AMPK-dependent and -independent coordination of mitochondrial function and muscle fiber type by FNIP1.

Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1Tg) mice. Fnip1Tg mice were crossed with Fnip1-knockout (Fnip1KO) mice to generate Fnip1TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness.

Cideb controls sterol-regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites.

SREBPs are master regulators of lipid homeostasis and undergo sterol-regulated export from ER to Golgi apparatus for processing and activation via COPII-coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet-associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP-Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet-induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.

Phosphatidylserine-Specific Phospholipase A1 Limits Aggressiveness of Lung Adenocarcinoma by Lysophosphatidylserine and Protein Kinase A-Dependent Pathway.

Lipid metabolic abnormalities in cancer cells are increasingly being studied. Several studies have reported that phosphatidylserine-specific phospholipase A1 (PLA1A) might be involved in the pathogenesis of cancers. Nevertheless, the function and mechanistic details of PLA1A in lung adenocarcinoma (LUAD) progression remain largely undefined. In the present study, low PLA1A expression was correlated with poor prognosis in patients with LUAD. Results from in vitro and in vivo animal studies showed that overexpressed PLA1A suppressed the proliferation of LUAD cells in vitro and tumor growth in vivo through regulation of cyclin abundance, thereby inducing S-phase arrest. Meanwhile, PLA1A overexpression attenuated migration and invasion of LUAD cells, including by inhibiting the epithelial-mesenchymal transition. Mechanistically, PLA1A overexpression inhibited aggressiveness of LUAD cells through elevated lysophosphatidylserine, which acts via G-protein-coupled receptor 174, further activating cAMP/protein kinase A pathway. Activating G-protein-coupled receptor 174/protein kinase A pathway may involve effects on cell cycle regulators and transcription factors-regulated epithelial-mesenchymal transition. The study uncovered the mechanism through which PLA1A regulates LUAD proliferation, invasion, and migration. These results demonstrate the potential use of PLA1A as a biomarker for diagnosing LUAD, which may therefore potentially serve as a therapeutic target for LUAD.

Ceramide d18:1/24:1 as a potential biomarker to differentiate obesity subtypes with unfavorable health outcomes.

BACKGROUND: The criteria for metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUO) remain controversial. This research aimed to identify a potential biomarker to differentiate the subtypes of obesity. METHODS: The study conducted a lipidomic evaluation of ceramide in the serum of 77 Chinese adults who had undergone hyperinsulinemic-euglycemic clamps. These adults were divided into three groups according to the clinical data: normal weight control group (N = 21), MHO (N = 20), and MUO (N = 36). RESULTS: The serum Cer d18:1/24:1 level in the MHO group was lower than that in the MUO group. As the Cer d18:1/24:1 level increased, insulin sensitivity decreased, and the unfavorable parameters increased in parallel. Multivariate logistic regression analysis revealed that serum Cer d18:1/24:1 levels were independently correlated with MUO in obesity. Individuals with higher levels of Cer d18:1/24:1 also had an elevated risk of cardiovascular disease. Most ceramide subtype levels increased in obesity compared to normal-weight individuals, but the levels of serum Cer d18:0/18:0 and Cer d18:1/16:0 decreased in obesity. CONCLUSIONS: The relationships between ceramide subtypes and metabolic profiles might be heterogeneous in populations with different body weights. Cer d18:1/24:1 could be a biomarker that can be used to differentiate MUO from MHO, and to better predict who will develop unfavorable health outcomes among obese individuals. TRIAL REGISTRATION: The First Affiliated Hospital of Nanjing Medical University's Institutional Review Board authorized this study protocol, and all participants provided written informed consent (2014-SR-003) prior to study entry.

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

BACKGROUND AND AIMS: 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. APPROACH AND RESULTS: 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. CONCLUSION: PNPLA7 plays a critical role in regulating hepatic VLDL secretion by modulating ApoE stability through its interaction with ApoE.

Chronic hepatitis C virus infection impairs insulin secretion by regulation of p38δ MAPK-dependent exocytosis in pancreatic ß-cells.

Chronic hepatitis C virus (HCV) infection has a close association with type 2 diabetes mellitus. Although the mechanisms of insulin resistance in chronic hepatitis C (CHC) patients have been extensively studied, little attention has been given to the role of ß-cell function in HCV-associated diabetes. Here, we analysed ß-cell function in CHC patients and HCV-infected mouse model and found in addition to insulin resistance, impaired pancreatic ß-cell function occurred in CHC patients and HCV-infected C/OTg mice, not only in diabetic individuals but also in individuals with impaired fasting glucose levels. Both first-phase and second-phase insulin secretion were impaired, at least partially due to the reduction of exocytosis of secretory insulin-containing granules following HCV infection. Up-regulated p38δ in HCV-infected ß-cells resulted in inactivation of protein kinase D (PKD), which was responsible for impaired insulin secretory capacity of ß-cells. Thus, impaired insulin secretion due to HCV infection in ß-cells contributes to HCV-associated type 2 diabetes. These findings provided a new inspiration for the important prognostic and therapeutic implications in the management of CHC patients with impaired fasting glucose.

Long non-coding RNA Bhmt-AS attenuates hepatic gluconeogenesis via modulation of Bhmt expression.

Dysregulation of gluconeogenesis contributes to the pathogenesis of metabolic disease, such as type-2 diabetes. The role of long non-coding RNAs (lncRNAs) in the pathogenesis of diabetes has recently received increased attention. In the present study, we identified a novel lncRNA, betaine-homocysteine methyltransferase-antisense (Bhmt-AS), and examined its expression patterns under pathophysiological conditions. Our results revealed that the expression of Bhmt-AS was significantly increased in the livers of fasted and db/db mice and was induced by gluconeogenic hormonal stimuli. The Bhmt-AS was also shown to be a concordant regulator of Bhmt expression. Functionally, depletion of Bhmt-AS suppressed hepatic glucose production both in vivo and in vitro. Adenovirus-mediated hepatic knockdown of Bhmt-AS improved pyruvate tolerance, glucose tolerance, and insulin sensitivity. Furthermore, overexpression of Bhmt restored the decreased glucose production caused by knockdown of Bhmt-AS in primary hepatocytes. Taken together, we uncovered a novel antisense lncRNA (Bhmt-AS) that is co-expressed with Bhmt and concordantly and specifically regulates Bhmt expression both in vitro and in vivo to regulate hepatic gluconeogenesis.

Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes.

Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.

A feed-forward loop amplifies nutritional regulation of PNPLA3.

The upsurge in prevalence of obesity has spawned an epidemic of nonalcoholic fatty liver disease (NAFLD). Previously, we identified a sequence variant (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) that confers susceptibility to both hepatic triglyceride (TG) deposition and liver injury. To glean insights into the biological role of PNPLA3, we examined the molecular mechanisms by which nutrient status controls hepatic expression of PNPLA3. PNPLA3 mRNA levels, which were low in fasting animals, increased approximately 90-fold with carbohydrate feeding. The increase was mimicked by treatment with a liver X receptor (LXR) agonist and required the transcription factor SREBP-1c. The site of SREBP-1c binding was mapped to intron 1 of Pnpla3 using chromatin immunoprecipitation and electrophoretic mobility shift assays. SREBP-1c also promotes fatty acid synthesis by activating several genes encoding enzymes in the biosynthetic pathway. Addition of fatty acids (C16:0, C18:1, and C18:2) to the medium of cultured hepatocytes (HuH-7) increased PNPLA3 protein mass without altering mRNA levels. The posttranslational increase in PNPLA3 levels persisted after blocking TG synthesis with triascin C. Oleate (400 muM) treatment prolonged the half-life of PNPLA3 from 2.4 to 6.7 h. These findings are consistent with nutritional control of PNPLA3 being effected by a feed-forward loop; SREBP-1c promotes accumulation of PNPLA3 directly by activating Pnpla3 transcription and indirectly by inhibiting PNPLA3 degradation through the stimulation of fatty acid synthesis.

Comprehensive Analysis of METTLs (METTL1/13/18/21A/23/25/2A/2B/5/6/9) and Associated mRNA Risk Signature in Hepatocellular Carcinoma.

Currently, 80%-90% of liver cancers are hepatocellular carcinomas (HCC). HCC patients develop insidiously and have an inferior prognosis. The methyltransferase-like (METTL) family principal members are strongly associated with epigenetic and tumor progression. The present study mainly analyzed the value of METTLs (METTL1/13/18/21A/23/25/2A/2B/5/6/9) and associated mRNA risk signature for HCC. METTLs expression is upregulated in HCC and is a poor prognostic factor in HCC. METTLs were upregulated in patients older than 60 and associated with grade. Except for METTL25, the remaining 10 genes were associated with the HCC stage, invasion depth (T). In addition, METTLs showed an overall alteration rate of 50%. Except for METTL13/2A/25/9, the expression of the other seven genes was significantly associated with overall survival, disease-specific survival, and progression-free survival. Multivariate studies have shown that METTL21A/6 can be an independent prognostic marker in HCC. A total of 664 mRNAs were selected based on Pearson correlation coefficient (R > 0.5), unsupervised consensus clustering, weighted coexpression network analysis, and univariate Cox analysis. These mRNAs were significantly associated with METTLs and were poor prognostic factors in HCC patients. The least absolute shrinkage and selection operator (lasso) was used to construct the best METTLs associated with mRNA risk signature. The mRNA risk signature was significantly associated with age, stage, and t grade. The mRNA high-risk group had higher TP53 and RB1 mutations. This study constructed a nomogram with the mRNA risk profile and clinicopathological features, which could better predict the OS of individuals with HCC. We also analyzed associations between METTLs and mRNA risk signatures in epithelial-mesenchymal transition, immune checkpoints, immune cell infiltration, tumor mutational burden, microsatellite instability, cancer stem cells, tumor pathways, and drug sensitivity. In addition, this study constructed a protein interaction network network including METTLs and mRNA risk signature genes related to tumor microenvironment remodeling based on single-cell sequencing. In conclusion, this study provides a theoretical basis for the mechanism, biomarker screening, and treatment of HCC.

Genetic analysis and functional study of a novel ABCG5 mutation in sitosterolemia with hematologic disease.

Sitosterolemia is a rare autosomal recessive hereditary disease caused by loss-of-function genetic mutations in either ATP-binding cassette subfamily G member 5 or member 8 (ABCG5 or ABCG8). Here, we investigate novel variants in ABCG5 and ABCG8 that are associated with the sitosterolemia phenotype. We describe a 32-year-old woman with hypercholesterolemia, tendon and hip xanthomas, autoimmune hemolytic anemia and macrothrombocytopenia from early life, which make us highly suspicious of the possibility of sitosterolemia. A novel homozygous variant in ABCG5 (c.1769C>A, p.S590X) was identified by genomic sequencing. We also examined the lipid profile, especially plant sterols levels, using gas chromatography-mass spectrometry. Functional studies, including western blotting and immunofluorescence staining, showed that the nonsense mutation ABCG5 1769C>A hinders the formation of ABCG5 and ABCG8 heterodimers and the function of transporting sterols. Our study expands the knowledge of variants in sitosterolemia and provides diagnosis and treatment recommendations.

Long noncoding RNA lnc_217 regulates hepatic lipid metabolism by modulating lipogenesis and fatty acid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is considered a major health epidemic with an estimated 32.4% worldwide prevalence. No drugs have yet been approved and therapeutic nodes remain a major unmet need. Long noncoding RNAs are emerging as an important class of novel regulators influencing multiple biological processes and the pathogenesis of NAFLD. Herein, we described a novel long noncoding RNA, lnc_217, which was liver enriched and upregulated in high-fat diet-fed mice, and a genetic animal model of NAFLD. We found that liver specific knockdown of lnc_217 was resistant to high-fat diet-induced hepatic lipid accumulation and decreased serum lipid in mice. Mechanistically, we demonstrated that knockdown of lnc_217 not only decreased de novo lipogenesis by inhibiting sterol regulatory element binding protein-1c cleavage but also increased fatty acid ß-oxidation through activation of peroxisome proliferator-activated receptor α and carnitine palmitoyltransferase-1α. Taken together, we conclude that lnc_217 may be a novel regulator of hepatic lipid metabolism and a potential therapeutic target for the treatment of hepatic steatosis and NAFLD-related metabolic disorders.

Functional Characterization of MC4R Variants in Chinese Morbid Obese Patients and Weight Loss after Bariatric Surgery.

Mutations in MC4R are the most common genetic cause of obesity. In the reported Chinese morbid obesity cohort, 10 out of 59 harbor six MC4R variants, including Y35C, T53I, V103I, R165W, G233S, and C277X, among which V103I has a relatively high frequency, while other five variants are rare in the population. The prevalence of MC4R carriers in Chinese morbid obese patients (body mass index ≥ 45 kg m-2 ) is detected as 16.9% in this study. R165W and C277X are loss-of-function variants. The patient with R165W achieves excess weight loss (%EWL) as high as 20.6% and 50.3% at 1 and 8 months after surgery, respectively. G233S is reported for the first time in Asia obese population. The patient harboring G233S has a %EWL as 23.3% one month postsurgery. It is concluded that morbid obese patients with rare MC4R variants can benefit from metabolic surgery. More importantly, the choice of surgery procedure and MC4R variant should be taken into consideration for personalized treatment. In the future, a larger size cohort, accompanied with regular and longer follow-up, would be helpful.

Rab8a as a mitochondrial receptor for lipid droplets in skeletal muscle.

Dynamic interaction between lipid droplets (LDs) and mitochondria controls the mobilization of long-chain fatty acids (LCFAs) from LDs for mitochondrial ß-oxidation in skeletal muscle in response to energy stress. However, little is known about the composition and regulation of the tethering complex mediating LD-mitochondrion interaction. Here, we identify Rab8a as a mitochondrial receptor for LDs forming the tethering complex with the LD-associated PLIN5 in skeletal muscle. In rat L6 skeletal muscle cells, the energy sensor AMPK increases the GTP-bound active Rab8a that promotes LD-mitochondrion interaction through binding to PLIN5 upon starvation. The assembly of the Rab8a-PLIN5 tethering complex also recruits the adipose triglyceride lipase (ATGL), which couples LCFA mobilization from LDs with its transfer into mitochondria for ß-oxidation. Rab8a deficiency impairs fatty acid utilization and decreases endurance during exercise in a mouse model. These findings may help to elucidate the regulatory mechanisms underlying the beneficial effects of exercise on lipid homeostasis control.

Downregulation of AMP-activated protein kinase by Cidea-mediated ubiquitination and degradation in brown adipose tissue.

We previously showed that Cidea(-/-) mice are resistant to diet-induced obesity through the upregulation of energy expenditure. The AMP-activated protein kinase (AMPK), consisting of catalytic alpha subunit and regulatory subunits beta and gamma, has a pivotal function in energy homoeostasis. We show here that AMPK protein levels and enzymatic activity were significantly increased in the brown adipose tissue of Cidea(-/-) mice. We also found that Cidea is colocalized with AMPK in the endoplasmic reticulum and forms a complex with AMPK in vivo through specific interaction with the beta subunit of AMPK, but not with the alpha or gamma subunit. When co-expressed with Cidea, the stability of AMPK-beta subunit was dramatically reduced due to increased ubiquitination-mediated degradation, which depends on a physical interaction between Cidea and AMPK. Furthermore, AMPK stability and enzymatic activity were increased in Cidea(-/-) adipocytes differentiated from mouse embryonic fibroblasts or preadipocytes. Our data strongly suggest that AMPK can be regulated by Cidea-mediated ubiquitin-dependent proteosome degradation, and provide a molecular explanation for the increased energy expenditure and lean phenotype in Cidea-null mice.

Long Noncoding RNA lncRHL Regulates Hepatic VLDL Secretion by Modulating hnRNPU/BMAL1/MTTP Axis.

Dysregulation of hepatic VLDL secretion contributes to the pathogenesis of metabolic diseases, such as nonalcoholic fatty liver disease (NAFLD) and hyperlipidemia. Accumulating evidence has suggested that long noncoding RNAs (lncRNAs) had malfunctioning roles in the pathogenesis of NAFLD. However, the function of lncRNAs in controlling hepatic VLDL secretion remains largely unillustrated. Here, we identified a novel lncRNA, lncRNA regulator of hyperlipidemia (lncRHL), which was liver-enriched, downregulated on high-fat diet feeding, and inhibited by oleic acid treatment in primary hepatocytes. With genetic manipulation in mice and primary hepatocytes, depletion of lncRHL induces hepatic VLDL secretion accompanied by decreased hepatic lipid contents. Conversely, lncRHL restoration reduces VLDL secretion with increased lipid deposition in hepatocytes. Mechanistic analyses indicate that lncRHL binds directly to heterogeneous nuclear ribonuclear protein U (hnRNPU), and thereby enhances its stability, and that hnRNPU can transcriptional activate Bmal1, leading to inhibition of VLDL secretion in hepatocytes. lncRHL deficiency accelerates the protein degradation of hnRNPU and suppresses the transcription of Bmal1, which in turn activates VLDL secretion in hepatocytes. With results taken together, we conclude that lncRHL is a novel suppressor of hepatic VLDL secretion. Activating the lncRHL/hnRNPU/BMAL1/MTTP axis represents a potential strategy for the maintenance of intrahepatic and plasma lipid homeostasis.

Impaired Insulin Clearance as the Initial Regulator of Obesity-Associated Hyperinsulinemia: Novel Insight Into the Underlying Mechanism Based on Serum Bile Acid Profiles.

OBJECTIVE: To investigate the roles of insulin clearance and insulin secretion in the development of hyperinsulinemia in obese subjects and to reveal the association between insulin clearance and bile acids (BAs). RESEARCH DESIGN AND METHODS: In cohort 1, insulin secretion, sensitivity, and endogenous insulin clearance were evaluated with an oral glucose tolerance test in 460 recruited participants. In cohort 2, 81 participants underwent an intravenous glucose tolerance test and a hyperinsulinemic-euglycemic clamp to assess insulin secretion, endogenous and exogenous insulin clearance, and insulin sensitivity. Based on insulin resistance levels ranging from mild to severe, obese participants without diabetes were further divided into 10 quantiles in cohort 1 and into tertiles in cohort 2. Forty serum BAs were measured in cohort 2 to examine the association between BAs and insulin clearance. RESULTS: All obese participants had impaired insulin clearance, and it worsened with additional insulin resistance in obese subjects without diabetes. However, insulin secretion was unchanged from quantile 1 to 3 in cohort 1, and no difference was found in cohort 2. After adjustments for all confounding factors, serum-conjugated BAs, especially glycodeoxycholic acid (GDCA; ß = -0.335, P = 0.004) and taurodeoxycholic acid (TDCA; ß = -0.333, P = 0.003), were negatively correlated with insulin clearance. The ratio of unconjugated to conjugated BAs (ß = 0.335, P = 0.002) was positively correlated with insulin clearance. CONCLUSIONS: Hyperinsulinemia in obese subjects might be primarily induced by decreased insulin clearance rather than increased insulin secretion. Changes in circulating conjugated BAs, especially GDCA and TDCA, might play an important role in regulating insulin clearance.

Pnpla3/Adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome.

PNPLA3 (adiponutrin, calcium-independent phospholipase A(2) epsilon [iPLA(2)ε]) is an adipose-enriched, nutritionally regulated protein that belongs to the patatin-like phospholipase domain containing (PNPLA) family of lipid metabolizing proteins. Genetic variations in the human PNPLA3 gene (i.e., the rs738409 I148M allele) has been strongly and repeatedly associated with fatty liver disease. Although human PNPLA3 has triacylglycerol (TAG) hydrolase and transacylase activities in vitro, its in vivo function and physiological relevance remain controversial. The objective of this study was to determine the metabolic consequences of global targeted deletion of the Pnpla3 gene in mice. We found that Pnpla3 mRNA expression is altered in adipose tissue and liver in response to acute and chronic nutritional challenges. However, global targeted deletion of the Pnpla3 gene in mice did not affect TAG hydrolysis, nor did it influence energy/glucose/lipid homoeostasis or hepatic steatosis/injury. Experimental interventions designed to increase Pnpla3 expression (refeeding, high-sucrose diet, diet-induced obesity, and liver X receptor agonism) likewise failed to reveal differences in the above-mentioned metabolic phenotypes. Expression of the Pnpla3 paralog, Pnpla5, was increased in adipose tissue but not in liver of Pnpla3-deficient mice, but compensatory regulation of genes involved in TAG metabolism was not identified. Together these data argue against a role for Pnpla3 loss-of-function in fatty liver disease or metabolic syndrome in mice.

A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis.

Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is associated with increased hepatic triglyceride content in humans. Here we examined the effect of the I148M substitution on the enzymatic activity and cellular location of PNPLA3. Structural modeling predicted that the substitution of methionine for isoleucine at residue 148 would restrict access of substrate to the catalytic serine at residue 47. In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. Expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content. Cell fractionation studies revealed that approximately 90% of wild type PNPLA3 partitioned between membranes and lipid droplets; substitution of isoleucine for methionine at position 148 did not alter the subcellular distribution of the protein. These data are consistent with PNPLA3-I148M promoting triglyceride accumulation by limiting triglyceride hydrolysis.

Control of cholesterol biosynthesis, uptake and storage in hepatocytes by Cideb.

Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases.