The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

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.

Endoplasmic Reticulum Protein TXNDC5 Interacts with PRDX6 and HSPA9 to Regulate Glutathione Metabolism and Lipid Peroxidation in the Hepatic AML12 Cell Line.

Non-alcoholic fatty liver disease or steatosis is an accumulation of fat in the liver. Increased amounts of non-esterified fatty acids, calcium deficiency, or insulin resistance may disturb endoplasmic reticulum (ER) homeostasis, which leads to the abnormal accumulation of misfolded proteins, activating the unfolded protein response. The ER is the primary location site for chaperones like thioredoxin domain-containing 5 (TXNDC5). Glutathione participates in cellular oxidative stress, and its interaction with TXNDC5 in the ER may decrease the disulfide bonds of this protein. In addition, glutathione is utilized by glutathione peroxidases to inactivate oxidized lipids. To characterize proteins interacting with TXNDC5, immunoprecipitation and liquid chromatography-mass spectrometry were used. Lipid peroxidation, reduced glutathione, inducible phospholipase A2 (iPLA2) and hepatic transcriptome were assessed in the AML12 and TXNDC5-deficient AML12 cell lines. The results showed that HSPA9 and PRDX6 interact with TXNDC5 in AML12 cells. In addition, TXNDC5 deficiency reduced the protein levels of PRDX6 and HSPA9 in AML12. Moreover, lipid peroxidation, glutathione and iPLA2 activities were significantly decreased in TXNDC5-deficient cells, and to find the cause of the PRDX6 protein reduction, proteasome suppression revealed no considerable effect on it. Finally, hepatic transcripts connected to PRDX6 and HSPA9 indicated an increase in the Dnaja3, Mfn2 and Prdx5 and a decrease in Npm1, Oplah, Gstp3, Gstm6, Gstt1, Serpina1a, Serpina1b, Serpina3m, Hsp90aa1 and Rps14 mRNA levels in AML12 KO cells. In conclusion, the lipid peroxidation system and glutathione mechanism in AML12 cells may be disrupted by the absence of TXNDC5, a novel protein-protein interacting partner of PRDX6 and HSPA9.

Interaction between estrogen receptor-α and PNPLA3 p.I148M variant drives fatty liver disease susceptibility in women.

Fatty liver disease (FLD) caused by metabolic dysfunction is the leading cause of liver disease and the prevalence is rising, especially in women. Although during reproductive age women are protected against FLD, for still unknown and understudied reasons some develop rapidly progressive disease at the menopause. The patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M variant accounts for the largest fraction of inherited FLD variability. In the present study, we show that there is a specific multiplicative interaction between female sex and PNPLA3 p.I148M in determining FLD in at-risk individuals (steatosis and fibrosis, P < 10-10; advanced fibrosis/hepatocellular carcinoma, P = 0.034) and in the general population (P < 10-7 for alanine transaminase levels). In individuals with obesity, hepatic PNPLA3 expression was higher in women than in men (P = 0.007) and in mice correlated with estrogen levels. In human hepatocytes and liver organoids, PNPLA3 was induced by estrogen receptor-α (ER-α) agonists. By chromatin immunoprecipitation and luciferase assays, we identified and characterized an ER-α-binding site within a PNPLA3 enhancer and demonstrated via CRISPR-Cas9 genome editing that this sequence drives PNPLA3 p.I148M upregulation, leading to lipid droplet accumulation and fibrogenesis in three-dimensional multilineage spheroids with stellate cells. These data suggest that a functional interaction between ER-α and PNPLA3 p.I148M variant contributes to FLD in women.

Long-term hypercaloric diet exacerbates metabolic liver disease in PNPLA3 I148M animals.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a major health burden associated with the metabolic syndrome leading to liver fibrosis, cirrhosis and ultimately liver cancer. In humans, the PNPLA3 I148M polymorphism of the phospholipase patatin-like phospholipid domain containing protein 3 (PNPLA3) has a well-documented impact on metabolic liver disease. In this study, we used a mouse model mimicking the human PNPLA3 I148M polymorphism in a long-term high fat diet (HFD) experiment to better define its role for NAFLD progression. METHODS: Male mice bearing wild-type Pnpla3 (Pnpla3WT ), or the human polymorphism PNPLA3 I148M (Pnpla3148M/M ) were subjected to HFD feeding for 24 and 52 weeks. Further analysis concerning basic phenotype, inflammation, proliferation and cell death, fibrosis and microbiota were performed in each time point. RESULTS: After 52 weeks HFD Pnpla3148M/M animals had more liver fibrosis, enhanced numbers of inflammatory cells as well as increased Kupffer cell activity. Increased hepatocyte cell turnover and ductular proliferation were evident in HFD Pnpla3148M/M livers. Microbiome diversity was decreased after HFD feeding, changes were influenced by HFD feeding (36%) and the PNPLA3 I148M genotype (12%). Pnpla3148M/M mice had more faecal bile acids. RNA-sequencing of liver tissue defined an HFD-associated signature, and a Pnpla3148M/M specific pattern, which suggests Kupffer cell and monocytes-derived macrophages as significant drivers of liver disease progression in Pnpla3148M/M animals. CONCLUSION: With long-term HFD feeding, mice with the PNPLA3 I148M genotype show exacerbated NAFLD. This finding is linked to PNPLA3 I148M-specific changes in microbiota composition and liver gene expression showing a stronger inflammatory response leading to enhanced liver fibrosis progression.

Squalene through Its Post-Squalene Metabolites Is a Modulator of Hepatic Transcriptome in Rabbits.

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 (PNPLA3), GCK, TFCP2L1, ASCL1, ACSS2, OST4, FAM91A1, MYH6, LRRC39, LOC108176846, GLT1D1 and TREH. A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2, Fam91a1 and Pnpla3. In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction.

Distinctive features of hepatocellular carcinoma in non-alcoholic fatty liver disease.

Hepatocellular carcinoma (HCC) is on the rise globally, causing more than 800 thousand deaths annually, with an estimated annual percent change of 0.51 for causes other than viral hepatitis, including nonalcoholic fatty liver disease (NAFLD). The incidence of NAFLD-related HCC is peaking in several Far East regions (6-12% vs. 2-3% in Western Europe and USA), HCC risk being mainly driven by the epidemic of obesity and diabetes, both favored by an unhealthy diet and sedentary lifestyle. Under inherited susceptibility outlined by such genetic markers as variants in PNPLA3, TM6SF2 and MBOAT7, neoplastic transformation of NAFLD is driven by sublethal lipotoxicity consequent to hepatocyte lipid overload, whereas a myriad of factors spanning from subverted circadian homeostasis and gut dysbiosis to alcohol abuse and tobacco may interact as risk modifiers. At variance with viral HCC, NAFLD-HCC shows a frequent association with cardiovascular co-morbidities, absence of cirrhosis in up to half of patients and an association with persistently normal transaminase values. All these misleading features of NAFLD-related HCC account for the low uptake of surveillance and linkage to curative treatments that has been reported in patients with this cancer, a downside that could be attenuated when scores for cost-effective risk stratification become available.

GWAS loci associated with Chagas cardiomyopathy influences DNA methylation levels.

A recent genome-wide association study (GWAS) identified a locus in chromosome 11 associated with the chronic cardiac form of Chagas disease. Here we aimed to elucidate the potential functional mechanism underlying this genetic association by analyzing the correlation among single nucleotide polymorphisms (SNPs) and DNA methylation (DNAm) levels as cis methylation quantitative trait loci (cis-mQTL) within this region. A total of 2,611 SNPs were tested against 2,647 DNAm sites, in a subset of 37 chronic Chagas cardiomyopathy patients and 20 asymptomatic individuals from the GWAS. We identified 6,958 significant cis-mQTLs (False Discovery Rate [FDR]<0.05) at 1 Mb each side of the GWAS leading variant, where six of them potentially modulate the expression of the SAC3D1 gene, the reported gene in the previous GWAS. In addition, a total of 268 cis-mQTLs showed differential methylation between chronic Chagas cardiomyopathy patients and asymptomatic individuals. The most significant cis-mQTLs mapped in the gene bodies of POLA2 (FDR = 1.04x10-11), PLAAT3 (FDR = 7.22x10-03), and CCDC88B (FDR = 1.89x10-02) that have been associated with cardiovascular and hematological traits in previous studies. One of the most relevant interactions correlated with hypermethylation of CCDC88B. This gene is involved in the inflammatory response, and its methylation and expression levels have been previously reported in Chagas cardiomyopathy. Our findings support the functional relevance of the previously associated genomic region, highlighting the regulation of novel genes that could play a role in the chronic cardiac form of the disease.

Bile acids induced hepatic lipid accumulation in mice by inhibiting mRNA expression of patatin-like phospholipase domain containing 3 and microsomal triglyceride transfer protein.

Preliminary studies have shown that a lithogenic diet (LG), which contains cholesterol and cholic acid, induces gallstones and hepatic lipid accumulation (HLA), and reduction of blood triglyceride in mice. We hypothesized that an LG induces HLA by diminishing hepatic triglyceride excretion; however, there is no clear understanding of the mechanism of LG-induced HLA. This study aimed to investigate transcript expression related to the synthesis, expenditure, and efflux of hepatic triglyceride, in mice fed an LG for 4 weeks. Results showed lower plasma concentrations of triglyceride in the LG group than in the control group, but no symptoms of hepatic injury were observed. Hepatic mRNA expressions of patatin-like phospholipase domain containing 3 (Pnpla3), microsomal triglyceride transfer protein (Mttp), and acyl-CoA oxidase 1 (Acox1) were also reduced in the LG group. Deoxycholic acid and lithocholic acid promoted intracellular lipid accumulation, reduced triglyceride concentration in media, and suppressed expression of PNPLA3 and MTTP in HepG2 human hepatoma cells. These findings suggest that deoxycholic acid and lithocholic acid promote HLA by inhibiting the expression of PNPLA3, ACOX1, and MTTP that are involved in lipid metabolism.

Risperidone Exacerbates Glucose Intolerance, Nonalcoholic Fatty Liver Disease, and Renal Impairment in Obese Mice.

Risperidone, a second-generation antipsychotic drug used for schizophrenia treatment with less-severe side effects, has recently been applied in major depressive disorder treatment. The mechanism underlying risperidone-associated metabolic disturbances and liver and renal adverse effects warrants further exploration. This research explores how risperidone influences weight, glucose homeostasis, fatty liver scores, liver damage, and renal impairment in high-fat diet (HFD)-administered C57BL6/J mice. Compared with HFD control mice, risperidone-treated obese mice exhibited increases in body, liver, kidney, and retroperitoneal and epididymal fat pad weights, daily food efficiency, serum triglyceride, blood urea nitrogen, creatinine, hepatic triglyceride, and aspartate aminotransferase, and alanine aminotransferase levels, and hepatic fatty acid regulation marker expression. They also exhibited increased insulin resistance and glucose intolerance but decreased serum insulin levels, Akt phosphorylation, and glucose transporter 4 expression. Moreover, their fatty liver score and liver damage demonstrated considerable increases, corresponding to increases in sterol regulatory element-binding protein 1 mRNA, fatty acid-binding protein 4 mRNA, and patatin-like phospholipid domain containing protein 3 expression. Finally, these mice demonstrated renal impairment, associated with decreases in glutathione peroxidase, superoxide dismutase, and catalase levels. In conclusion, long-term administration of risperidone may exacerbate diabetes syndrome, nonalcoholic fatty liver disease, and kidney injury.

RORα regulates hepatic lipolysis by inducing transcriptional expression of PNPLA3 in mice.

Nonalcoholic fatty liver diseases (NAFLDs) are characterized by excessive triacylglycerol (TAG) accumulation in the liver which contributes to hepatocyte dysfunction, inflammation, and fibrosis. Patatin-like phospholipase domain-containing 3 (PNPLA3; also known as adiponutrin) has emerged as an important enzyme leading to hepatic TAG hydrolysis. Because the I148M substitution in the PNPLA3 gene markedly reduces hepatic TAG hydrolase activity, this genetic variation is strongly associated with increased hepatic TAG in the full spectrum of NAFLDs. The Retinoic acid-related orphan receptor α (RORα) regulates various target genes related to lipid metabolism. Here, we investigated the role of RORα on PNPLA3-mediated hepatic lipid hydrolysis. With blockade of lipid esterification and ß-oxidation, RORα enhanced TAG hydrolysis, resulting in increased free glycerol levels. We found a putative RORα response element on the upstream of PNPLA3 gene that was activated by RORα. Furthermore, the inhibitory action of cJUN on the RORα/PNPLA3 axis was enhanced under lipid stress and contributed to hepatic lipid accumulation. In summary, we showed for the first time that RORα activates the transcription of PNPLA3, which suggests that RORα and its ligands represent potential precision therapeutic approaches for NAFLDs.

Impaired Hepatic Vitamin A Metabolism in NAFLD Mice Leading to Vitamin A Accumulation in Hepatocytes.

BACKGROUND & AIMS: Systemic retinol (vitamin A) homeostasis is controlled by the liver, involving close collaboration between hepatocytes and hepatic stellate cells (HSCs). Genetic variants in retinol metabolism (PNPLA3 and HSD17B13) are associated with non-alcoholic fatty liver disease (NAFLD) and disease progression. Still, little mechanistic details are known about hepatic vitamin A metabolism in NAFLD, which may affect carbohydrate and lipid metabolism, inflammation, oxidative stress and the development of fibrosis and cancer, e.g. all risk factors of NAFLD. METHODS: Here, we analyzed vitamin A metabolism in 2 mouse models of NAFLD; mice fed a high-fat, high-cholesterol (HFC) diet and Leptinob mutant (ob/ob) mice. RESULTS: Hepatic retinol and retinol binding protein 4 (RBP4) levels were significantly reduced in both mouse models of NAFLD. In contrast, hepatic retinyl palmitate levels (the vitamin A storage form) were significantly elevated in these mice. Transcriptome analysis revealed a hyperdynamic state of hepatic vitamin A metabolism, with enhanced retinol storage and metabolism (upregulated Lrat, Dgat1, Pnpla3, Raldh's and RAR/RXR-target genes) in fatty livers, in conjunction with induced hepatic inflammation (upregulated Cd68, Tnfα, Nos2, Il1ß, Il-6) and fibrosis (upregulated Col1a1, Acta2, Tgfß, Timp1). Autofluorescence analyses revealed prominent vitamin A accumulation in hepatocytes rather than HSC in HFC-fed mice. Palmitic acid exposure increased Lrat mRNA levels in primary rat hepatocytes and promoted retinyl palmitate accumulation when co-treated with retinol, which was not detected for similarly-treated primary rat HSCs. CONCLUSION: NAFLD leads to cell type-specific rearrangements in retinol metabolism leading to vitamin A accumulation in hepatocytes. This may promote disease progression and/or affect therapeutic approaches targeting nuclear receptors.

Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), rs738409, encoding the missense mutation I148M. This aminoacidic substitution interferes with the normal remodeling of lipid droplets in hepatocytes. It is also thought to play a key role in promoting liver fibrosis by inhibiting the release of retinol from hepatic stellate cells. Reducing PNPLA3 levels in individuals homozygous for 148M may be an effective treatment for the entire spectrum of NAFLD, based on gene dosage analysis in the human population, as well as the protective effect of another naturally occurring SNP (rs2294918) in PNPLA3 which, when co-inherited, reduces PNPLA3 mRNA levels to 50% and counteracts disease risk. By screening a clinical compound library targeting specific signaling pathways active in primary human hepatocytes, we identified momelotinib, a drug evaluated in clinical trials to treat myelofibrosis, as a potent down-regulator of PNPLA3 expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in PNPLA3 mRNA in human primary hepatocytes and stellate cells, as well as in vivo via acute and chronic treatment of WT mice. Using a human multilineage 3D spheroid model of NASH homozygous for the PNPLA3 mutant protein, we additionally show that it decreases PNPLA3 mRNA as well as intracellular lipid content. Furthermore, we show that the effects on PNPLA3 coincide with changes in chromatin accessibility within regulatory regions of the PNPLA3 locus, consistent with inhibition occurring at the level of transcription. In addition to its primary reported targets, the JAK kinases, momelotinib inhibits several non-JAK kinases, including ACVR1. Using a combination of targeted siRNA knockdowns and signaling pathway perturbations, we show that momelotinib reduces the expression of the PNPLA3 gene largely through the inhibition of BMP signaling rather than the JAK/STAT pathway. Overall, our work identified momelotinib as a potential NASH therapeutic and uncovered previously unrecognized connections between signaling pathways and PNPLA3. These pathways may be exploited by drug modalities to "tune down" the level of gene expression, and therefore offer a potential therapeutic benefit to a high at-risk subset of NAFLD/NASH patients.

PLA2G16 is a mutant p53/KLF5 transcriptional target and promotes glycolysis of pancreatic cancer.

PLA2G16 is a member of the phospholipase family that catalyses the generation of lysophosphatidic acids (LPAs) and free fatty acids (FFAs) from phosphatidic acid. In the current study, we explored the functional role of PLA2G16 in pancreatic adenocarcinoma (PAAD) and the genetic/epigenetic alterations leading to its dysregulation. Bioinformatic analysis was performed using data from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and the Human Protein Atlas (HPA). Then, PANC-1 and MIA-PaCa-2 cells harbouring TP53 mutations were used for cellular and animal studies. Results showed that PL2G16 expression was significantly up-regulated in PAAD tissue and was associated with unfavourable survival. PLA2G16 inhibition suppressed pancreatic cell growth in vitro and in vivo and also inhibited aerobic glycolysis. Bioinformatic analysis indicated that KLF5 was positively correlated with PLA2G16 expression in PAAD tumours with TP53 mutation. TP53 or KLF5 inhibition significantly reduced PLA2G16 expression at both mRNA and protein levels. Dual-luciferase and chromatin Immunoprecipitation-quantitative polymerase chain reaction assays showed that KLF5 directly bound to the PLA2G16 promoter and activated its transcription. Co-immunoprecipitation assay indicated that mutant p53 had a physical interaction with KLF5. Inhibition of mutant p53 impaired the transcriptional activating effects of KLF5. In PAAD cases in TCGA, PLA2G16 expression was positively correlated with its copy number (Pearson's r = 0.51, P < 0.001), but was strongly and negatively correlated with the methylation level of cg09518969 (Pearson's r = -0.64, P < 0.001), a 5'-cytosine-phosphodiester bond-guanine-3' site within its gene locus. In conclusion, this study revealed a novel mutant p53/KLF5-PLA2G16 regulatory axis on tumour growth and glycolysis in PAAD.

Genetic variant c.711A>T in the hepatobiliary phospholipid transporter ABCB4 is associated with significant liver fibrosis.

Liver fibrosis is the common consequence of chronic liver diseases (CLD). Recently liver stiffness measurements (LSM) ≥ 9.1 kPa, as determined by transient elastography (TE), were demonstrated to predict significant fibrosis (stages ≥ F2) in a population-based setting. The PNPLA3 (adiponutrin) p.I148M polymorphism enhances the risk of liver injury. The aim of our study was to investigate the association between the procholestatic ABCB4 polymorphism c.711A>T and LSM ≥ 9.1 kPa in humans as well as the interaction between ABCB4 and PNPLA3 in a mouse model of chronic cholestasis. Prospectively, we recruited 712 patients with CLD (278 women, age 50 ± 13 years) with available TE results; liver biopsy results were available in 165 individuals. The ABCB4 c.711 genotype was determined by PCR-based assays. PNPLA3 expression and liver injury were studied in Abcb4-/- mice and wild-type controls. Overall, median LSM in our cohort was 6.7 kPa, and 226 individuals had LSM ≥ 9.1 kPa. Carriers of the ABCB4 variant c.711A presented more frequently with LSM ≥ 9.1 kPa (OR = 1.33, P = 0.020) and FIB-4 score ≥ 2.67 (OR = 1.38, P = 0.040). The presence of the risk allele was associated (P = 0.002) with FIB-4. In a multivariate model, the ABCB4 variant (OR = 1.43, P = 0.047) as well as BMI (P = 0.043, OR = 1.04) and age (OR = 1.02, P < 0.010) were independent risk factors for fibrosis stage ≥ F2. Abcb4 deficiency in mice led to enhanced liver injury, coupled with a decrease (P = 0.020) of hepatic PNPLA3 expression. To conclude, the procholestatic variant ABCB4 c.711A>T might represent a new genetic risk factor for clinically significant liver fibrosis. Lower expression of PNPLA3 in fibrotic Abcb4-/- livers points to the interaction between phospholipid metabolism and PNPLA3 in progressive liver injury.

Skeletal muscle expression of adipose-specific phospholipase in peripheral artery disease.

Flow-limiting atherosclerotic lesions of arteries supplying the limbs are a cause of symptoms in patients with peripheral artery disease (PAD). Musculoskeletal metabolic factors also contribute to the pathophysiology of claudication, which is manifest as leg discomfort that impairs walking capacity. Accordingly, we conducted a case-control study to determine whether skeletal muscle metabolic gene expression is altered in PAD. Calf skeletal muscle gene expression of patients with PAD and healthy subjects was analyzed using microarrays. The top-ranking gene differentially expressed between PAD and controls (FDR < 0.001) was PLA2G16, which encodes adipose-specific phospholipase A2 (AdPLA) and is implicated in the maintenance of insulin sensitivity and regulation of lipid metabolism. Differential expression was confirmed by qRT-PCR; PLA2G16 was downregulated by 68% in patients with PAD (p < 0.001). Expression of Pla2g16 was then measured in control (db/+) and diabetic (db/db) mice that underwent unilateral femoral artery ligation. There was significantly reduced expression of Pla2g16 in the ischemic leg of both control and diabetic mice (by 51%), with significantly greater magnitude of reduction in the diabetic mice (by 79%). We conclude that AdPLA is downregulated in humans with PAD and in mice with hindlimb ischemia. Reduced AdPLA may contribute to impaired walking capacity in patients with PAD via its effects on skeletal muscle metabolism. Further studies are needed to fully characterize the role of AdPLA in PAD and to investigate its potential as a therapeutic target for alleviating symptoms of claudication.

The Contribution of Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s to Adrenic Acid Mobilization in Murine Macrophages.

Adrenic acid (AA), the 2-carbon elongation product of arachidonic acid, is present at significant levels in membrane phospholipids of mouse peritoneal macrophages. Despite its abundance and structural similarity to arachidonic acid, very little is known about the molecular mechanisms governing adrenic acid mobilization in cells of the innate immune system. This contrasts with the wide availability of data on arachidonic acid mobilization. In this work, we used mass-spectrometry-based lipidomic procedures to define the profiles of macrophage phospholipids that contain adrenic acid and their behavior during receptor activation. We identified the phospholipid sources from which adrenic acid is mobilized, and compared the data with arachidonic acid mobilization. Taking advantage of the use of selective inhibitors, we also showed that cytosolic group IVA phospholipase A2 is involved in the release of both adrenic and arachidonic acids. Importantly, calcium independent group VIA phospholipase A2 spared arachidonate-containing phospholipids and hydrolyzed only those that contain adrenic acid. These results identify separate mechanisms for regulating the utilization of adrenic and arachidonic acids, and suggest that the two fatty acids may serve non-redundant functions in cells.

Hepatocyte ELOVL Fatty Acid Elongase 6 Determines Ceramide Acyl-Chain Length and Hepatic Insulin Sensitivity in Mice.

BACKGROUND AND AIMS: Dysfunctional hepatic lipid metabolism is a cause of nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disorder worldwide, and is closely associated with insulin resistance and type 2 diabetes. ELOVL fatty acid elongase 6 (Elovl6) is responsible for converting C16 saturated and monounsaturated fatty acids (FAs) into C18 species. We have previously shown that Elovl6 contributes to obesity-induced insulin resistance by modifying hepatic C16/C18-related FA composition. APPROACH AND RESULTS: To define the precise molecular mechanism by which hepatic Elovl6 affects energy homeostasis and metabolic disease, we generated liver-specific Elovl6 knockout (LKO) mice. Unexpectedly, LKO mice were not protected from high-fat diet-induced insulin resistance. Instead, LKO mice exhibited higher insulin sensitivity than controls when consuming a high-sucrose diet (HSD), which induces lipogenesis. Hepatic patatin-like phospholipase domain-containing protein 3 (Pnpla3) expression was down-regulated in LKO mice, and adenoviral Pnpla3 restoration reversed the enhancement in insulin sensitivity in HSD-fed LKO mice. Lipidomic analyses showed that the hepatic ceramide(d18:1/18:0) content was lower in LKO mice, which may explain the effect on insulin sensitivity. Ceramide(d18:1/18:0) enhances protein phosphatase 2A (PP2A) activity by interfering with the binding of PP2A to inhibitor 2 of PP2A, leading to Akt dephosphorylation. Its production involves the formation of an Elovl6-ceramide synthase 4 (CerS4) complex in the endoplasmic reticulum and a Pnpla3-CerS4 complex on lipid droplets. Consistent with this, liver-specific Elovl6 deletion in ob/ob mice reduced both hepatic ceramide(d18:1/18:0) and PP2A activity and ameliorated insulin resistance. CONCLUSIONS: Our study demonstrates the key role of hepatic Elovl6 in the regulation of the acyl-chain composition of ceramide and that C18:0-ceramide is a potent regulator of hepatic insulin signaling linked to Pnpla3-mediated NAFLD.

Bypassing pan-enterovirus host factor PLA2G16.

Enteroviruses are a major cause of human disease. Adipose-specific phospholipase A2 (PLA2G16) was recently identified as a pan-enterovirus host factor and potential drug target. In this study, we identify a possible mechanism of PLA2G16 evasion by employing a dual glycan receptor-binding enterovirus D68 (EV-D68) strain. We previously showed that this strain does not strictly require the canonical EV-D68 receptor sialic acid. Here, we employ a haploid screen to identify sulfated glycosaminoglycans (sGAGs) as its second glycan receptor. Remarkably, engagement of sGAGs enables this virus to bypass PLA2G16. Using cryo-EM analysis, we reveal that, in contrast to sialic acid, sGAGs stimulate genome release from virions via structural changes that enlarge the putative openings for genome egress. Together, we describe an enterovirus that can bypass PLA2G16 and identify additional virion destabilization as a potential mechanism to circumvent PLA2G16.

iPLA2 Activation Mediates Granular Exocytosis and Corrects Microbicidal Defects in ROS-Deficient and CGD Human Neutrophils.

PURPOSE: The ubiquitous calcium-independent phospholipase A2 enzyme (iPLA2) is inhibited by calmodulin binding and known to be responsible for phospholipid remodeling housekeeping functions including granule exocytosis-associated membrane fusion in normal human neutrophils. We evaluate in human neutrophils the iPLA2 secretagogue effects using normal neutrophils, where reactive oxygen species (ROS) generation has been blocked by diphenyleneiodonium, as well as in neutrophils from chronic granulomatous disease (CGD) patients. METHODS: Neutrophils were pretreated with W7, a calmodulin inhibitor known to activate iPLA2 and exocytosis of granules, and vesicles as well as intra- and extra-microbicidal activity against Staphylococcus aureus and Aspergillus fumigatus were evaluated. RESULTS: W7 increases exocytosis of primary, secondary, and tertiary granules and vesicles and improves neutrophil microbicidal activity against S. aureus and A. fumigatus. CONCLUSIONS: In neutrophils, calmodulin-mediated iPLA2 inhibition controls granule and vesicle exocytosis in the phagosome and in the extracellular microenvironment. Relieving iPLA2 inhibition results in increased exocytosis of primary, secondary, and tertiary granules and secretory vesicles with correction of defective intracellular and extracellular microbicidal activity. In CGD patients presenting ROS defective production, this increase in the non-oxidative killing pathway partially corrects their microbicidal defects.

Identification of Phf16 and Pnpla3 as new adipogenic factors regulated by phytochemicals.

Adipocyte differentiation is controlled by multiple signaling pathways. To identify new adipogenic factors, C3H10T1/2 adipocytes were treated with previously known antiadipogenic phytochemicals (resveratrol, butein, sulfuretin, and fisetin) for 24 hours. Commonly regulated genes were then identified by transcriptional profiling analysis. Three genes (chemokine (C-X-C motif) ligand 1 [ Cxcl1], heme oxygenase 1 [ Hmox1], and PHD (plant homeo domain) finger protein 16 [ Phf16]) were upregulated while two genes (G0/G1 switch gene 2 [ G0s2] and patatin-like phospholipase domain containing 3 [ Pnpla3]) were downregulated by these four antiadipogenic compounds. Tissue expression profiles showed that the G0s2 and Pnpla3 expressions were highly specific to adipose depots while the other three induced genes were ubiquitously expressed with significantly higher expression in adipose tissues. While Cxcl1 expression was decreased, expressions of the other four genes were significantly increased during adipogenic differentiation of C3H10T1/2 cells. Small interfering RNA-mediated knockdown including Phf16 and Pnpla3 indicated that these genes might play regulatory roles in lipid accumulation and adipocyte differentiation. Specifically, the silencing of two newly identified adipogenic genes, Phf16 or Pnpla3, suppressed lipid accumulation and expression of adipocyte markers in both 3T3-L1 and C3H10T1/2 cells. Taken together, these data showed previously uncovered roles of Phf16 and Pnpla3 in adipogenesis, highlighting the potential of using phytochemicals for further investigation of adipocyte biology.