Search

Members of the carboxylesterase 2 (Ces2/CES2) family have been studied intensively with respect to their hydrolytic function on (pro)drugs, whereas their physiological role in lipid and energy metabolism has been realized only within the last few years. Humans have one CES2 gene which is highly expressed in liver, intestine, and kidney. Interestingly, eight homologous Ces2 (Ces2a to Ces2h) genes exist in mice and the individual roles of the corresponding proteins are incompletely understood. Mouse Ces2c (mCes2c) is suggested as potential ortholog of human CES2. Therefore, we aimed at its structural and biophysical characterization. Here, we present the first crystal structure of mCes2c to 2.12 Å resolution. The overall structure of mCes2c resembles that of the human CES1 (hCES1). The core domain adopts an α/β hydrolase-fold with S230, E347, and H459 forming a catalytic triad. Access to the active site is restricted by the cap, the flexible lid, and the regulatory domain. The conserved gate (M417) and switch (F418) residues might have a function in product release similar as suggested for hCES1. Biophysical characterization confirms that mCes2c is a monomer in solution. Thus, this study broadens our understanding of the mammalian carboxylesterase family and assists in delineating the similarities and differences of the different family members.

Cancer-associated cachexia (CAC) is a wasting syndrome drastically reducing efficacy of chemotherapy and life expectancy of patients. CAC affects up to 80% of cancer patients, yet the mechanisms underlying the disease are not well understood and no approved disease-specific medication exists. As a multiorgan disorder, CAC can only be studied on an organismal level. To cover the diverse aetiologies of CAC, researchers rely on the availability of a multifaceted pool of cancer models with varying degrees of cachexia symptoms. So far, no tumour model syngeneic to C57BL/6 mice exists that allows direct comparison between cachexigenic- and non-cachexigenic tumours.MCA207 and CHX207 fibrosarcoma cells were intramuscularly implanted into male or female, 10-11-week-old C57BL/6J mice. Tumour tissues were subjected to magnetic resonance imaging, immunohistochemical-, and transcriptomic analysis. Mice were analysed for tumour growth, body weight and -composition, food- and water intake, locomotor activity, OCHX207, but not MCA207-tumour-bearing mice exhibited major clinical features of CAC, including systemic inflammation, increased plasma IL-6 concentrations (190 pg/mL, P ≤ 0.0001), increased energy expenditure (+28%, P ≤ 0.01), adipose tissue loss (-47%, P ≤ 0.0001), skeletal muscle wasting (-18%, P ≤ 0.001), and body weight reduction (-13%, P ≤ 0.01) 13 days after cancer cell inoculation. Adipose tissue loss resulted from reduced lipid uptake and -synthesis combined with increased lipolysis but was not associated with elevated beta-adrenergic signalling or adipose tissue browning. Muscle atrophy was evident by reduced myofibre cross sectional area (-21.8%, P ≤ 0.001), increased catabolic- and reduced anabolic signalling. Deletion of IL-6 from CHX207 cancer cells completely protected CHX207In this study, we present CHX207 fibrosarcoma cells as a novel tool to investigate the mediators and metabolic consequences of CAC in C57BL/6 mice in comparison to non-cachectic MCA207-tumour-bearing mice. IL-6 represents an essential trigger for CAC development in CHX207-tumour-bearing mice.

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endolysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and parenchymal and nonparenchymal cells. Tissue samples were obtained from fed, fasted, 2 h refed, and insulin-treated mice, as well as from mice housed at 5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles that were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels, indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue- and cell-type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP plays a role in the functional adaption to nutrient starvation and ambient temperature.

Obesity and diabetes have strong heritable components, yet the genetic contributions to these diseases remain largely unexplained. In humans, a missense variant in Creb3 regulatory factor (CREBRF) [rs373863828 (p.Arg457Gln); CREBRFR457Q] is strongly associated with increased odds of obesity but decreased odds of diabetes. Although virtually nothing is known about CREBRF’s mechanism of action, emerging evidence implicates it in the adaptive transcriptional response to nutritional stress downstream of TORC1. The objectives of this study were to generate a murine model with knockin of the orthologous variant in mice (CREBRFR458Q) and to test the hypothesis that this CREBRF variant promotes obesity and protects against diabetes by regulating energy and glucose homeostasis downstream of TORC1. To test this hypothesis, we performed extensive phenotypic analysis of CREBRFR458Q knockin mice at baseline and in response to acute (fasting/refeeding), chronic (low- and high-fat diet feeding), and extreme (prolonged fasting) nutritional stress as well as with pharmacological TORC1 inhibition, and aging to 52 weeks. The results demonstrate that the murine CREBRFR458Q model of the human CREBRFR457Q variant does not influence energy/glucose homeostasis in response to these interventions, with the exception of possible greater loss of fat relative to lean mass with age. Alternative preclinical models and/or studies in humans will be required to decipher the mechanisms linking this variant to human health and disease.

Aims Lipotoxic cardiomyopathy in diabetic and obese patients typically encompasses increased cardiac fatty acid (FA) uptake eventually surpassing the mitochondrial oxidative capacity. Lowering FA utilization via inhibition of lipolysis represents a strategy to counteract the development of lipotoxic heart dysfunction. However, defective cardiac triacylglycerol (TAG) catabolism and FA oxidation in humans (and mice) carrying mutated ATGL alleles provokes lipotoxic heart dysfunction questioning a therapeutic approach to decrease cardiac lipolysis. Interestingly, decreased lipolysis via cardiac overexpression of Perilipin 5 (Plin5), a binding partner of ATGL, is compatible with normal heart function and lifespan despite massive cardiac lipid accumulation. Herein, we decipher mechanisms that protect Plin5 transgenic mice from the development of heart dysfunction. Methods and results We generated mice with cardiac-specific overexpression of Plin5 encoding a serine-155 to alanine exchange (Plin5-S155A) of the protein kinase A phosphorylation site, which has been suggested as a prerequisite to stimulate lipolysis and may play a crucial role in the preservation of heart function. Plin5-S155A mice showed a substantial increase in cardiac TAG and ceramide levels, which was comparable to mice overexpressing non-mutated Plin5. Lipid accumulation was compatible with normal heart function even under mild stress. Plin5-S155A mice showed reduced cardiac FA oxidation but normal ATP production and changes in the Plin5-S155A phosphoproteome compared to Plin5 transgenic mice. Interestingly, mitochondrial recruitment of dynamin-related protein 1 (Drp1) was markedly reduced in cardiac muscle of Plin5-S155A and Plin5 transgenic mice accompanied by decreased phosphorylation of mitochondrial fission factor, a mitochondrial receptor of Drp1. Conclusions This study suggests that low cardiac lipolysis is associated with reduced mitochondrial fission and may represent a strategy to combat the development of lipotoxic heart dysfunction.

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl–coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue–specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.—Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

Obesity and diabetes have strong heritable components, yet the genetic contributions to these diseases remain largely unexplained. In humans, a missense variant in Creb3 regulatory factor (CREBRF) [rs373863828 (p.Arg457Gln); CREBRFR457Q] is strongly associated with increased odds of obesity but decreased odds of diabetes. Although virtually nothing is known about CREBRF’s mechanism of action, emerging evidence implicates it in the adaptive transcriptional response to nutritional stress downstream of TORC1. The objectives of this study were to generate a murine model with knockin of the orthologous variant in mice (CREBRFR458Q) and to test the hypothesis that this CREBRF variant promotes obesity and protects against diabetes by regulating energy and glucose homeostasis downstream of TORC1. To test this hypothesis, we performed extensive phenotypic analysis of CREBRFR458Q knockin mice at baseline and in response to acute (fasting/refeeding), chronic (low- and high-fat diet feeding), and extreme (prolonged fasting) nutritional stress as well as with pharmacological TORC1 inhibition. The results demonstrate that the murine CREBRFR458Q model of the human CREBRFR457Q variant does not influence energy/glucose homeostasis in response to these interventions. Alternative preclinical models and/or studies in humans will be required to decipher the mechanisms linking this variant to human health and disease.

International audience; In hepatocytes, peroxisome proliferator-activated receptor α (PPARα) orchestrates a genomic and metabolic response required for homeostasis during fasting. This includes the biosynthesis of ketone bodies and of fibroblast growth factor 21 (FGF21). Here we show that in the absence of adipose triglyceride lipase (ATGL) in adipocytes, ketone body and FGF21 production is impaired upon fasting. Liver gene expression analysis highlights a set of fasting-induced genes sensitive to both ATGL deletion in adipocytes and PPARα deletion in hepatocytes. Adipose tissue lipolysis induced by activation of the β3-adrenergic receptor also triggers such PPARα-dependent responses not only in the liver but also in brown adipose tissue (BAT). Intact PPARα activity in hepatocytes is required for the cross-talk between adipose tissues and the liver during fat mobilization.

Triacylglycerol (TG) and steryl ester (SE) lipid storage is a universal strategy to maintain organismal energy and membrane homeostasis. Cycles of building and mobilizing storage fat are fundamental in (re)distributing lipid substrates between tissues or to progress ontogenetic transitions. In this study, we show that Hormone-sensitive lipase (Hsl) specifically controls SE mobilization to initiate intergenerational sterol transfer inDrosophila melanogaster. Tissue-autonomous Hsl functions in the maternal fat body and germline coordinately prevent adult SE overstorage and maximize sterol allocation to embryos. While Hsl-deficiency is largely dispensable for normal development on sterol-rich diets, animals depend on adipocyte Hsl for optimal fecundity when dietary sterol becomes limiting. Notably, accumulation of SE but not of TG is a characteristic of Hsl-deficient cells across phyla including murine white adipocytes. In summary, we identified Hsl as an ancestral regulator of SE degradation, which improves intergenerational sterol transfer and reproductive success in flies.

ObjectiveIn hepatocytes, peroxisome proliferator-activated receptor α (PPARα) acts as a lipid sensor that regulates hepatic lipid catabolism during fasting and orchestrates a genomic response required for whole-body homeostasis. This includes the biosynthesis of ketone bodies and the secretion of the starvation hormone fibroblast growth factor 21 (FGF21). Several lines of evidence suggest that adipose tissue lipolysis contributes to this specific process. However, whether adipose tissue lipolysis is a dominant signal for the extensive remodeling of liver gene expression dependent on PPARα has not been investigated.MethodsFirst, using mice lacking adipose tissue lipolysis through adipocyte-specific deletion of adipose triglyceride lipase (ATGL), we characterized the responses dependent on adipocyte ATGL during fasting. Next, we performed liver whole genome expression analysis in fasted mice upon deletion of adipocyte ATGL or hepatocyte PPARα. Finally, we tested the consequences of hepatocyte-specific PPARα deficiency during pharmacological induction of adipocyte lipolysis with a β3-adrenergic receptor agonist.ResultsIn the absence of ATGL in adipocytes, ketone body and FGF21 productions were impaired in response to starvation. Liver transcriptome analysis revealed that adipocyte ATGL is critical for regulation of hepatic gene expression during fasting and highlighted a strong enrichment in PPARα target genes in this condition. Genome expression analysis confirmed that a large set of fasting-induced genes are sensitive to both ATGL and PPARα. Adipose tissue lipolysis induced by acute activation of the β3-adrenergic receptor also triggered PPARα-dependent responses in the liver, supporting a role for adipocyte-derived fatty acids as dominant signals for hepatocyte PPARα activity. In addition, the absence of hepatocyte PPARα altered brown adipose tissue (BAT) morphology and reduced UCP1 expression upon stimulation of the β3-adrenergic receptor. In agreement with this finding, mice lacking hepatocyte PPARα showed decreased tolerance to acute cold exposure.ConclusionsThese results underscore the central role of hepatocyte PPARα in the sensing of adipocyte-derived fatty acids and reveal that its activity is essential for full activation of BAT. Intact PPARα activity in hepatocytes is required for cross-talk between adipose tissues and the liver during fat mobilization during fasting and cold exposure.

Modulation of adipocyte lipolysis represents an attractive approach to treat metabolic diseases. Lipolysis mainly depends on two enzymes: adipose triglyceride lipase and hormone-sensitive lipase (HSL). Here, we investigated the short- and long-term impact of adipocyte HSL on energy homeostasis using adipocyte-specific HSL knockout (AHKO) mice. AHKO mice fed high-fat-diet (HFD) progressively developed lipodystrophy accompanied by excessive hepatic lipid accumulation. The increased hepatic triglyceride deposition was due to induced de novo lipogenesis driven by increased fatty acid release from adipose tissue during refeeding related to defective insulin signaling in adipose tissue. Remarkably, the fatty liver of HFD-fed AHKO mice reversed with advanced age. The reversal of fatty liver coincided with a pronounced lipodystrophic phenotype leading to blunted lipolytic activity in adipose tissue. Overall, we demonstrate that impaired adipocyte HSL-mediated lipolysis affects systemic energy homeostasis in AHKO mice, whereby with older age, these mice reverse their fatty liver despite advanced lipodystrophy., Pajed et al. investigate the role of adipocyte hormone-sensitive lipase (HSL) in whole body energy homeostasis. They show that adipocyte-specific HSL Knockout (AHKO) mice fed high fat diet develop fatty liver. Interestingly, this phenotype reverses in aged AHKO mice, possibly due to blunted lipolytic activity in adipose tissue with pronounced lipodystrophy.

Background: Tumor cells display metabolic changes that correlate with malignancy, including an elevated hydrolysis of monoacylglycerol (MAG) in various cancer types. However, evidence is absent for the relationship between MAG lipolysis and NSCLC. Methods: MAG hydrolase activity assay, migration, invasion, proliferation, lipids quantification, and transactivation assays were performed in vitro. Tumor xenograft studies and lung metastasis assays were examined in vivo. The correlations of MAGL/ABHD6 expression in cancerous tissues with the clinicopathological characteristics and survival of NSCLC patients were validated. Findings: ABHD6 functions as the primary MAG lipase and an oncogene in NSCLC. MAG hydrolase activities were more than 11-fold higher in cancerous lung tissues than in paired non-cancerous tissues derived from NSCLC patients. ABHD6, instead of MAGL, was significantly associated with advanced tumor node metastasis (TNM) stage (HR, 1.382; P = 0.004) and had a negative impact on the overall survival of NSCLC patients (P = 0.001). ABHD6 silencing reduced migration and invasion of NSCLC cells in vitro as well as metastatic seeding and tumor growth in vivo. Conversely, ectopic overexpression of ABHD6 provoked the pathogenic potential. ABHD6 blockade significantly induced intracellular MAG accumulation which activated PPARα/γ signaling and inhibited cancer pathophysiology. Interpretation: The present study provide evidence for a previously uncovered pro-oncogenic function of ABHD6 in NSCLC, with the outlined metabolic mechanisms shedding light on new potential strategies for anticancer therapy. Fund: This work was supported by the Project for Major New Drug Innovation and Development (2015ZX09501010 and 2018ZX09711001-002-003). Keywords: NSCLC, ABHD6, Aggressiveness, MAG

Murine hepatic carboxylesterase 2c (Ces2c) and the presumed human ortholog carboxylesterase 2 (CES2) have been implicated in the development of nonalcoholic fatty liver disease (NAFLD) in mice and obese humans. These studies demonstrated that Ces2c hydrolyzes triglycerides (TGs) in hepatocytes. Interestingly, Ces2c/CES2 is most abundantly expressed in the intestine, indicating a role of Ces2c/CES2 in intestinal TG metabolism. Here we show that Ces2c is an important enzyme in intestinal lipid metabolism in mice. Intestine-specific Ces2c overexpression (Ces2c(int)) provoked increased fatty acid oxidation (FAO) in the small intestine accompanied by enhanced chylomicron clearance from the circulation. As a consequence, high-fat diet-fed Ces2c(int) mice were resistant to excessive diet-induced weight gain and adipose tissue expansion. Notably, intestinal Ces2c overexpression increased hepatic insulin sensitivity and protected mice from NAFLD development. Although lipid absorption was not affected in Ces2c(int) mice, fecal energy content was significantly increased. Mechanistically, we demonstrate that Ces2c is a potent neutral lipase, which efficiently hydrolyzes TGs and diglycerides (DGs) in the small intestine, thereby generating fatty acids (FAs) for FAO and monoglycerides (MGs) and DGs for potential re-esterification. Consequently, the increased availability of MGs and DGs for re-esterification and primordial apolipoprotcin B(48 )particle lipidation may increase chylomicron size, ultimately mediating more efficient chylomicron clearance from the circulation. Conclusion: This study suggests a critical role for Ces2c in intestinal lipid metabolism and highlights the importance of intestinal lipolysis to protect mice from the development of hepatic insulin resistance, NAFLD, and excessive diet-induced weight gain during metabolic stress.

Aberrant fatty acid (FA) metabolism is a hallmark of proliferating cells, including untransformed fibroblasts or cancer cells. Lipolysis of intracellular triglyceride (TG) stores by adipose triglyceride lipase (ATGL) provides an important source of FAs serving as energy substrates, signaling molecules, and precursors for membrane lipids. To investigate if ATGL-mediated lipolysis impacts cell proliferation, we modified ATGL activity in murine embryonic fibroblasts (MEFs) and in five different cancer cell lines to determine the consequences on cell growth and metabolism. Genetic or pharmacological inhibition of ATGL in MEFs causes impaired FA oxidation, decreased ROS production, and a substrate switch from FA to glucose leading to decreased AMPK-mTOR signaling and higher cell proliferation rates. ATGL expression in these cancer cells is low when compared to MEFs. Additional ATGL knockdown in cancer cells did not significantly affect cellular lipid metabolism or cell proliferation whereas the ectopic overexpression of ATGL increased lipolysis and reduced proliferation. In contrast to ATGL silencing, pharmacological inhibition of ATGL by Atglistatin© impeded the proliferation of diverse cancer cell lines, which points at an ATGL-independent effect. Our data indicate a crucial role of ATGL-mediated lipolysis in the regulation of cell proliferation. The observed low ATGL activity in cancer cells may represent an evolutionary selection process and mechanism to sustain high cell proliferation rates. As the increasing ATGL activity decelerates proliferation of five different cancer cell lines this may represent a novel therapeutic strategy to counteract uncontrolled cell growth., Highlights • ATGL deficiency increases cell proliferation and decreases phosphorylation of AMPK in murine embryonic fibroblasts. • ATGL silencing does not reduce lipolysis or increase proliferation of diverse cancer cells. • Increasing ATGL activity represses cell proliferation in a panel of cancer cells. • Atglistatin© exhibits an ATGL independent tumor suppressive effect.

Metabolic reprogramming is a hallmark of malignancy. In addition to well-established changes in glycolysis and lipogenesis, recent evidence suggests that lipolysis is also implicated in the pathophysiology of cancer. However, mechanistic details remain largely unknown. Here, we show that the monoacylglycerol (MAG) lipase α/β-hydrolase-domain-containing 6 (ABHD6) functions as an oncogene in regulating the aggressiveness of non-small cell lung cancer (NSCLC). Deficiency of ABHD6 limits migration, invasion, metastatic seeding, and tumor growth. Conversely, overexpression of ABHD6 promotes the pathophysiology of NSCLC. ABHD6 deficiency causes accumulation of MAGs, which act as intrinsic cell-autonomous signals that activate PPARα/γ target gene expression and limit cancer pathophysiology. Clinical analyses demonstrate that ABHD6 is highly expressed in NSCLC and significantly associated with poor prognosis of disease progression. Our work suggests that ABHD6 has significant potential as diagnostic marker and therapeutic target in NSCLC. ABHD6 inhibitors and MAG mimetic may represent drug candidates for the treatment of NSCLC.

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl ester (CE) and retinyl ester (RE) and triglyceride (TG). Mice globally lacking LAL accumulate CE most prominently in the liver. The severity of the CE accumulation phenotype progresses with age and is accompanied by hepatomegaly and hepatic cholesterol crystal deposition. In contrast, hepatic TG accumulation is much less pronounced in these mice, and hepatic RE levels are even decreased. To dissect the functional role of LAL for neutral lipid ester mobilization in the liver, we generated mice specifically lacking LAL in hepatocytes (hep-LAL-ko). On a standard chow diet, hep-LAL-ko mice exhibited increased hepatic CE accumulation but unaltered TG and RE levels. Feeding the hep-LAL-ko mice a vitamin A excess/high-fat diet (VitA/HFD) further increased hepatic cholesterol levels, but hepatic TG and RE levels in these mice were lower than in control mice. Performing in vitro activity assays with lysosome-enriched fractions from livers of mice globally lacking LAL, we detected residual acid hydrolytic activities against TG and RE. Interestingly, this non-LAL acid TG hydrolytic activity was elevated in lysosome-enriched fractions from livers of hep-LAL-ko mice upon VitA/HFD feeding. In conclusion, the neutral lipid ester phenotype in livers from hep-LAL-ko mice indicates that LAL is limiting for CE turnover, but not for TG and RE turnovers. Furthermore, in vitro hydrolase activity assays revealed the existence of non-LAL acid hydrolytic activities for TG and RE. The corresponding acid lipase(s) catalyzing these reactions remains to be identified.

Accumulation of myeloid cells in the liver, notably dendritic cells (DCs) and monocytes/macrophages (MCs), is a major component of the metainflammation of obesity. However, the mechanism(s) stimulating hepatic DC/MC infiltration remain ill defined. Herein, we addressed the hypothesis that adipose tissue (AT) free fatty acids (FFAs) play a central role in the initiation of hepatic DC/MC accumulation, using a number of mouse models of altered FFA supply to the liver. In two models of acute FFA elevation (lipid infusion and fasting) hepatic DC/MC and triglycerides (TGs) but not AT DC/MC were increased without altering plasma cytokines (PCs; TNFα and monocyte chemoattractant protein 1) and with variable effects on oxidative stress (OxS) markers. However, fasting in mice with profoundly reduced AT lipolysis (AT-specific deletion of adipose TG lipase; AAKO) failed to elevate liver DC/MC, TG, or PC, but liver OxS increased. Livers of obese AAKO mice that are known to be resistant to steatosis were similarly protected from inflammation. In high-fat feeding studies of 1, 3, 6, or 20-wk duration, liver DC/MC accumulation dissociated from PC and OxS but tracked with liver TGs. Furthermore, decreasing OxS by ~80% in obese mice failed to decrease liver DC/MC. Therefore, FFA and more specifically AT-derived FFA stimulate hepatic DC/MC accumulation, thus recapitulating the pathology of the obese liver. In a number of cases the effects of FFA can be dissociated from OxS and PC but match well with liver TG, a marker of FFA oversupply.

The transient activation of inflammatory networks is required for adipose tissue remodeling including the “browning” of white fat in response to stimuli such as β3-adrenergic receptor activation. In this process, white adipose tissue acquires thermogenic characteristics through the recruitment of so-called beige adipocytes. We investigated the downstream signaling pathways impinging on adipocyte progenitors that promote de novo formation of adipocytes. We showed that the Jak family of kinases controlled TGFβ signaling in the adipose tissue microenvironment through Stat3 and thereby adipogenic commitment, a function that was required for beige adipocyte differentiation of murine and human progenitors. Jak/Stat3 inhibited TGFβ signaling to the transcription factors Srf and Smad3 by repressing local Tgfb3 and Tgfb1 expression before the core transcriptional adipogenic cascade was activated. This pathway cross-talk was triggered in stromal cells by ATGL-dependent adipocyte lipolysis and a transient wave of IL-6 family cytokines at the onset of adipose tissue remodeling induced by β3-adrenergic receptor stimulation. Our results provide insight into the activation of adipocyte progenitors and are relevant for the therapeutic targeting of adipose tissue inflammatory pathways.

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triacylglycerol hydrolysis in virtually all cells, including adipocytes and skeletal myocytes, and hence, plays a critical role in mobilizing fatty acids. Global ATGL deficiency promotes skeletal myopathy and exercise intolerance in mice and humans, and yet the tissue-specific contributions to these phenotypes remain unknown. The goal of this study was to determine the relative contribution of ATGL-mediated triacylglycerol hydrolysis in adipocytes vs. skeletal myocytes to acute exercise performance. To achieve this goal, we generated murine models with adipocyte- and skeletal myocyte-specific targeted deletion of ATGL. We then subjected untrained mice to acute peak and submaximal exercise interventions and assessed exercise performance and energy substrate metabolism. Impaired ATGL-mediated lipolysis within adipocytes reduced peak and submaximal exercise performance, reduced peripheral energy substrate availability, shifted energy substrate preference toward carbohydrate oxidation, and decreased HSL Ser660 phosphorylation and mitochondrial respiration within skeletal muscle. In contrast, impaired ATGL-mediated lipolysis within skeletal myocytes was not sufficient to reduce peak and submaximal exercise performance or peripheral energy substrate availability and instead tended to enhance metabolic flexibility during peak exercise. Furthermore, the expanded intramyocellular triacylglycerol pool in these mice was reduced following exercise in association with preserved HSL phosphorylation, suggesting that HSL may compensate for impaired ATGL action in skeletal muscle during exercise. These data suggest that adipocyte rather than skeletal myocyte ATGL-mediated lipolysis plays a greater role during acute exercise in part because of compensatory mechanisms that maintain lipolysis in muscle, but not adipose tissue, when ATGL is absent.

Summary Elevated circulating fatty acids (FAs) contribute to obesity-associated metabolic complications, but the mechanisms by which insulin suppresses lipolysis are poorly understood. We show that α/β-hydrolase domain-containing 15 (ABHD15) is required for the anti-lipolytic action of insulin in white adipose tissue (WAT). Neither insulin nor glucose treatments can suppress FA mobilization in global and conditional Abhd15-knockout (KO) mice. Accordingly, insulin signaling is impaired in Abhd15-KO adipocytes, as indicated by reduced AKT phosphorylation, glucose uptake, and de novo lipogenesis. In vitro data reveal that ABHD15 associates with and stabilizes phosphodiesterase 3B (PDE3B). Accordingly, PDE3B expression is decreased in the WAT of Abhd15-KO mice, mechanistically explaining increased protein kinase A (PKA) activity, hormone-sensitive lipase (HSL) phosphorylation, and undiminished FA release upon insulin signaling. Ultimately, Abhd15-KO mice develop insulin resistance. Notably, ABHD15 expression is decreased in humans with obesity and diabetes compared to humans with obesity and normal glucose tolerance, identifying ABHD15 as a potential therapeutic target to mitigate insulin resistance.

Intramyocellular triacylglycerol (IMTG) accumulation is highly associated with insulin resistance and metabolic complications of obesity (lipotoxicity), whereas comparable IMTG accumulation in endurance-trained athletes is associated with insulin sensitivity (the athlete’s paradox). Despite these findings, it remains unclear whether changes in IMTG accumulation and metabolism per se influence muscle-specific and systemic metabolic homeostasis and insulin responsiveness. By mediating the rate-limiting step in triacylglycerol hydrolysis, adipose triglyceride lipase (ATGL) has been proposed to influence the storage/production of deleterious as well as essential lipid metabolites. However, the physiological relevance of ATGL-mediated triacylglycerol hydrolysis in skeletal muscle remains unknown. To determine the contribution of IMTG hydrolysis to tissue-specific and systemic metabolic phenotypes in the context of obesity, we generated mice with targeted deletion or transgenic overexpression of ATGL exclusively in skeletal muscle. Despite dramatic changes in IMTG content on both chow and high-fat diets, modulation of ATGL-mediated IMTG hydrolysis did not significantly influence systemic energy, lipid, or glucose homeostasis, nor did it influence insulin responsiveness or mitochondrial function. These data argue against a role for altered IMTG accumulation and lipolysis in muscle insulin resistance and metabolic complications of obesity.

Liver X receptors (LXRs) are transcription factors essential for cholesterol homeostasis and lipogenesis. LXRα has been implicated in regulating hepatic triglyceride (TG) accumulation upon both influx of adipose-derived fatty acids (FAs) during fasting and stimulation of de novo FA synthesis by chemical agonism of LXR. However, whether or not a convergent mechanism is employed to drive deposition of FAs from these 2 different sources in TGs is undetermined. Here, we report that the G0/G1 Switch Gene 2 (G0S2), a selective inhibitor of intracellular TG hydrolysis/lipolysis, is a direct target gene of LXRα. Transcriptional activation is conferred by LXRα binding to a direct repeat 4 (DR4) motif in the G0S2 promoter. While LXRα–/– mice exhibited decreased hepatic G0S2 expression, adenoviral expression of G0S2 was sufficient to restore fasting-induced TG storage and glycogen depletion in the liver of these mice. In response to LXR agonist T0901317, G0S2 ablation prevented hepatic steatosis and hypertriglyceridemia without affecting the beneficial effects on HDL. Thus, the LXRα-G0S2 axis plays a distinct role in regulating hepatic TG during both fasting and pharmacological activation of LXR.

Lipid accumulation is a pathological feature of every type of kidney injury. Despite this striking histological feature, physiological accumulation of lipids in the kidney is poorly understood. We studied whether the accumulation of lipids in the fasted kidney are derived from lipoproteins or NEFAs. With overnight fasting, kidneys accumulated triglyceride, but had reduced levels of ceramide and glycosphingolipid species. Fasting led to a nearly 5-fold increase in kidney uptake of plasma [14C]oleic acid. Increasing circulating NEFAs using a β adrenergic receptor agonist caused a 15-fold greater accumulation of lipid in the kidney, while mice with reduced NEFAs due to adipose tissue deficiency of adipose triglyceride lipase had reduced triglycerides. Cluster of differentiation (Cd)36 mRNA increased 2-fold, and angiopoietin-like 4 (Angptl4), an LPL inhibitor, increased 10-fold. Fasting-induced kidney lipid accumulation was not affected by inhibition of LPL with poloxamer 407 or by use of mice with induced genetic LPL deletion. Despite the increase in CD36 expression with fasting, genetic loss of CD36 did not alter fatty acid uptake or triglyceride accumulation. Our data demonstrate that fasting-induced triglyceride accumulation in the kidney correlates with the plasma concentrations of NEFAs, but is not due to uptake of lipoprotein lipids and does not involve the fatty acid transporter, CD36.

SummaryNumerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate genes involved in energy metabolism and inflammation. For biological activity, PPARs require cognate lipid ligands, heterodimerization with retinoic X receptors, and coactivation by PPAR-gamma coactivator-1alpha or PPAR-gamma coactivator-1beta (PGC-1alpha or PGC-1beta, encoded by Ppargc1a and Ppargc1b, respectively). Here we show that lipolysis of cellular triglycerides by adipose triglyceride lipase (patatin-like phospholipase domain containing protein 2, encoded by Pnpla2; hereafter referred to as Atgl) generates essential mediator(s) involved in the generation of lipid ligands for PPAR activation. Atgl deficiency in mice decreases mRNA levels of PPAR-alpha and PPAR-delta target genes. In the heart, this leads to decreased PGC-1alpha and PGC-1beta expression and severely disrupted mitochondrial substrate oxidation and respiration; this is followed by excessive lipid accumulation, cardiac insufficiency and lethal cardiomyopathy. Reconstituting normal PPAR target gene expression by pharmacological treatment of Atgl-deficient mice with PPAR-alpha agonists completely reverses the mitochondrial defects, restores normal heart function and prevents premature death. These findings reveal a potential treatment for the excessive cardiac lipid accumulation and often-lethal cardiomyopathy in people with neutral lipid storage disease, a disease marked by reduced or absent ATGL activity.

In mammals, excess energy is stored in the form of triacylglycerol primarily in lipid droplets of white adipose tissue. The first step of lipolysis (i.e. the mobilization of fat stores) is catalyzed by adipose triglyceride lipase (ATGL). The enzymatic activity of ATGL is strongly enhanced by CGI-58 (comparative gene identification-58), and the loss of either ATGL or CGI-58 function causes systemic triglyceride accumulation in humans and mice. However, the mechanism by which CGI-58 stimulates ATGL activity is unknown. To gain insight into CGI-58 function using structural features of the protein, we generated a three-dimensional homology model based on sequence similarity with other proteins. Interestingly, the model of CGI-58 revealed that the N terminus forms an extension of the otherwise compact structure of the protein. This N-terminal region (amino acids 1-30) harbors a lipophilic tryptophan-rich stretch, which affects the localization of the protein. (1)H NMR experiments revealed strong interaction between the N-terminal peptide and dodecylphosphocholine micelles as a lipid droplet-mimicking system. A role for this N-terminal region of CGI-58 in lipid droplet binding was further strengthened by localization studies in cultured cells. Although wild-type CGI-58 localizes to the lipid droplet, the N-terminally truncated fragments of CGI-58 are dispersed in the cytoplasm. Moreover, CGI-58 lacking the N-terminal extension loses the ability to stimulate ATGL, implying that the ability of CGI-58 to activate ATGL is linked to correct localization. In summary, our study shows that the N-terminal, Trp-rich region of CGI-58 is essential for correct localization and ATGL-activating function of CGI-58.

FAs are mobilized from triglyceride (TG) stores during exercise to supply the working muscle with energy. Mice deficient for adipose triglyceride lipase (ATGL-ko) exhibit defective lipolysis and accumulate TG in adipose tissue and muscle, suggesting that ATGL deficiency affects energy availability and substrate utilization in working muscle. In this study, we investigated the effect of moderate treadmill exercise on blood energy metabolites and liver glycogen stores in mice lacking ATGL. Because ATGL-ko mice exhibit massive accumulation of TG in the heart and cardiomyopathy, we also investigated a mouse model lacking ATGL in all tissues except cardiac muscle (ATGL-ko/CM). In contrast to ATGL-ko mice, these mice did not accumulate TG in the heart and had normal life expectancy. Exercise experiments revealed that ATGL-ko and ATGL-ko/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. Together, our studies suggest that ATGL-ko mice cannot adjust circulating FA levels to the increased energy requirements of the working muscle, resulting in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required for proper energy supply of the skeletal muscle during exercise.

Adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL) are essential for efficient lipolysis in adipose tissue and skeletal muscle. Herein, we utilized whole body knockout mice to address the importance of ATGL and HSL for metabolic function and exercise performance. ATGL deletion severely disrupts whole-body substrate partitioning at rest; reducing plasma free fatty acid (FFA) availability (WT: 0.49 ± 0.06 vs. ATGL−/− 0.34 ± 0.03 mM), which in turn enhances carbohydrate oxidation during fasting (mean RER, WT: 0.86 ± 0.02, ATGL−/− 0.90 ± 0.01) and is associated with depleted muscle and liver glycogen stores. While plasma FFA was modestly reduced (23%) and whole body carbohydrate metabolism increased in HSL−/− mice, resting glycogen storage was not compromised. Studies in isolated muscles revealed that the capacity of ATGL and HSL−/− muscle to transport exogenous fatty acids is not compromised and the capacity to oxidize fatty acids is actually increased (3.7- and 1.3-fold above WT for ATGL and HSL). The exercise-induced increase in plasma FFA and glycerol was blunted with ATGL or HSL deletion, demonstrating an impaired capacity for exercise-induced lipolysis in these mice. Carbohydrate oxidation was increased concomitantly during exercise in ATGL−/− and HSL−/− mice, resulting in more muscle and liver glycogen depletion. Maximal running velocity and endurance capacity were reduced by 42% and 46% in ATGL−/− mice, but not in HSL−/− mice. The reduction in performance in ATGL−/− mice was not due to defective muscle contractile performance. These results demonstrate an essential role for both ATGL and HSL in maintaining adequate FFA supply to sustain normal substrate metabolism at rest and during exercise.

Emerging evidence suggests that impaired regulation of adipocyte lipolysis contributes to the proinflammatory immune cell infiltration of metabolic tissues in obesity, a process that is proposed to contribute to the development and exacerbation of insulin resistance. To test this hypothesis in vivo, we generated mice with adipocyte-specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme catalyzing triacylglycerol hydrolysis. In contrast to previous models, adiponectin-driven Cre expression was used for targeted ATGL deletion. The resulting adipocyte-specific ATGL knockout (AAKO) mice were then characterized for metabolic and immune phenotypes. Lean and diet-induced obese AAKO mice had reduced adipocyte lipolysis, serum lipids, systemic lipid oxidation, and expression of peroxisome proliferator-activated receptor alpha target genes in adipose tissue (AT) and liver. These changes did not increase overall body weight or fat mass in AAKO mice by 24 weeks of age, in part due to reduced expression of genes involved in lipid uptake, synthesis, and adipogenesis. Systemic glucose and insulin tolerance were improved in AAKO mice, primarily due to enhanced hepatic insulin signaling, which was accompanied by marked reduction in diet-induced hepatic steatosis as well as hepatic immune cell infiltration and activation. In contrast, although adipocyte ATGL deletion reduced AT immune cell infiltration in response to an acute lipolytic stimulus, it was not sufficient to ameliorate, and may even exacerbate, chronic inflammatory changes that occur in AT in response to diet-induced obesity.

Summary Adipose triglyceride lipase (ATGL) was recently identified as an important triacylglycerol (TG) hydrolase promoting the catabolism of stored fat in adipose and nonadipose tissues. We now demonstrate that efficient ATGL enzyme activity requires activation by CGI-58. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman Syndrome (CDS), a rare genetic disease where TG accumulates excessively in multiple tissues. CGI-58 interacts with ATGL, stimulating its TG hydrolase activity up to 20-fold. Alleles of CGI-58 carrying point mutations associated with CDS fail to activate ATGL. Moreover, CGI-58/ATGL coexpression attenuates lipid accumulation in COS-7 cells. Antisense RNA-mediated reduction of CGI-58 expression in 3T3-L1 adipocytes inhibits TG mobilization. Finally, expression of functional CGI-58 in CDS fibroblasts restores lipolysis and reverses the abnormal TG accumulation typical for CDS. These data establish an important biochemical function for CGI-58 in the lipolytic degradation of fat, implicating this lipolysis activator in the pathogenesis of CDS.

Mobilization of fatty acids from triglyceride stores in adipose tissue requires lipolytic enzymes. Dysfunctional lipolysis affects energy homeostasis and may contribute to the pathogenesis of obesity and insulin resistance. Until now, hormone-sensitive lipase (HSL) was the only enzyme known to hydrolyze triglycerides in mammalian adipose tissue. Here, we report that a second enzyme, adipose triglyceride lipase (ATGL), catalyzes the initial step in triglyceride hydrolysis. It is interesting that ATGL contains a “patatin domain” common to plant acyl-hydrolases. ATGL is highly expressed in adipose tissue of mice and humans. It exhibits high substrate specificity for triacylglycerol and is associated with lipid droplets. Inhibition of ATGL markedly decreases total adipose acyl-hydrolase activity. Thus, ATGL and HSL coordinately catabolize stored triglycerides in adipose tissue of mammals.

Summary Fatty acids (FAs) activate and fuel UCP1-mediated non-shivering thermogenesis (NST) in brown adipose tissue (BAT). Release of FAs from intracellular fat stores by adipose triglyceride lipase (ATGL) is considered a key step in NST. Accordingly, the severe cold intolerance of global ATGL knockout (AKO) mice has been attributed to defective BAT lipolysis. Here we show that this conclusion is incorrect. We demonstrate that although the BAT-specific loss of ATGL impairs BAT lipolysis and alters BAT morphology, it does not compromise the β3-adrenergic thermogenic response or cold-induced NST. Instead, NST depends on nutrient supply or lipolysis in white adipose tissue during fasting, suggesting that circulating energy substrates are sufficient to fuel NST. Cold intolerance in AKO mice is not caused by BAT dysfunction as previously suspected but by severe cardiomyopathy. We conclude that functional NST requires adequate substrate supply and cardiac function, but does not depend on ATGL-mediated lipolysis in BAT., Graphical Abstract, Highlights • ATGL in BAT is not a prerequisite for cold-induced thermogenesis in vivo • ATGL in WAT is crucial to liberate fatty acids for energy combustion during fasting • Loss of ATGL in the heart induces hypothermia due to insufficient cardiac reserve • Adaptive thermogenesis is normal in mice lacking ATGL in BAT, Schreiber et al. demonstrate that ATGL-mediated lipolysis in brown adipose tissue is not a prerequisite for cold-induced non-shivering thermogenesis in vivo. Instead, ATGL in white adipose tissue and the heart is essential for supplying fatty acids during fasting and for meeting increased demands on the cardiovascular system in the cold, respectively.