Cardiac-specific CGI-58 deficiency activates the ER stress pathway to promote heart failure in mice.

Excess myocardial triacylglycerol accumulation (i.e., cardiac steatosis) impairs heart function, suggesting that enzymes promoting triacylglycerol metabolism exert essential regulatory effects on heart function. Comparative gene identification 58 (CGI-58) is a key enzyme that promotes the hydrolysis of triglycerides by activating adipose triglyceride lipase and plays a protective role in maintaining heart function. In this study, the effects of CGI-58 on heart function and the underlying mechanism were investigated using cardiac-specific CGI58-knockout mice (CGI-58cko mice). Echocardiography and pathological staining were performed to detect changes in the structure and function of the heart. Proteomic profiling, immunofluorescent staining, western blotting, and real-time PCR were used to evaluate molecular changes. In CGI-58cko mice, we detected cardiac hypertrophic remodeling and heart failure associated with excessive cardiac lipid accumulation, ROS production, and decreased expression of regulators of fatty acid metabolism. These changes were markedly attenuated in CGI-58cko mice injected with rAAV9-CGI58. A quantitative proteomics analysis revealed significant increases in the expression of ER stress-related proteins and decreases in proteins related to fatty acid and amino acid metabolism in the hearts of CGI-58cko mice. Furthermore, the inhibition of ER stress by the inhibitor 4-PBA improved mitochondrial dysfunction, reduced oxidative stress, and reversed cardiac remodeling and dysfunction in cultured cardiomyocytes or in CGI-58cko mice. Our results suggested that CGI-58 is essential for the maintenance of heart function by reducing lipid accumulation and ER stress in cardiomyocytes, providing a new therapeutic target for cardiac steatosis and dysfunction.

Western diet induces severe nonalcoholic steatohepatitis, ductular reaction, and hepatic fibrosis in liver CGI-58 knockout mice.

Humans and rodents with Comparative Gene Identification-58 (CGI-58) mutations manifest nonalcoholic fatty liver disease (NAFLD). Here we show that liver CGI-58 knockout (LivKO) mice fed a Western diet rapidly develop advanced NAFLD, including nonalcoholic steatohepatitis (NASH) and hepatic fibrosis. After 14 weeks of diet challenge, starting at 6 weeks of age, LivKO mice showed increased inflammatory cell infiltration and proinflammatory gene expression in the liver, which was associated with elevated plasma levels of aminotransferases. Hepatic ductular reactions, pericellular fibrosis, and bridging fibrosis were observed only in the LivKO mice. Consistently, the KO mice had a significant increase in hepatic mRNAs for fibrogenic genes. In addition, LivKO mice displayed massive accumulation of lipid droplets (LDs) in hepatocytes. LDs were also observed in the cholangiocytes of the LivKO mice, but not the floxed controls. Four of the five LD coat proteins, including perilipins 2, 3, 4, and 5, were increased in the CGI-58 KO liver. CRISPR/Cas9-mediated knockout of CGI-58 in Huh7 human hepatoma cells induced LD deposition and perilipin expression, suggesting a cell autonomous effect. Our findings establish the Western diet-fed LivKO mice as an animal model of NASH and hepatic fibrosis. These animals may facilitate preclinical screening of therapeutic agents that counter against NAFLD progression.

Loss of ABHD5 promotes the aggressiveness of prostate cancer cells.

The accumulation of neutral lipids in intracellular lipid droplets has been associated with the formation and progression of many cancers, including prostate cancer (PCa). Alpha-beta Hydrolase Domain Containing 5 (ABHD5) is a key regulator of intracellular neutral lipids that has been recently identified as a tumor suppressor in colorectal cancer, yet its potential role in PCa has not been investigated. Through mining publicly accessible PCa gene expression datasets, we found that ABHD5 gene expression is markedly decreased in metastatic castration-resistant PCa (mCRPC) samples. We further demonstrated that RNAi-mediated ABHD5 silencing promotes, whereas ectopic ABHD5 overexpression inhibits, the invasion and proliferation of PCa cells. Mechanistically, we found that ABHD5 knockdown induces epithelial to mesenchymal transition, increasing aerobic glycolysis by upregulating the glycolytic enzymes hexokinase 2 and phosphofrucokinase, while decreasing mitochondrial respiration by downregulating respiratory chain complexes I and III. Interestingly, knockdown of ATGL, the best-known molecular target of ABHD5, impeded the proliferation and invasion, suggesting an ATGL-independent role of ABHD5 in modulating PCa aggressiveness. Collectively, these results provide evidence that ABHD5 acts as a metabolic tumor suppressor in PCa that prevents EMT and the Warburg effect, and indicates that ABHD5 is a potential therapeutic target against mCRPC, the deadly aggressive PCa.

PNPLA1 has a crucial role in skin barrier function by directing acylceramide biosynthesis.

Mutations in patatin-like phospholipase domain-containing 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, but the mechanism involved remains unclear. Here we show that PNPLA1, an enzyme expressed in differentiated keratinocytes, plays a crucial role in the biosynthesis of ω-O-acylceramide, a lipid component essential for skin barrier. Global or keratinocyte-specific Pnpla1-deficient neonates die due to epidermal permeability barrier defects with severe transepidermal water loss, decreased intercellular lipid lamellae in the stratum corneum, and aberrant keratinocyte differentiation. In Pnpla1-/- epidermis, unique linoleate-containing lipids including acylceramides, acylglucosylceramides and (O-acyl)-ω-hydroxy fatty acids are almost absent with reciprocal increases in their putative precursors, indicating that PNPLA1 catalyses the ω-O-esterification with linoleic acid to form acylceramides. Moreover, acylceramide supplementation partially rescues the altered differentiation of Pnpla1-/- keratinocytes. Our findings provide valuable insight into the skin barrier formation and ichthyosis development, and may contribute to novel therapeutic strategies for treatment of epidermal barrier defects.

Macrophage CGI-58 deficiency promotes IL-1ß transcription by activating the SOCS3-FOXO1 pathway.

Over-nutrition induces low-grade inflammation that dampens insulin sensitivity, but the underlying molecular mediators are not fully understood. Comparative gene identification-58 (CGI-58) is an intracellular lipolytic activator. In the present study, we show that in mouse visceral fat-derived macrophages or human peripheral blood monocytes, CGI-58 negatively and interleukin (IL)-1ß positively correlate with obesity. Saturated non-esterified fatty acid (NEFA) suppresses CGI-58 expression in macrophages and this suppression activates FOXO1 (forkhead box-containing protein O subfamily-1) through inhibition of FOXO1 phosphorylation. Activated FOXO1 binds to an insulin-responsive element in IL-1ß promoter region to potentiate IL-1ß transcription. Gain- and loss-of-function studies demonstrate that NEFA-induced CGI-58 suppression activates FOXO1 to augment IL-1ß transcription by dampening insulin signalling through induction of SOCS3 (suppressor of cytokine signalling 3) expression. CGI-58 deficiency-induced SOCS3 expression is NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome-dependent. Our data thus identified a vicious cycle (IL-1ß-SOCS3-FOXO1-IL-1ß) that amplifies IL-1ß secretion and is initiated by CGI-58 deficiency-induced activation of the NLRP3 inflammasome in macrophages. We further show that blocking this cycle with a FOXO1 inhibitor, an antioxidant that inhibits FOXO1 or IL-1 receptor antagonist alleviates chronic inflammation and insulin resistance in high-fat diet (HFD)-fed mice. Collectively, our data suggest that obesity-associated factors such as NEFA and lipopolysaccharide (LPS) probably adopt this vicious cycle to promote inflammation and insulin resistance.

Macrophage CGI-58 deficiency activates ROS-inflammasome pathway to promote insulin resistance in mice.

Overnutrition activates a proinflammatory program in macrophages to induce insulin resistance (IR), but its molecular mechanisms remain incompletely understood. Here, we show that saturated fatty acid and lipopolysaccharide, two factors implicated in high-fat diet (HFD)-induced IR, suppress macrophage CGI-58 expression. Macrophage-specific CGI-58 knockout (MaKO) in mice aggravates HFD-induced glucose intolerance and IR, which is associated with augmented systemic/tissue inflammation and proinflammatory activation of adipose tissue macrophages. CGI-58-deficient macrophages exhibit mitochondrial dysfunction due to defective peroxisome proliferator-activated receptor (PPAR)γ signaling. Consequently, they overproduce reactive oxygen species (ROS) to potentiate secretion of proinflammatory cytokines by activating NLRP3 inflammasome. Anti-ROS treatment or NLRP3 silencing prevents CGI-58-deficient macrophages from oversecreting proinflammatory cytokines and from inducing proinflammatory signaling and IR in the cocultured fat slices. Anti-ROS treatment also prevents exacerbation of inflammation and IR in HFD-fed MaKO mice. Our data thus establish CGI-58 as a suppressor of overnutrition-induced NLRP3 inflammasome activation in macrophages.

Intestinal Cgi-58 deficiency reduces postprandial lipid absorption.

Comparative Gene Identification-58 (CGI-58), a lipid droplet (LD)-associated protein, promotes intracellular triglyceride (TG) hydrolysis in vitro. Mutations in human CGI-58 cause TG accumulation in numerous tissues including intestine. Enterocytes are thought not to store TG-rich LDs, but a fatty meal does induce temporary cytosolic accumulation of LDs. Accumulated LDs are eventually cleared out, implying existence of TG hydrolytic machinery in enterocytes. However, identities of proteins responsible for LD-TG hydrolysis remain unknown. Here we report that intestine-specific inactivation of CGI-58 in mice significantly reduces postprandial plasma TG concentrations and intestinal TG hydrolase activity, which is associated with a 4-fold increase in intestinal TG content and large cytosolic LD accumulation in absorptive enterocytes during the fasting state. Intestine-specific CGI-58 knockout mice also display mild yet significant decreases in intestinal fatty acid absorption and oxidation. Surprisingly, inactivation of CGI-58 in intestine significantly raises plasma and intestinal cholesterol, and reduces hepatic cholesterol, without altering intestinal cholesterol absorption and fecal neutral sterol excretion. In conclusion, intestinal CGI-58 is required for efficient postprandial lipoprotein-TG secretion and for maintaining hepatic and plasma lipid homeostasis. Our animal model will serve as a valuable tool to further define how intestinal fat metabolism influences the pathogenesis of metabolic disorders, such as obesity and type 2 diabetes.

Hepatic gluconeogenesis is enhanced by phosphatidic acid which remains uninhibited by insulin in lipodystrophic Agpat2-/- mice.

In this study we examined the role of phosphatidic acid (PA) in hepatic glucose production (HGP) and development of hepatic insulin resistance in mice that lack 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2). Liver lysophosphatidic acid and PA levels were increased ∼2- and ∼5-fold, respectively, in male Agpat2(-/-) mice compared with wild type mice. In the absence of AGPAT2, the liver can synthesize PAs by activating diacylglycerol kinase or phospholipase D, both of which were elevated in the livers of Agpat2(-/-) mice. We found that PAs C16:0/18:1 and C18:1/20:4 enhanced HGP in primary WT hepatocytes, an effect that was further enhanced in primary hepatocytes from Agpat2(-/-) mice. Lysophosphatidic acids C16:0 and C18:1 failed to increase HGP in primary hepatocytes. The activation of HGP was accompanied by an up-regulation of the key gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This activation was suppressed by insulin in the WT primary hepatocytes but not in the Agpat2(-/-) primary hepatocytes. Thus, the lack of normal insulin signaling in Agpat2(-/-) livers allows unrestricted PA-induced gluconeogenesis significantly contributing to the development of hyperglycemia in these mice.

Leptin ameliorates insulin resistance and hepatic steatosis in Agpat2-/- lipodystrophic mice independent of hepatocyte leptin receptors.

Leptin is essential for energy homeostasis and regulation of food intake. Patients with congenital generalized lipodystrophy (CGL) due to mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) and the CGL murine model (Agpat2(-/-) mice) both have severe insulin resistance, diabetes mellitus, hepatic steatosis, and low plasma leptin levels. In this study, we show that continuous leptin treatment of Agpat2(-/-) mice for 28 days reduced plasma insulin and glucose levels and normalized hepatic steatosis and hypertriglyceridemia. Leptin also partially, but significantly, reversed the low plasma thyroxine and high corticosterone levels found in Agpat2(-/-) mice. Levels of carbohydrate response element binding protein (ChREBP) were reduced, whereas lipogenic gene expression were increased in the livers of Agpat2(-/-) mice, suggesting that deregulated ChREBP contributed to the development of fatty livers in these mice and that this transcription factor is a target of leptin's beneficial metabolic action. Leptin administration did not change hepatic fatty acid oxidation enzymes mRNA levels in Agpat2(-/-) mice. The selective deletion of leptin receptors only in hepatocytes did not prevent the positive metabolic actions of leptin in Agpat2(-/-) mice, supporting the notion that the majority of metabolic actions of leptin are dependent on its action in nonhepatocyte cells and/or the central nervous system.

Deficiency of liver Comparative Gene Identification-58 causes steatohepatitis and fibrosis in mice.

Triglyceride (TG) accumulation in hepatocytes (hepatic steatosis) preludes the development of advanced nonalcoholic fatty liver diseases (NAFLDs) such as steatohepatitis, fibrosis, and cirrhosis. Mutations in human Comparative Gene Identification-58 (CGI-58) cause cytosolic TG-rich lipid droplets to accumulate in almost all cell types including hepatocytes. However, it is unclear if CGI-58 mutation causes hepatic steatosis locally or via altering lipid metabolism in other tissues. To directly address this question, we created liver-specific CGI-58 knockout (LivKO) mice. LivKO mice on standard chow diet displayed microvesicular and macrovesicular panlobular steatosis, and progressed to advanced NAFLD stages over time, including lobular inflammation and centrilobular fibrosis. Compared with CGI-58 floxed control littermates, LivKO mice showed 8-fold and 52-fold increases in hepatic TG content, which was associated with 40% and 58% decreases in hepatic TG hydrolase activity at 16 and 42 weeks, respectively. Hepatic cholesterol also increased significantly in LivKO mice. At 42 weeks, LivKO mice showed increased hepatic oxidative stress, plasma aminotransferases, and hepatic mRNAs for genes involved in fibrosis and inflammation, such as α-smooth muscle actin, collagen type 1 α1, tumor necrosis factor α, and interleukin-1ß. In conclusion, CGI-58 deficiency in the liver directly causes not only hepatic steatosis but also steatohepatitis and fibrosis.

Regulation of skeletal muscle lipolysis and oxidative metabolism by the co-lipase CGI-58.

We investigated here the specific role of CGI-58 in the regulation of energy metabolism in skeletal muscle. We first examined CGI-58 protein expression in various muscle types in mice, and next modulated CGI-58 expression during overexpression and knockdown studies in human primary myotubes and evaluated the consequences on oxidative metabolism. We observed a preferential expression of CGI-58 in oxidative muscles in mice consistent with triacylglycerol hydrolase activity. We next showed by pulse-chase that CGI-58 overexpression increased by more than 2-fold the rate of triacylglycerol (TAG) hydrolysis, as well as TAG-derived fatty acid (FA) release and oxidation. Oppositely, CGI-58 silencing reduced TAG hydrolysis and TAG-derived FA release and oxidation (-77%, P < 0.001), whereas it increased glucose oxidation and glycogen synthesis. Interestingly, modulations of CGI-58 expression and FA release are reflected by changes in pyruvate dehydrogenase kinase 4 gene expression. This regulation involves the activation of the peroxisome proliferator activating receptor-δ (PPARδ) by lipolysis products. Altogether, these data reveal that CGI-58 plays a limiting role in the control of oxidative metabolism by modulating FA availability and the expression of PPARδ-target genes, and highlight an important metabolic function of CGI-58 in skeletal muscle.

CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance.

Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ∼80-95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ∼4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.

Higher adiponectin levels in patients with Berardinelli-Seip congenital lipodystrophy due to seipin as compared with 1-acylglycerol-3-phosphate-o-acyltransferase-2 deficiency.

CONTEXT: Human lipodystrophies are characterized by loss of adipose tissue, insulin resistance, and metabolic complications. The mechanisms linking fat loss to severe insulin resistance remain unclear. Adipokines may have important roles as intermediary players in metabolism. OBJECTIVE: We sought to determine the plasma concentrations of leptin and adiponectin in patients with Berardinelli-Seip congenital lipodystrophy (BSCL) harboring mutations in the genes encoding either 1-acylglycerol-3-phosphate-O-acyltransferase-2 (AGPAT2) or BSCL2/seipin, in comparison with patients with other forms of inherited or acquired lipodystrophies or insulin receptor alterations. DESIGN: Leptin and total and high-molecular-weight adiponectin were measured in plasma of 16 BSCL1/AGPAT2 and 19 BSCL2/seipin patients and compared with heterozygous (n = 22) or nonmutated relatives (controls, n = 30); patients with Dunnigan-type partial lipodystrophy due to lamin A/C mutations (n = 23), HIV-related lipodystrophy (n = 124), and insulin receptor dysfunctions caused by mutations or autoantibodies (n = 17). RESULTS: Leptin was dramatically decreased in BSCL patients as compared with other subgroups. Adiponectin was decreased in BSCL as compared with controls and patients with altered insulin receptor but was discrepant between the two BSCL subgroups. Whereas total and high-molecular-weight adiponectin levels were almost undetectable in BSCL1/AGPAT2 patients, higher levels were detected in BSCL2/seipin patients, comparable with those of patients with partial lipodystrophy. Adiponectin greater than 1.6 mg/liter had a 100% negative predictive value for AGPAT2 mutations in inherited lipodystrophies. CONCLUSIONS: The presence of circulating adiponectin in BSCL2/seipin patients with near absence of adipose tissue outlines the complexity of adiponectin biology. Use of circulating adiponectin might be helpful to guide the genetic investigations in BSCL.

Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6-/- mice.

Elucidation of the metabolic pathways of triacylglycerol (TAG) synthesis is critical to the understanding of chronic metabolic disorders such as obesity, cardiovascular disease, and diabetes. sn-Glycerol-3-phosphate acyltransferase (GPAT) and sn-1-acylglycerol-3-phosphate acyltransferase (AGPAT) catalyze the first and second steps in de novo TAG synthesis. AGPAT6 is one of eight AGPAT isoforms identified through sequence homology, but the enzyme activity for AGPAT6 has not been confirmed. We found that in liver and brown adipose tissue from Agpat6-deficient (Agpat6(-/-)) mice, N-ethylmaleimide (NEM)-sensitive GPAT specific activity was 65% lower than in tissues from wild-type mice, but AGPAT specific activity was similar. Overexpression of Agpat6 in Cos-7 cells increased an NEM-sensitive GPAT specific activity, but AGPAT specific activity was not increased. Agpat6 and Gpat1 overexpression in Cos-7 cells increased the incorporation of [(14)C]oleate into diacylglycerol (DAG) or into DAG and TAG, respectively, suggesting that the lysophosphatidic acid, phosphatidic acid, and DAG intermediates initiated by each of these isoforms lie in different cellular pools. Together, these data show that "Agpat6(-/-) mice" are actually deficient in a novel NEM-sensitive GPAT, GPAT4, and indicate that the alterations in lipid metabolism in adipose tissue, liver, and mammary epithelium of these mice are attributable to the absence of GPAT4.