate obesity-related, moderate age-related and severe autoimmune diabetes clusters (all p&amp;lt;0.05). HCL was negatively associated with eGFR (r=-0.287, p&amp;lt;0.01) across all groups. Further stratification by PNPLA3 G-allele carrier status did not reveal any association between HCL and eGFR in any of the diabetes types, irrespective of G-allele carrier status. However, with declining eGFR the proportion of G-allele carriers increased from 44% for eGFR &amp;gt;60 ml/min to 52% for eGFR &amp;lt;60 ml/min (p&amp;lt;0.05). In conclusion, increased hepatic lipid content is associated with reduced kidney function across all diabetes endotypes. This association is independent of the presence of the PNPLA3 polymorphism in newly diagnosed diabetes, but there might be role for PNPLA3 for the severity of CKD. Disclosure O.P. Zaharia: None. K. Strassburger: None. B. Knebel: None. Y. Kupriyanova: None. J. Kotzka: None. K. Bódis: None. M. Schön: None. M. Bombrich: None. C. Möser: None. K. Prystupa: Other Relationship; Berlin-Chemie AG. H. Al-Hasani: None. V. Schrauwen-Hinderling: None. K. Jandeleit-Dahm: None. R. Wagner: Speaker's Bureau; Novo Nordisk, Sanofi. Advisory Panel; Daiichi Sankyo. M. Roden: Advisory Panel; Eli Lilly and Company. Research Support; Boehringer-Ingelheim, Novo Nordisk, Novartis. Consultant; TARGET PharmaSolutions, Inc. Research Support; Sanofi. Funding German Research Foundation; European Foundation for the Study of Diabetes; German Diabetes Association; German Federal Ministry of Education and Research; Heinrich Heine University

Prospects of evolution-based artificial intelligence models in genome-wide studies to stratify genetic risk variants in nonalcoholic fatty liver disease.

The emergence of genome-wide association studies (GWAS) has identified genetic traits and polymorphisms that are associated with the progression of nonalcoholic fatty liver disease (NAFLD). Phospholipase domain-containing 3 and transmembrane 6 superfamily member 2 are genes commonly associated with NAFLD phenotypes. However, there are fewer studies and replicability in lesser-known genes such as LYPLAL1 and glucokinase regulator (GCKR). With the advent of artificial intelligence (AI) in clinical genetics, studies have utilized AI algorithms to identify phenotypes through electronic health records and utilize convolution neural networks to improve the accuracy of variant identification, predict the deleterious effects of variants, and conduct phenotype-to-genotype mapping. Natural language processing (NLP) and machine-learning (ML) algorithms are popular tools in GWAS studies and connect electronic health record phenotypes to genetic diagnoses using a combination of international classification disease (ICD)-based approaches. However, there are still limitations to machine-learning - and NLP-based models, such as the lack of replicability in larger cohorts and underpowered sample sizes, which prevent the accurate prediction of genetic variants that may increase the risk of NAFLD and its progression to advanced-stage liver fibrosis. This may be largely due to the lack of understanding of the clinical consequence in the majority of pathogenic variants. Though the concept of evolution-based AI models and evolutionary algorithms is relatively new, combining current international classification disease -based NLP models with phylogenetic and evolutionary data can improve prediction accuracy and create valuable connections between variants and their pathogenicity in NAFLD. Such developments can improve risk stratification within clinical genetics and research while overcoming limitations in GWAS studies that prevent community-wide interpretations.

P204 CARDIAC INVOLVEMENT IN NEUTRAL LIPID STORAGE DISEASE (NLSD): A CASE REPORT

Abstract Introduction Neutral Lipid Storage Diseases (NLSDs) are rare diseases with autosomal recessive transmission characterized by a defect in the metabolism of triglycerides that accumulate in many tissues. Vacuoles containing lipids in white blood cells (Jordans anomaly) are considered a diagnostic marker. In particular, NLSD–M (NLSD–myopathy) is caused by mutations in the PNPLA2 gene (Patatin Like Phospholipase Domain Containing 2, chromosome 11p15.5) that codes for an enzyme involved in the first phase of triglyceride hydrolysis in adipose tissue. In this patient the mutation of the PNPLA2 gene is c.542delCA, truncating the protein with loss of the hydrophobic domain. This mutation is associated with more severe infiltrative and arrhythmic cardiac involvement than those known in the literature. Case Report 52–year–old man with NLSD–M. The first symptoms of the disease appeared when he turned 35: weakness and atrophy of axial and proximal muscles, increased CK, scoliosis, fatty liver, electromyographic changes, arterial hypertension, dyslipidemia, renal failure and hypertransaminasemia. At 49, ECG revealed sinus rhythm, absence of ventricular repolarization abnormalities and echocardiogram revealed wall hypertrophy with a trabecular appearance of the left ventricle, sigmoid interventricular septum, preserved EF (58%), moderate diastolic dysfunction, left atrial enlargement, absence of significant valvulopathies. Cardiac MRI revealed lipid infiltration. An ICD was implanted for sustained ventricular tachycardia. At 50, atrial fibrillation was detected. EF was 53% and there was a small partially organized circumferential pericardial effusion. The last medical check, performed at 52 during hospitalization for atrial fibrillation with high ventricular response associated with hypokalemia and hypomagnesaemia, revealed ST–T alterations on ECG and a further reduction of EF to 46% on echocardiogram. Conclusions There is currently no medical treatment. A dietary supplement with medium–chain fatty acids (CNT–02) can be used to slow down the accumulation of lipids. The search for specific mutation is important to stratify cardiac risk.

A PNPLA8 frameshift variant in Australian shepherd dogs with hereditary ataxia.

Hereditary ataxias are common among canine breeds with various molecular etiology. We identified a hereditary ataxia in young‐adult Australian Shepherd dogs characterized by uncoordinated movements and spasticity, worsening progressively and leading to inability to walk. Pedigree analysis suggested an autosomal recessive transmission. By whole genome sequencing and variant filtering of an affected dog we identified a PNPLA8:c.1169_1170dupTT variant. This variant, located in PNPLA8 (Patatin Like Phospholipase Domain Containing 8), was predicted to induce a PNPLA8:p.(His391PhefsTer394) frameshift, leading to a premature stop codon in the protein. The truncated protein was predicted to lack the functional patatin catalytic domain of PNPLA8, a calcium‐independent phospholipase. PNPLA8 is known to be essential for maintaining mitochondrial energy production through tailoring mitochondrial membrane lipid metabolism and composition. The Australian Shepherd ataxia shares molecular and clinical features with Weaver syndrome in cattle and the mitochondrial‐related neurodegeneration associated with PNPLA8 loss‐of‐function variants in humans. By genotyping a cohort of 85 control Australian Shepherd dogs sampled in France, we found a 4.7% carrier frequency. The PNPLA8:c.[1169_1170dupTT] allele is easily detectable with a genetic test to avoid at‐risk matings.

Effects of PNPLA3 I148M on hepatic lipid and very-low-density lipoprotein metabolism in humans.

The phospholipase domain-containing 3 gene (PNPLA3)-148M variant is associated with liver steatosis but its influence on the metabolism of triglyceride-rich lipoproteins remains unclear. Here, we investigated the kinetics of large, triglyceride-rich very-low-density lipoprotein (VLDL), (VLDL1 ), and smaller VLDL2 in homozygotes for the PNPLA3-148M variant. The kinetics of apolipoprotein (apo) B100 (apoB100) and triglyceride in VLDL subfractions were analysed in nine subjects homozygous for PNPLA3-148M and nine subjects homozygous for PNPLA3-148I (controls). Liver fat was >3-fold higher in the 148M subjects. Production rates for apoB100 and triglyceride in VLDL1 did not differ significantly between the two groups. Likewise, production rates for VLDL2 -apoB100 and -triglyceride, and fractional clearance rates for both apoB100 and triglyceride in VLDL1 and VLDL2 , were not significantly different. Despite the higher liver fat content in PNPLA3 148M homozygotes, there was no increase in VLDL production. Equally, VLDL production was maintained at normal levels despite the putative impairment in cytosolic lipid hydrolysis in these subjects.

The Patatin-Like Phospholipase Domain Containing Protein 7 Facilitates VLDL Secretion by Modulating ApoE Stability.

The regulation of hepatic very-low-density lipoprotein (VLDL) secretion is vital for lipid metabolism whose pathogenetic status is involved in fatty liver disease and dyslipidemia seen in hepatic steatosis. Accumulated evidence suggest that apolipoprotein E (ApoE) is closely related to hepatic VLDL secretion. Here, we report that the expression of patatin-like phospholipase domain containing protein 7 (PNPLA7) is strongly induced by hepatic steatosis and positively correlates with plasma triacylglycerol (TAG) levels in the human subjects, whereas the role of PNPLA7 in hepatic VLDL secretion is unknown. Herein, with genetic manipulation in the mice, the deficiency of hepatic PNPLA7 expression resulted in reduced VLDL secretion accompanied by enhanced hepatic lipid accumulation and decreased hepatic ApoE expression. Furthermore, knockdown of PNPLA7 in the livers of the db/db mice also resulted in significant reduction in plasma TAG level but aggravated hepatic steatosis. Importantly, we observed that PNPLA7 interacted with ApoE and presumably at the site of endoplasmic reticulum. Mechanistically, we have shown that PNPLA7 could modulate polyubiquitination and proteasomal-mediated degradation of ApoE. Overexpressed ApoE restored the impaired VLDL-TAG metabolism in PNPLA7-knockdown primary hepatocytes. PNPLA7 plays a critical role in regulating hepatic VLDL secretion by modulating ApoE stability through its interaction with ApoE.

Triptolide enhances lipolysis of adipocytes by enhancing ATGL transcription via upregulation of p53.

Lipolysis is an essential physiological activity of adipocytes. The Patatin Like Phospholipase Domain Containing 2 (PNPLA2) gene encodes the enzyme adipose triglyceride lipase (ATGL) responsible for triglyceride hydrolysis, the first step in lipolysis. In this study, we investigated the potential of triptolide (TP), a natural plant extract, to induce weight loss by examining its effect on ATGL expression. We found that long- and short-term TP administration reduced body weight and fat weight and increased heat production in brown adipose tissue in wild-type C57BL/6 mice. In 3T3-L1 fibroblasts and porcine adipocytes, TP treatment reduced the number of lipid droplets as determined by Oil Red O and BODIPY staining, with concomitant increases in free fatty acid and triglyceride levels in the culture medium. Combined treatment with TP and p53 inhibitor reversed these lipolytic effects. We next amplified the ATGL promoter region and identified conserved p53 binding sites in the sequence by in silico analysis. The results of the dual-luciferase reporter assay using a construct containing the ATGL promoter harboring the p53 binding site showed that p53 induces ATGL promoter activity and consequently, ATGL transcription. These results demonstrate that TP has therapeutic value as an anti-obesity agent and acts by promoting lipolysis via upregulation of p53 and ATGL transcription.

Role of Patatin-Like Phospholipase Domain-Containing 3 Gene for Hepatic Lipid Content and Insulin Resistance in Diabetes.

The rs738409(G) single nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene associates with increased risk and progression of nonalcoholic fatty liver disease (NAFLD). As the recently described severe insulin-resistant diabetes (SIRD) cluster specifically relates to NAFLD, this study examined whether this SNP differently associates with hepatic lipid content (hepatocellular lipids [HCL]) and insulin sensitivity in recent-onset diabetes. A total of 917 participants in the German Diabetes Study (GDS) underwent genotyping, hyperinsulinemic-euglycemic clamps with stable isotopic tracer dilution, and MRS. The G allele associated positively with HCL (β = 0.36, P < 0.01), independent of age, sex, and BMI across the whole cohort, but not in the individual clusters. Those with SIRD exhibited lowest whole-body insulin sensitivity compared with those with severe insulin-deficient (SIDD), moderate obesity-related (MOD), moderate age-related (MARD), and severe autoimmune diabetes (SAID) clusters (all P < 0.001). Interestingly, the SIRD group presented with higher prevalence of the rs738409(G) SNP compared with other clusters and the glucose-tolerant control group (P < 0.05). HCL was higher in the SIRD group (median 13.6% [1st quartile 5.8; 3rd quartile 19.1] compared with the MOD (6.4 % [2.1; 12.4], P < 0.05), MARD (3.0% [1.0; 7.9], P < 0.001), SAID (0.4% [0.0; 1.5], P < 0.001), and glucose-tolerant (0.9% [0.4; 4.9), P < 0.001) group. Although the PNPLA3 polymorphism did not directly associate with whole-body insulin sensitivity in SIRD, the G-allele carriers had higher circulating free fatty acid concentrations and greater adipose tissue insulin resistance compared with noncarriers (both P < 0.001). Members of the SIRD cluster are more frequently carriers of the rs738409(G) variant. The SNP-associated adipose tissue insulin resistance and excessive lipolysis may contribute to their NAFLD.

Gene Expression in LEW.1WR1 Rats Indicate Insulin Resistance in Muscle and Liver Tissue

FAT10 is a ubiquitin‐like protein and type 1 diabetes (T1D) susceptibility gene that may play a role in age‐related inflammation, adiposity, cancer, and kidney disease. The young T1D susceptible, LEW.1WR1 rat overexpresses FAT10 and has higher fasting concentrations of blood insulin. It is unclear how the overexpression of FAT10 directly affects insulin sensitivity. It is also unclear if the initial insulin sensitivity of this animal model plays a role in disease susceptibility. We measured the relative expression of lipid and glucose metabolism genes in skeletal muscle and liver tissue in LEW.1WR1 and LEW/ssNHsd rats to determine if 12‐week old LEW.1WR1 rats were insulin resistant. We also tested the gene expression in pancreatic islets to determine if there were changes in endocrine pancreas cell health and hormone production. The results indicate that gene expression increased in the islets for glucagon, insulin, and PLIN5 (Perilipin‐5), and cyclin D expression decreased in LEW.1WR1 rats, suggesting reduced compensation and a potentially lipotoxic environment for beta cells. The increase in glucagon and insulin also supports peripheral insulin resistance. In liver tissue, UBD (Ubiquitin‐D), a gene suspected to regulate beta‐oxidation, was increased. INSIG (Insulin‐induced gene 1) and GLUT2 (Glucose transporter 2) gene expression decreased, indicating decreased expression of insulin‐regulated gene and glucose transporters, respectively. In muscle tissues, ATGL (Adipose triglyceride lipase), GAPDH (Glyceraldehyde‐3‐Phosphate Dehydrogenase), and PNPLA2 (Patatin Like Phospholipase Domain Containing 2) gene expression decreased, indicating a reduced ability to burn fat as energy and glucose intolerance in muscle tissue. These results, in tandem with previous work, suggest that fat storage in organs may account for the difference in weight previously observed between LEW.1WR1 and LEW/ssNHsd rats, and that LEW.1WR1 rats were developing insulin resistance which further suggests these rats are a novel animal model for pre‐diabetes.Support or Funding InformationThe authors would like to thank the UAH Office of the Vice President for Research and Economic Development, The Alabama Space Grant Consortium, ALSAMP, Adriel D. Johnson Sr. Fellowship, College of Science, Department of Chemistry, Department of Biology, Honors College, and Office of the Provost for the financial support for the resources, supplies, and student salaries.

The Role of Genetic Predisposition, Programing During Fetal Life, Family Conditions, and Post-natal Diet in the Development of Pediatric Fatty Liver Disease

To evaluate, in patients with nonalcoholic fatty liver disease (NAFLD), the role of lifetime exposures associated with genetic predisposition, family history (parental obesity, economic income), programming during fetal life (gestational age, birthweight), being breastfed or not, and later biomarkers of dietary habits and lifestyle in the development of fibrosis. In total, 182 children with overweight/obesity diagnosed with NAFLD proven by biopsy results were enrolled in our study and evaluated for liver fibrosis. We estimated prevalence ORs of fibrosis according to genetics, parental obesity, occupational socioeconomic status (SES), birth weight, breastfeeding, fructose intake (indicator of junk food consumption), and vitamin D status (inflammatory indicator) using logistic regression models, adjusted for age and children's body mass. One hundred thirty-seven patients (75.3%) had liver fibrosis, and 45 patients (24.7%) did not have liver fibrosis. The ORs of fibrosis were significant (P<.05) for patatin like phospholipase domain-containing 3-GG genotype (OR 2.1), parental obesity (OR 2.9), not being breastfed (OR 3.1), vitamin D status (<20mg/dL) (OR 1.24), and fructose consumption (OR 1.6 per 1g/day increase), whereas a high SES maternal occupation was inversely associated with fibrosis (OR 0.30). Our results show independent roles of the patatin like phospholipase domain-containing 3 gene, parental obesity, maternal SES, and postnatal diet and lifestyle in the development of progressive liver disease secondary to NAFLD.

Dynamic interactions of ABHD5 with PNPLA3 regulate triacylglycerol metabolism in brown adipocytes.

Patatin-Like Phospholipase Domain Containing 2 (PNPLA2)/Adipose Triglyceride Lipase (ATGL) and PNPLA3/Adiponutrin are close paralogs that appear to have opposite functions on triacylglycerol (TAG) mobilization and storage. PNPLA2/ATGL is a major triglyceride lipase in adipose tissue and liver, whereas a common human variant of PNPLA3, I148M, greatly increases risk of hepatosteatosis. Nonetheless, the function of PNPLA3 and the mechanism by which the I148M variant promotes TAG accumulation are poorly understood. Here we demonstrate that PNPLA3 strongly interacts with α/β hydrolase domain-containing 5 (ABHD5/CGI-58), an essential co-activator of PNPLA2/ATGL. Molecular imaging experiments demonstrate that PNPLA3 effectively competes with PNPLA2/ATGL for ABHD5, and that PNPLA3 I148M is more effective in this regard. Inducible overexpression of PNPLA3 I148M greatly suppressed PNPLA2/ATGL-dependent lipolysis and triggered massive TAG accumulation in brown adipocytes, and these effects were dependent on ABHD5. The interaction of PNPLA3 and ABHD5 can be regulated by fatty acid supplementation and synthetic ABHD5 ligands, raising the possibility that this interaction might be targeted for treatment of fatty liver disease.

Validation of PNPLA3 polymorphisms as risk factor for NAFLD and liver fibrosis in an admixed population

Introduction and aimStudies have shown that two polymorphisms were associated with steatosis and progression of non-alcoholic fatty liver disease (NAFLD) in different populations: the Patatin-like Phospholipase Domain Containing 3 (PNPLA3) and Transmembrane 6 Superfamily Member 2 (TM6SF2). However, the frequency and significance of these polymorphisms in an admixed population, i.e., Brazilian, is unknown. Therefore, we aimed to evaluate them in healthy subjects in comparison to patients with NAFLD. Material and methodsThis was a multicenter cross-sectional study in 248 patients with biopsy-proven NAFLD and in 134 healthy controls from two tertiary centers in Brazil. PNPLA3 (rs738409 c.444C>G) and TM6SF2 (rs58542926 c.449C>T) polymorphisms were evaluated. ResultsIn controls, the frequencies of PNPLA3 CC and CG+GG were 49.25% and 50.74%, respectively; in NAFLD patients, this was 31.05% and 68.88% (p=0.0044, 95% CI 1.037–2.977). PNPLA3 GG subjects had an increased risk (3.29-fold) of having NAFLD when compared to CC subjects (p=0.0044, 95% CI 1.504–7.225). In patients with nonalcoholic steatohepatitis (NASH), PNPLA3 GG compared to CC was associated with higher AST levels [38.4±25.3 versus 36.7±40.1IU/L, p=0.0395)] and with the presence of liver fibrosis (≥F2 fibrosis, p=0.0272). TM6SF2 polymorphisms were not in Hardy-Weinberg equilibrium in our NAFLD group precluding further analysis. ConclusionWe demonstrated for the first time that PNPLA3 CG+GG increase the risk of NAFLD among Brazilian subjects. Moreover, PNPLA3 GG was associated with liver enzyme elevation and fibrosis in NASH patients.

Association of the PNPLA2, SCD1 and Leptin Expression with Fat Distribution in Liver and Adipose Tissue From Obese Subjects

The expansion of adipose tissue is regulated by insulin and leptin through sterol regulatory element-binding protein-1c (SREBP-1c), up-regulating lipogenesis in tissues by Stearoylcoenzyme A desaturase 1 (SCD1) enzyme, while adipose triglyceride lipase (ATGL) enzyme is key in lipolysis. The research objective was to evaluate the expression of Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1), SCD1, Patatin Like Phospholipase Domain Containing 2 (PNPLA2), and leptin (LEP) genes in hepatic-adipose tissue, and related them with the increment and distribution of fat depots of individuals without insulin resistance. Thirty-eight subjects undergoing elective cholecystectomy with liver and adipose tissue biopsies (subcutaneous-omental) are included. Tissue gene expression was assessed by qPCR and biochemical parameters determined. Individuals are classified according to the body mass index, classified as lean (control group, n=12), overweight (n=11) and obesity (n=15). Abdominal adiposity was determined by anthropometric and histopathological study of the liver. Increased SCD1 expression in omental adipose tissue (p=0.005) and PNPLA2 in liver (p=0.01) were found in the obesity group. PNPLA2 decreased expression in subcutaneous adipose tissue was significant in individuals with abdominal adiposity (p=0.017). Anthropometric parameters positively correlated with liver PNPLA2 and the expression of liver PNPLA2 with serum leptin. SCD1 increased levels may represent lipid storage activity in omental adipose tissue. Liver PNPLA2 increased expression could function as a primary compensatory event of visceral fat deposits associated to the leptin hormone related to the increase of adipose tissue.

Systematic review with meta-analysis: the I148M variant of patatin-like phospholipase domain-containing 3 gene (PNPLA3) is significantly associated with alcoholic liver cirrhosis.

Several studies have reported an association between alcoholic liver cirrhosis (ALC) or other forms of alcoholic liver disease (ALD) and the genetic variant rs738409 (C>G) in adiponutrin/patatin-like phospholipase domain-containing 3 gene (PNPLA3). To evaluate the influence of this variant on ALC and other forms of ALD. We performed a systematic review of previous studies on the relationship between rs738409 of PNPLA3 and ALD and meta-analysis was conducted in a random-effects model. Calculations of the odds ratios (ORs) and their confidence intervals (CIs), tests for heterogeneity and sensitivity analyses were performed. Database search identified 11 previous studies available for inclusion with a total of 3495 patients with ALD (2087 with ALC) and 5038 controls (4007 healthy subjects and 1031 alcoholics without ALD). Patients with ALC compared to controls had a significantly higher prevalence of the G allele when comparing GG vs. CC (OR 4.30, 95% CI 3.25-5.69; P < 0.00001) or GC vs. CC genotypes (GC vs. CC: OR 1.91, 95% CI 1.67-2.17) or under a recessive or dominant model. Similar results were found when comparing separately patients with ALC vs. alcoholics without ALD or healthy subjects. An association of the G allele with ALD emerged when comparing ALD patients vs. alcoholics without ALD and/or healthy subjects although moderate to large heterogeneity was observed. Our data suggested an additive genetic model for this variant in ALD. Our meta-analysis shows that the rs738409 variant of PNPLA3 is clearly associated with alcoholic liver cirrhosis.

PNPLA3 I148M polymorphism is associated with elevated alanine transaminase levels in Mexican Indigenous and Mestizo populations

The patatin like phospholipase domain-containing (PNPLA3) I148M variant is the strongest genetic factor associated with elevated alanine transaminase (ALT) levels in different populations, particularly in Hispanics who have the highest 148M risk allele frequency reported to date. It has been suggested that Indigenous ancestry is associated with higher ALT levels in Mexicans. The aim of the present study was to assess the frequency of the PNPLA3 148M risk allele in Mexican indigenous and Mestizo individuals, and to examine its association with serum ALT levels. The study included a total of 1624 Mexican individuals: 919 Indigenous subjects from five different native groups and 705 Mexican Mestizo individuals (141 cases with ALT levels ≥ 40 U/L and 564 controls with ALT <40 U/L). The I148M polymorphism was genotyped by TaqMan assays. The frequency of elevated ALT levels in Indigenous populations was 18.7%, and varied according to obesity status: 14.4% in normal weight, 19.9% in overweight and 24.5% in obese individuals. The Mexican indigenous populations showed the highest reported frequency of the PNPLA3 148M risk allele (mean 0.73). The M148M genotype was significantly associated with elevated ALT levels in indigenous individuals (OR = 3.15, 95 % CI 1.91-5.20; P = 7.1 × 10(-6)) and this association was confirmed in Mexican Mestizos (OR = 2.24, 95% CI 1.50-3.33; P = 8.1 × 10(-5)). This is the first study reporting the association between M148M genotype and elevated ALT levels in Indigenous Mexican populations. The 148M allele risk may be considered an important risk factor for liver damage in Mexican indigenous and Mestizo populations.

Laminin Receptor Involvement in the Anti-angiogenic Activity of Pigment Epithelium-derived Factor

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A(2)/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44-77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120-210). A 25-mer peptide named P46 (aa 46-70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.

Identification of a Lipase-linked Cell Membrane Receptor for Pigment Epithelium-derived Factor

Pigment epithelium-derived factor (PEDF) is an extracellular multifunctional protein belonging to the serpin superfamily with demonstrable neurotrophic, gliastatic, neuronotrophic, antiangiogenic, and antitumorigenic properties. We have previously provided biochemical evidence for high affinity PEDF-binding sites and proteins in plasma membranes of retina, retinoblastoma, and CNS cells. This study was designed to reveal a receptor involved in the biological activities of PEDF. Using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDF-binding partner, which we term PEDF-R. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. Recently, PEDF-R (TTS-2.2/independent phospholipase A(2) (PLA(2))zeta and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA(2)/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. Here we describe the PEDF-R gene expression in the retina and its heterologous expression by bacterial and eukaryotic systems, and we demonstrate that its protein product has specific and high binding affinity for PEDF, has a potent phospholipase A(2) activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. In conclusion, we have identified a novel PEDF-R gene in the retina for a phospholipase-linked membrane protein with high affinity for PEDF, suggesting a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal.