Patatin-like Phospholipase Domain Containing 2 Research Articles

The lipase cofactor CGI58 controls placental lipolysis.

In eutherians, the placenta plays a critical role in the uptake, storage, and metabolism of lipids. These processes govern the availability of fatty acids to the developing fetus, where inadequate supply has been associated with substandard fetal growth. Whereas lipid droplets are essential for the storage of neutral lipids in the placenta and many other tissues, the processes that regulate placental lipid droplet lipolysis remain largely unknown. To assess the role of triglyceride lipases and their cofactors in determining placental lipid droplet and lipid accumulation, we assessed the role of patatin like phospholipase domain containing 2 (PNPLA2) and comparative gene identification-58 (CGI58) in lipid droplet dynamics in the human and mouse placenta. While both proteins are expressed in the placenta, the absence of CGI58, not PNPLA2, markedly increased placental lipid and lipid droplet accumulation. These changes were reversed upon restoration of CGI58 levels selectively in the CGI58-deficient mouse placenta. Using co-immunoprecipitation, we found that, in addition to PNPLA2, PNPLA9 interacts with CGI58. PNPLA9 was dispensable for lipolysis in the mouse placenta yet contributed to lipolysis in human placental trophoblasts. Our findings establish a crucial role for CGI58 in placental lipid droplet dynamics and, by extension, in nutrient supply to the developing fetus.

G0S2 inhibits adipogenesis and unsaturated fatty acid biosynthesis by repressing the expression of SCD in chicken

Fatty acids metabolic products and intramuscular fat (IMF) content determine meat quality in chickens. In this study, the mechanisms of G0/G1 switch gene 2 (G0S2) underlying modulation of chicken IMF content as well as fatty acid synthesis was investigated. Several advanced techniques including Gas Chromatography–Mass Spectrometer (GC–MS), RNA-sequencing and lentiviral transfection were utilised. RNA-sequencing showed that 2242 differentially expressed genes were significantly different, and many of these were related to lipid catabolism, such as stearoyl-CoA desaturase 1 (SCD), acyl-CoA synthetase bubblegum family member 2 (ACSBG2), and fatty acid desaturase 2 (FADS2), etc. Knocking down of G0S2 also affected the biological activities of signal transduction, catalytic activity, lipid metabolism, cell cycle and death, cell differentiation and immune response. GC–MS revealed that decreased lipid accumulation and lower unsaturated fatty acid percentage were observed in G0S2 knockdown adipocytes. Our results showed that lower expression of G0S2 decreased lipid accumulation mainly via down-regulated SCD, then regulated mRNA levels of patatin-like phospholipase domain containing 2 (PNPLA2), which was shown as a major triglyceride lipase in the liver and adipose tissue. Importantly, we further verified the regulation network of G0S2 in the thigh tissues of 12 Huangshan Black chickens, which was consistent with previous IMF results. Thus, our study highlights that G0S2 modulated chicken meat quality traits by the G0S2–SCD–PNPLA2 axis in lipid metabolism in vitro and in vivo. These findings are expected to provide a strategy from a genetic perspective for improving the meat quality.

Ablation of pigment epithelium-derived factor receptor (PEDF-R/Pnpla2) causes photoreceptor degeneration

Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2−/− mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA2 enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2−/− photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2−/− and Pnpla2+/− mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.

PNPLA2 mobilizes retinyl esters from retinosomes and promotes the generation of 11-cis-retinal in the visual cycle.

Retinosomes are intracellular lipid bodies found in the retinal pigment epithelium (RPE). They contain retinyl esters (REs) and are thought to be involved in visual chromophore regeneration during dark adaptation and in case of chromophore depletion. However, key enzymes in chromophore regeneration, retinoid isomerase (RPE65), and lecithin:retinol acyltransferase (LRAT) are located in the endoplasmic reticulum (ER). The mechanism and the enzyme responsible for mobilizing REs from retinosomes remained unknown. Our study demonstrates that patatin-like phospholipase domain containing 2 (PNPLA2) mobilizes all-trans-REs from retinosomes. The absence of PNPLA2 in mouse eyes leads to a significant accumulation of lipid droplets in RPE cells, declined electroretinography (ERG) response, and delayed dark adaptation compared with those of WT control mouse. Our work suggests a function of PNPLA2 as an RE hydrolase in the RPE, mobilizing REs from lipid bodies and functioning as an essential component of the visual cycle.

ALOX12B and PNPLA1 Have Distinct Roles in Epidermal Lipid Lamellar Organization

The stratum corneum (SC) has remarkable permeability barrier properties that are attributable to highly ordered lipid lamellae (LL) that fill the extracellular spaces between corneocytes (Ribaud et al., 1994). Corneocytes also have a lipid monolayer covalently bound to their surfaces, called the corneocyte lipid envelope (CLE)(Crumrine et al., 2019). LL composition influences their organization and permeability function; for example, esterified omega-hydroxy (EO)-ceramides, which are synthesized by patatin-like phospholipase domain-containing 1 (PNPLA1), have been shown in vitro to mediate formation of the “long periodicity phase” triplet repeat pattern that characterizes normal LL (Opálka et al., 2016).

Identification of Differential Expression Genes between Volume and Pressure Overloaded Hearts Based on Bioinformatics Analysis.

Volume overload (VO) and pressure overload (PO) are two common pathophysiological conditions associated with cardiac disease. VO, in particular, often occurs in a number of diseases, and no clinically meaningful molecular marker has yet been established. We intend to find the main differential gene expression using bioinformatics analysis. GSE97363 and GSE52796 are the two gene expression array datasets related with VO and PO, respectively. The LIMMA algorithm was used to identify differentially expressed genes (DEGs) of VO and PO. The DEGs were divided into three groups and subjected to functional enrichment analysis, which comprised GO analysis, KEGG analysis, and the protein–protein interaction (PPI) network. To validate the sequencing data, cardiomyocytes from AR and TAC mouse models were used to extract RNA for qRT-PCR. The three genes with random absolute values of LogFC and indicators of heart failure (natriuretic peptide B, NPPB) were detected: carboxylesterase 1D (CES1D), whirlin (WHRN), and WNK lysine deficient protein kinase 2 (WNK2). The DEGs in VO and PO were determined to be 2761 and 1093, respectively, in this study. Following the intersection, 305 genes were obtained, 255 of which expressed the opposing regulation and 50 of which expressed the same regulation. According to the GO and pathway enrichment studies, DEGs with opposing regulation are mostly common in fatty acid degradation, propanoate metabolism, and other signaling pathways. Finally, we used Cytoscape’s three techniques to identify six hub genes by intersecting 255 with the opposite expression and constructing a PPI network. Peroxisome proliferator-activated receptor (PPARα), acyl-CoA dehydrogenase medium chain (ACADM), patatin-like phospholipase domain containing 2 (PNPLA2), isocitrate dehydrogenase 3 (IDH3), heat shock protein family D member 1 (HSPD1), and dihydrolipoamide S-acetyltransferase (DLAT) were identified as six potential genes. Furthermore, we predict that the hub genes PPARα, ACADM, and PNPLA2 regulate VO myocardial changes via fatty acid metabolism and acyl-Coa dehydrogenase activity, and that these genes could be employed as basic biomarkers for VO diagnosis and treatment.

ATGL is a biosynthetic enzyme for fatty acid esters of hydroxy fatty acids

Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4–7. The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80–90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.

ω-O-Acylceramides but not ω-hydroxy ceramides are required for healthy lamellar phase architecture of skin barrier lipids

Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1+/+ and Pnpla1-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.

Hepatocyte-specific deletion of adipose triglyceride lipase (adipose triglyceride lipase/patatin-like phospholipase domain containing 2) ameliorates dietary induced steatohepatitis in mice.

Increased fatty acid (FA) flux from adipose tissue to the liver contributes to the development of NAFLD. Because free FAs are key lipotoxic triggers accelerating disease progression, inhibiting adipose triglyceride lipase (ATGL)/patatin-like phospholipase domain containing 2 (PNPLA2), the main enzyme driving lipolysis, may attenuate steatohepatitis. Hepatocyte-specific ATGL knockout (ATGL LKO) mice were challenged with methionine-choline-deficient (MCD) or high-fat high-carbohydrate (HFHC) diet. Serum biochemistry, hepatic lipid content and liver histology were assessed. Mechanistically, hepatic gene and protein expression of lipid metabolism, inflammation, fibrosis, apoptosis, and endoplasmic reticulum (ER) stress markers were investigated. DNA binding activity for peroxisome proliferator-activated receptor (PPAR) α and PPARδ was measured. After short hairpin RNA-mediated ATGL knockdown, HepG2 cells were treated with lipopolysaccharide (LPS) or oleic acid:palmitic acid 2:1 (OP21) to explore the direct role of ATGL in inflammation in vitro. On MCD and HFHC challenge, ATGL LKO mice showed reduced PPARα and increased PPARδ DNA binding activity when compared with challenged wild-type (WT) mice. Despite histologically and biochemically pronounced hepatic steatosis, dietary-challenged ATGL LKO mice showed lower hepatic inflammation, reflected by the reduced number of Galectin3/MAC-2 and myeloperoxidase-positive cells and low mRNA expression levels of inflammatory markers (such as IL-1β and F4/80) when compared with WT mice. In line with this, protein levels of the ER stress markers protein kinase R-like endoplasmic reticulum kinase and inositol-requiring enzyme 1α were reduced in ATGL LKO mice fed with MCD diet. Accordingly, pretreatment of LPS-treated HepG2 cells with the PPARδ agonist GW0742 suppressed mRNA expression of inflammatory markers. Additionally, ATGL knockdown in HepG2 cells attenuated LPS/OP21-induced expression of proinflammatory cytokines and chemokines such as chemokine (C-X-C motif) ligand 5, chemokine (C-C motif) ligand (Ccl) 2, and Ccl5. Low hepatic lipolysis and increased PPARδ activity in ATGL/PNPLA2 deficiency may counteract hepatic inflammation and ER stress despite increased steatosis. Therefore, lowering hepatocyte lipolysis through ATGL inhibition represents a promising therapeutic strategy for the treatment of steatohepatitis.

The Association Between PNPLA2 Methylation in Peripheral Blood and Early-Stage Lung Cancer in a Case-Control Study.

PurposeLung cancer (LC) brings great burden to the society worldwide. Exploring novel biomarkers in vitro for the early detection of LC would be of great importance.Patients and MethodsWe measured DNA methylation levels of 21 CpG sites within Patatin-like phospholipase domain containing 2 (PNPLA2) gene in the peripheral blood of 168 early-stage LC cases (94.0% LC at stage I) and 187 age- and gender-matched cancer-free controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression adjusted for covariates. Non-parametric tests were applied for the comparisons of stratified groups.ResultsHypomethylation of PNPLA2_CpG_8,10 and hypermethylation of PNPLA2_CpG_9 were correlated to the early-stage LC with the ORs of 1.44 (95% CI: 1.06–1.96, P = 0.018) and 0.82 (95% CI: 0.69–0.98, P = 0.029), respectively. The associations were still significant for the very early-stage LC patients (stage I). Further gender- and age-stratified analyses indicated that the association between hypomethylation of PNPLA2_CpG_8,10 and LC existed only in females and in subjects younger than 55 years. In addition, the association between LC and hypermethylation of PNPLA2_CpG_6 and PNPLA2_CpG_9 was also observed in the younger population.ConclusionTaken together, our study has proved the hypothesis that the altered methylation in the peripheral blood may be correlated with the burden of cancer at an early stage. Here, we find a novel association between blood-based aberrant PNPLA2 methylation and LC at a very early stage and particularly for women at a younger age.

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.

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.

Adipocyte lipolysis: from molecular mechanisms of regulation to disease and therapeutics.

Fatty acids (FAs) are stored safely in the form of triacylglycerol (TAG) in lipid droplet (LD) organelles by professional storage cells called adipocytes. These lipids are mobilized during adipocyte lipolysis, the fundamental process of hydrolyzing TAG to FAs for internal or systemic energy use. Our understanding of adipocyte lipolysis has greatly increased over the past 50 years from a basic enzymatic process to a dynamic regulatory one, involving the assembly and disassembly of protein complexes on the surface of LDs. These dynamic interactions are regulated by hormonal signals such as catecholamines and insulin which have opposing effects on lipolysis. Upon stimulation, patatin-like phospholipase domain containing 2 (PNPLA2)/adipocyte triglyceride lipase (ATGL), the rate limiting enzyme for TAG hydrolysis, is activated by the interaction with its co-activator, alpha/beta hydrolase domain-containing protein 5 (ABHD5), which is normally bound to perilipin 1 (PLIN1). Recently identified negative regulators of lipolysis include G0/G1 switch gene 2 (G0S2) and PNPLA3 which interact with PNPLA2 and ABHD5, respectively. This review focuses on the dynamic protein-protein interactions involved in lipolysis and discusses some of the emerging concepts in the control of lipolysis that include allosteric regulation and protein turnover. Furthermore, recent research demonstrates that many of the proteins involved in adipocyte lipolysis are multifunctional enzymes and that lipolysis can mediate homeostatic metabolic signals at both the cellular and whole-body level to promote inter-organ communication. Finally, adipocyte lipolysis is involved in various diseases such as cancer, type 2 diabetes and fatty liver disease, and targeting adipocyte lipolysis is of therapeutic interest.

Neutral lipid storage disease with myopathy in China: a large multicentric cohort study

BackgroundNeutral lipid storage disease with myopathy (NLSDM) is a rare clinical heterogeneous disorder caused by mutations in the patatin-like phospholipase domain-containing 2 (PNPLA2) gene. NLSDM usually presents skeletal myopathy, cardiomyopathy and the multiple organs dysfunction. Around 50 cases of NLSDM have been described worldwide, whereas the comprehensive understanding of this disease are still limited. We therefore recruit NLSDM patients from 10 centers across China, summarize the clinical, muscle imaging, pathological and genetic features, and analyze the genotype-phenotype relationship.ResultsA total of 45 NLSDM patients (18 men and 27 women) were recruited from 40 unrelated families. Thirteen patients were born from consanguineous parents. The phenotypes were classified as asymptomatic hyperCKemia (2/45), pure skeletal myopathy (18/45), pure cardiomyopathy (4/45), and the combination of skeletal myopathy and cardiomyopathy (21/45). Right upper limb weakness was the early and prominent feature in 61.5% of patients. On muscle MRI, the long head of the biceps femoris, semimembranosus and adductor magnus on thighs, the soleus and medial head of the gastrocnemius on lower legs showed the most severe fatty infiltration. Thirty-three families were carrying homozygous mutations, while seven families were carrying compound heterozygous mutations. A total of 23 mutations were identified including 11 (47.8%) point mutations, eight (34.8%) deletions and four (17.4%) insertions. c.757 + 1G > T, c.245G > A and c.187 + 1G > A were the three most frequent mutations. Among four groups of phenotypes, significant differences were shown in disease onset (< 20 years versus ≥20 years old, p = 0.003) and muscle pathology (with rimmed vacuoles versus without rimmed vacuoles, p = 0.001). PNPLA2 mutational type or functional defects did not show great impact on phenotypes.ConclusionWe outline the clinical and genetic spectrum in a large cohort of NLSDM patients. Selective muscle fatty infiltration on posterior compartment of legs are characteristic of NLSDM. Chinese patients present with distinctive and relative hotspot PNPLA2 mutations. The disease onset age and pathological appearance of rimmed vacuoles are proved to be related with the clinical manifestations. The phenotypes are not strongly influenced by genetic defects, suggesting the multiple environmental risk factors in the development of NLSDM.

Molecular Genetic Study of a Large Inbred Pakistani Family Affected with Autosomal Recessive Congenital Ichthyosis Through Whole Exome Sequencing.

Background: Autosomal recessive congenital ichthyoses (ARCI) are a group of rare nonsyndromic genodermatoses characterized by generalized scaly appearance of the epidermis with markedly impaired cutaneous barriers owing to defects in keratinization related genes. In this study, we ascertained a consanguineous Pakistani family affected with ARCI. Aims: To investigate genetic defect underlying disease phenotype in the affected family. Methods: All available members of the family (affected and unaffected) were sampled. Whole exome sequencing (WES) was performed on DNA of the proband and the data were analyzed for probable pathogenic variants. Segregation of the identified variant was validated by Sanger sequencing. Results: Analysis of the WES data identified a novel nonsense mutation, c.762C>G, in the PNPLA1 (patatin-like phospholipase domain containing 1) gene. The protein product of of this gene is involved in lipid organization during cornified cell envelope formation. The variant is predicted to result in the generation of a premature truncation site at amino acid position 254 (p.Tyr254*). This would result in the loss of a large C-terminal portion of the protein suggesting it to be rendered nonfunctional. In silico protein structure modeling confirmed a detrimental effect of the variation on protein structure. Conclusions: The study supports the evidence for the prevalence of PNPLA1 mutations in distant ethnic groups. Despite the significant number of reported ARCI cases with PNPLA1 variants, a straightforward genotype-phenotype correlation cannot be established.

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.

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.

ABHD5 stimulates PNPLA1-mediated ω-O-acylceramide biosynthesis essential for a functional skin permeability barrier

Mutations in the genes coding for patatin-like phospholipase domain-containing 1 (PNPLA1) and α/β-hydrolase domain-containing 5 (ABHD5), also known as comparative gene identification 58, are causative for ichthyosis, a severe skin barrier disorder. Individuals with mutations in either of these genes show a defect in epidermal ω-O-acylceramide (AcylCer) biosynthesis, suggesting that PNPLA1 and ABHD5 act in the same metabolic pathway. In this report, we identified ABHD5 as a coactivator of PNPLA1 that stimulates the esterification of ω-hydroxy ceramides with linoleic acid for AcylCer biosynthesis. ABHD5 interacts with PNPLA1 and recruits the enzyme to its putative triacylglycerol substrate onto cytosolic lipid droplets. Conversely, alleles of ABHD5 carrying point mutations associated with ichthyosis in humans failed to accelerate PNPLA1-mediated AcylCer biosynthesis. Our findings establish an important biochemical function of ABHD5 in interacting with PNPLA1 to synthesize crucial epidermal lipids, emphasizing the significance of these proteins in the formation of a functional skin permeability barrier.

Patients with neutral lipid storage disease with myopathy (NLSDM) in Southwestern China

ObjectivesNeutral lipid storage disease with myopathy (NLSDM) is a rare metabolic myopathy occurring owing to mutations in the patatin like phospholipase domain containing 2 (PNPLA2) gene. Till date, less than 50 patients with PNPLA2 mutations have been reported. In this study, we describe the clinical, pathological, and genetic findings, and muscle magnetic resonance imaging (MRI) changes in four Chinese patients with NLSDM. Patients and methodsPeripheral blood smears were stained using Wright’s stain. Muscle biopsies, muscle MRI, and sequence analysis of PNPLA2 gene were performed. ResultsAll patients exhibited slowly progressive myopathy during adulthood. Cardiomyopathy, sensorineural hearing loss, hepatic adipose infiltration, and hypertriglyceridemia were observed in some patients. Jordan’s anomaly was detected in the blood smears of all patients. Muscle biopsies revealed the presence of massive lipid droplets and rimmed vacuoles in two patients. MR images of the lower lumbar, pelvis, and lower extremities showed the involvement of posterior compartment muscles. The anterior compartment muscles were found to be less affected. Gene analysis for PNPLA2 revealed an identical homozygous mutation c.757 + 1G > T in all patients. ConclusionPatients with NLSDM display clinical heterogeneities despite sharing the same mutation (c.757 + 1G > T) of the PNPLA2 gene, may suggest a founder effect in the region.

MRNA m6A plays opposite role in regulating UCP2 and PNPLA2 protein expression in adipocytes.

N6-methyladenosine (m6A) modification of mRNA plays an important role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Using Jinhua and Landrace pigs as fat and lean models, we presented a comprehensive transcriptome-wide m6A profiling in adipose tissues from these two pig breeds. Two differentially methylated genes were selected to explore the mechanisms of m6A-mediated regulation of gene function. The ratio of m6A/A in the layer of backfat (LB) was significantly higher in Landrace than that in Jinhua. Transcriptome-wide m6A profiling revealed that m6A modification on mRNA occurs in the conserved sequence motif of RRACH and that the pig transcriptome contains 0.53-0.91 peak per actively expressed transcript. The relative density of m6A peaks in the 3'UTR were higher than in 5'UTR. Genes with common m6A peaks from both Landrace (L-LB) and Jinhua (J-LB) were enriched in RNA splicing and cellular lipid metabolic process. The unique m6A peak genes (UMGs) from L-LB were mainly enriched in the extracellular matrix (ECM) and collagen catabolic process, whereas the UMGs from J-LB are mainly involved in RNA splicing, etc. Lipid metabolism processes were not significantly enriched in the UMGs from L-LB or J-LB. Uncoupling protein-2 (UCP2) and patatin-like phospholipase domain containing 2 (PNPLA2) were two of the UMGs in L-LB. Synonymous mutations (MUT) were conducted to reduce m6A level of UCP2 and PNPLA2 mRNAs. Adipogenesis test showed that UCP2-MUT further inhibited adipogenesis, while PNPLA2-MUT promoted lipid accumulation compared with UCP2-WT and PNPLA2-WT, respectively. Further study showed m6A negatively mediates UCP2 protein expression and positively mediates PNPLA2 protein expression. m6A modification affects the translation of PNPLA2 most likely through YTHDF1, whereas UCP2 is likely neither the target of YTHDF2 nor the target of YTHDF1. Our data demonstrated a conserved and yet dynamically regulated m6A methylome in pig transcriptomes and provided an important resource for studying the function of m6A epitranscriptomic modification in obesity development.