Search

BACKGROUND AND AIMS: Increased fatty acid (FA) flux from adipose tissue to the liver contributes to the development of NAFLD. Because free FAs are key lipotoxic triggers accelerating disease progression, inhibiting adipose triglyceride lipase (ATGL)/patatin-like phospholipase domain containing 2 (PNPLA2), the main enzyme driving lipolysis, may attenuate steatohepatitis. APPROACH AND RESULTS: Hepatocyte-specific ATGL knockout (ATGL LKO) mice were challenged with methionine-choline-deficient (MCD) or high-fat high-carbohydrate (HFHC) diet. Serum biochemistry, hepatic lipid content and liver histology were assessed. Mechanistically, hepatic gene and protein expression of lipid metabolism, inflammation, fibrosis, apoptosis, and endoplasmic reticulum (ER) stress markers were investigated. DNA binding activity for peroxisome proliferator-activated receptor (PPAR) α and PPARδ was measured. After short hairpin RNA-mediated ATGL knockdown, HepG2 cells were treated with lipopolysaccharide (LPS) or oleic acid:palmitic acid 2:1 (OP21) to explore the direct role of ATGL in inflammation in vitro. On MCD and HFHC challenge, ATGL LKO mice showed reduced PPARα and increased PPARδ DNA binding activity when compared with challenged wild-type (WT) mice. Despite histologically and biochemically pronounced hepatic steatosis, dietary-challenged ATGL LKO mice showed lower hepatic inflammation, reflected by the reduced number of Galectin3/MAC-2 and myeloperoxidase-positive cells and low mRNA expression levels of inflammatory markers (such as IL-1β and F4/80) when compared with WT mice. In line with this, protein levels of the ER stress markers protein kinase R-like endoplasmic reticulum kinase and inositol-requiring enzyme 1α were reduced in ATGL LKO mice fed with MCD diet. Accordingly, pretreatment of LPS-treated HepG2 cells with the PPARδ agonist GW0742 suppressed mRNA expression of inflammatory markers. Additionally, ATGL knockdown in HepG2 cells attenuated LPS/OP21-induced expression of proinflammatory cytokines and chemokines such as chemokine (C-X-C motif) ligand 5, chemokine (C-C motif) ligand (Ccl) 2, and Ccl5. CONCLUSIONS: Low hepatic lipolysis and increased PPARδ activity in ATGL/PNPLA2 deficiency may counteract hepatic inflammation and ER stress despite increased steatosis. Therefore, lowering hepatocyte lipolysis through ATGL inhibition represents a promising therapeutic strategy for the treatment of steatohepatitis. ispartof: HEPATOLOGY vol:75 issue:1 pages:125-139 ispartof: location:United States status: published

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.

Catecholamines stimulate the first step of lipolysis by PKA-dependent release of the lipid droplet-associated protein ABHD5 from perilipin to co-activate the lipase ATGL. Here, we unmask a yet unrecognized proteolytic and cardioprotective function of ABHD5. ABHD5 acts in vivo and in vitro as a serine protease cleaving HDAC4. Through the production of an N-terminal polypeptide of HDAC4 (HDAC4-NT), ABHD5 inhibits MEF2-dependent gene expression and thereby controls glucose handling. ABHD5-deficiency leads to neutral lipid storage disease in mice. Cardiac-specific gene therapy of HDAC4-NT does not protect from intra-cardiomyocyte lipid accumulation but strikingly from heart failure, thereby challenging the concept of lipotoxicity-induced heart failure. ABHD5 levels are reduced in failing human hearts and murine transgenic ABHD5 expression protects from pressure-overload induced heart failure. These findings represent a conceptual advance by connecting lipid with glucose metabolism through HDAC4 proteolysis and enable new translational approaches to treat cardiometabolic disease.

Disturbances in lipid homeostasis can cause mitochondrial dysfunction and lipotoxicity. Perilipin 5 (PLIN5) decorates intracellular lipid droplets (LDs) in oxidative tissues and controls triacylglycerol (TG) turnover via its interactions with adipose triglyceride lipase and the adipose triglyceride lipase coactivator, comparative gene identification-58. Furthermore, PLIN5 anchors mitochondria to the LD membrane via the outermost part of the carboxyl terminus. However, the role of this LD-mitochondria coupling (LDMC) in cellular energy catabolism is less established. In this study, we investigated the impact of PLIN5-mediated LDMC in comparison to disrupted LDMC on cellular TG homeostasis, FA oxidation, mitochondrial respiration, and protein interaction. To do so, we established PLIN5 mutants deficient in LDMC whilst maintaining normal interactions with key lipolytic players. Radiotracer studies with cell lines stably overexpressing wild-type or truncated PLIN5 revealed that LDMC has no significant impact on FA esterification upon lipid loading or TG catabolism during stimulated lipolysis. Moreover, we demonstrated that LDMC exerts a minor if any role in mitochondrial FA oxidation. In contrast, LDMC significantly improved the mitochondrial respiratory capacity and metabolic flexibility of lipid-challenged cardiomyocytes, which was corroborated by LDMC-dependent interactions of PLIN5 with mitochondrial proteins involved in mitochondrial respiration, dynamics, and cristae organization. Taken together, this study suggests that PLIN5 preserves mitochondrial function by adjusting FA supply via the regulation of TG hydrolysis and that LDMC is a vital part of mitochondrial integrity.

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.

Enterocytes of the small intestine (SI) play an important role in maintaining systemic lipid levels by regulating dietary lipid absorption and postprandial lipoprotein secretion. An excessive amount of dietary-derived triglycerides (TGs) taken up by the apical side of enterocytes or basolaterally internalized lipoprotein remnants can be transiently stored in cytosolic lipid droplets (cLDs). As mice lacking adipose TG lipase (ATGL) in the SI display massive accumulation of cLDs but also delayed cholesterol absorption, we hypothesized that SI-specific overexpression of ATGL (Atgl iTg) might have beneficial effects on lipid homeostasis in the gut and possibly throughout the body. Here, we demonstrate that Atgl iTg mice had only modestly increased enzymatic activity despite drastically elevated Atgl mRNA levels (up to 120-fold) on chow diet, and was highly induced upon high-fat/high-cholesterol diet (HF/HCD) feeding. Atgl iTg mice showed markedly reduced intestinal TG concentrations after acute and chronic lipid challenge without affecting chylomicron TG secretion. Circulating plasma cholesterol levels were significantly lower in Atgl iTg mice under different feeding conditions, contrasting the accelerated uptake of dietary cholesterol into the circulation after HF/HCD feeding. In the fasted state, gene expression analysis revealed modulation of PPARα and liver X receptor (LXR) target genes by an increased fatty acid release, whereas the decreased plasma cholesterol concentrations in refed mice were more likely due to changes in HDL synthesis and secretion. We conclude that ATGL, in addition to its role in TG catabolism, plays a critical role in whole-body cholesterol homeostasis by modulating PPARα and LXR signaling in intestinal enterocytes.

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.

Physical contacts between organelles play a pivotal role in intracellular trafficking of metabolites. Monitoring organelle interactions in living cells using fluorescence microscopy is a powerful approach to functionally assess these cellular processes. However, detailed target acquisition is typically limited due to light diffraction. Furthermore, subcellular compartments such as lipid droplets and mitochondria are highly dynamic and show significant subcellular movement. Thus, high-speed acquisition of these organelles with extended-resolution is appreciated. Here, we present an imaging informatics pipeline enabling spatial and time-resolved analysis of the dynamics and interactions of fluorescently labeled lipid droplets and mitochondria in a fibroblast cell line. The imaging concept is based on multispectral confocal laser scanning microscopy and includes high-speed resonant scanning for fast spatial acquisition of organelles. Extended-resolution is achieved by the recording of images at minimized pinhole size and by post-processing of generated data using a computational image restoration method. Computation of inter-organelle contacts is performed on basis of segmented spatial image data. We show limitations of the image restoration and segmentation part of the imaging informatics pipeline. Since both image processing methods are implemented in other related methodologies, our findings will help to identify artifacts and the false-interpretation of obtained morphometric data. As a proof-of-principle, we studied how lipid load and overexpression of PLIN5, considered to be involved in the tethering of LDs and mitochondria, affects organelle association.

We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.

SummaryAdipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) are critical regulators of triacylglycerol (TAG) turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG) and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes.

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa−/−) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa−/− mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa−/− mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa−/− mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.

Excessive accumulation of triacylglycerol is the common denominator of a wide range of clinical pathologies of liver diseases, termed non-alcoholic fatty liver disease. Such excessive triacylglycerol deposition in the liver is also referred to as hepatic steatosis. Although liver steatosis often resolves over time, it eventually progresses to steatohepatitis, liver fibrosis and cirrhosis, with associated complications, including liver failure, hepatocellular carcinoma and ultimately death of affected individuals. From the disease etiology it is obvious that a tight regulation between lipid uptake, triacylglycerol synthesis, hydrolysis, secretion and fatty acid oxidation is required to prevent triacylglycerol deposition in the liver. In addition to triacylglycerol, also a tight control of other neutral lipid ester classes, i.e. cholesteryl esters and retinyl esters, is crucial for the maintenance of a healthy liver. Excessive cholesteryl ester accumulation is a hallmark of cholesteryl ester storage disease or Wolman disease, which is associated with premature death. The loss of hepatic vitamin A stores (retinyl ester stores of hepatic stellate cells) is incidental to the onset of liver fibrosis. Importantly, this more advanced stage of liver disease usually does not resolve but progresses to life threatening stages, i.e. liver cirrhosis and cancer. Therefore, understanding the enzymes and pathways that mobilize hepatic neutral lipid esters is crucial for the development of strategies and therapies to ameliorate pathophysiological conditions associated with derangements of hepatic neutral lipid ester stores, including liver steatosis, steatohepatitis, and fibrosis. This review highlights the physiological roles of enzymes governing the mobilization of neutral lipid esters at different sites in liver cells, including cytosolic lipid droplets, endoplasmic reticulum, and lysosomes. This article is part of a Special Issue entitled Molecular Basis of Disease: Animal models in liver disease.

In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, WAT undergoes profound changes; it expands, becomes dysfunctional, and develops a low-grade inflammatory state. Importantly, BAT content and activity decline in obese subjects, mainly as a result of the conversion of brown adipocytes to white-like unilocular cells. Here, we show that BAT “whitening” is induced by multiple factors, including high ambient temperature, leptin receptor deficiency, β-adrenergic signaling impairment, and lipase deficiency, each of which is capable of inducing macrophage infiltration, brown adipocyte death, and crown-like structure (CLS) formation. Brown-to-white conversion and increased CLS formation were most marked in BAT from adipose triglyceride lipase (Atgl)-deficient mice, where, according to transmission electron microscopy, whitened brown adipocytes contained enlarged endoplasmic reticulum, cholesterol crystals, and some degenerating mitochondria, and were surrounded by an increased number of collagen fibrils. Gene expression analysis showed that BAT whitening in Atgl-deficient mice was associated to a strong inflammatory response and NLRP3 inflammasome activation. Altogether, the present findings suggest that converted enlarged brown adipocytes are highly prone to death, which, by promoting inflammation in whitened BAT, may contribute to the typical inflammatory state seen in obesity.

Keratinocyte differentiation is essential for skin development and the formation of the skin permeability barrier. This process involves an orchestrated remodeling of lipids. The cleavage of precursor lipids from lamellar bodies by β-glucocerebrosidase, sphingomyelinase, phospholipases and sterol sulfatase generates ceramides, non-esterified fatty acids and cholesterol for the lipid-containing extracellular matrix, the lamellar membranes in the stratum corneum. The importance of triacylglycerol (TAG) hydrolysis for the formation of a functional permeability barrier was only recently appreciated. Mice with defects in TAG synthesis (acyl-CoA:diacylglycerol acyltransferase-2-knock-out) or TAG catabolism (comparative gene identification-58, -CGI-58-knock-out) develop severe permeability barrier defects and die soon after birth because of desiccation. In humans, mutations in the CGI-58 gene also cause (non-lethal) neutral lipid storage disease with ichthyosis. As a result of defective TAG synthesis or catabolism, humans and mice lack ω-(O)-acylceramides, which are essential lipid precursors for the formation of the corneocyte lipid envelope. This structure plays an important role in linking the lipid-enriched lamellar membranes to highly cross-linked corneocyte proteins. This review focuses on the current knowledge of biochemical mechanisms that are essential for epidermal neutral lipid metabolism and the formation of a functional skin permeability barrier.

We showed earlier that nutritional stress like starvation or high-fat diet resulted in phenotypic changes in the lipidomes of hepatocyte lipid droplets (LDs), representative for the pathophysiological status of the mouse model. Here we extend our former study by adding genetic stress due to knockout (KO) of adipocyte triglyceride lipase (ATGL), the rate limiting enzyme in LD lipolysis. An intervention trial for 6 weeks with male wild-type (WT) and ATGL-KO mice was carried out; both genotypes were fed lab chow or were exposed to short-time starvation. Isolated LDs were analyzed by LC-MS/MS. Triacylglycerol, diacylglycerol, and phosphatidylcholine lipidomes, in that order, provided the best phenotypic signatures characteristic for respective stresses applied to the animals. This was evidenced at lipid species level by principal component analysis, calculation of average values for chain-lengths and numbers of double bonds, and by visualization in heat maps. Structural backgrounds for analyses and metabolic relationships were elaborated at lipid molecular species level. Relating our lipidomic data to nonalcoholic fatty liver diseases of nutritional and genetic etiologies with or without accompanying insulin resistance, phenotypic distinction in hepatocyte LDs dependent on insulin status emerged. Taken together, lipidomes of hepatocyte LDs are sensitive responders to nutritional and genetic stress.

Although diabetic cardiomyopathy is associated with enhanced intramyocardial triacylglycerol (TAG) levels, the role of TAG catabolizing enzymes in this process is unclear. Because the TAG hydrolase, adipose triglyceride lipase (ATGL), regulates baseline cardiac metabolism and function, we examined whether alterations in cardiomyocyte ATGL impact cardiac function during uncontrolled type 1 diabetes. In genetic (Akita) and pharmacological (streptozotocin) murine models of type 1 diabetes, cardiac ATGL protein expression and TAG content were significantly increased. To determine whether increased ATGL expression during diabetes is detrimental or beneficial to cardiac function, we studied streptozotocin-diabetic mice with heterozygous ATGL deficiency and cardiomyocyte-specific ATGL overexpression. After diabetes, streptozotocin-diabetic mice with heterozygous ATGL deficiency displayed increased TAG accumulation, lipotoxicity, and diastolic dysfunction comparable to wild-type mice. In contrast, myosin heavy chain promoter (MHC)-ATGL mice were resistant to diabetes-induced increases in intramyocardial TAG levels, lipotoxicity, and cardiac dysfunction. Moreover, hearts from diabetic MHC-ATGL mice exhibited decreased reliance on palmitate oxidation and blunted peroxisome proliferator--activated receptor-α activation. Collectively, this study shows that after diabetes, increased cardiac ATGL expression is an adaptive, albeit insufficient, response to compensate for the accumulation of myocardial TAG, and that overexpression of ATGL is sufficient to ameliorate diabetes-induced cardiomyopathy.

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular degradation of triacylglycerols. It was the aim of this study to elucidate how the deficiency in one of these proteins affects the residual lipolytic proteome in adipose tissue. For this purpose, we compared the lipase patterns of brown and white adipose tissue from ATGL (-/-) and HSL (-/-) mice using differential activity-based gel electrophoresis. This method is based on activity-recognition probes possessing the same substrate analogous structure but carrying different fluorophores for specific detection of the enzyme patterns of two different tissues in one electrophoresis gel. We found that ATGL-deficiency in brown adipose tissue had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in brown adipose tissue. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in white adipose tissue. Enzyme activities of mouse tissues on acylglycerol substrates were analyzed as well, showing that ATGL-and HSL-deficiencies can be compensated for at least in part by other enzymes. The proteins that responded to ATGL-deficiency in brown adipose tissue were overexpressed and their activities on acylglycerols were analyzed. Among these enzymes, Es1, Es10, and Es31-like represent lipase candidates as they catalyze the hydrolysis of long-chain acylglycerols.

Mutations in PNPLA1 have been identified as causative for autosomal recessive congenital ichthyosis in humans and dogs. So far, the underlying molecular mechanisms are unknown. In this study, we generated and characterized PNPLA1-deficient mice and found that PNPLA1 is crucial for epidermal sphingolipid synthesis. The absence of functional PNPLA1 in mice impaired the formation of omega-O-acylceramides and led to an accumulation of nonesterified omega-hydroxy-ceramides. As a consequence, PNPLA1-deficient mice lacked a functional corneocyte-bound lipid envelope leading to a severe skin barrier defect and premature death of newborn animals. Functional analyses of differentiated keratinocytes from a patient with mutated PNPLA1 demonstrated an identical defect in omega-O-acylceramide synthesis in human cells, indicating that PNPLA1 function is conserved among mammals and indispensable for normal skin physiology. Notably, topical application of epidermal lipids from wild-type onto Pnpla1-mutant mice promoted rebuilding of the corneocyte-bound lipid envelope, indicating that supplementation of ichthyotic skin with omega-O-acylceramides might be a therapeutic approach for the treatment of skin symptoms in individuals affected by omega-O-acylceramide deficiency.

Adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) are limiting in cellular triglyceride catabolism. Although ATGL deficiency is compatible with normal skin development, mice globally lacking CGI-58 die postnatally and exhibit a severe epidermal permeability barrier defect, which may originate from epidermal and/or peripheral changes in lipid and energy metabolism. Here, we show that epidermis-specific disruption of CGI-58 is sufficient to provoke a defect in the formation of a functional corneocyte lipid envelope linked to impaired ω-O-acylceramide synthesis. As a result, epidermis-specific CGI-58-deficient mice show severe skin dysfunction, arguing for a tissue autonomous cause of disease development. Defective skin permeability barrier formation in global CGI-58-deficient mice could be reversed via transgenic restoration of CGI-58 expression in differentiated but not basal keratinocytes suggesting that CGI-58 is essential for lipid metabolism in suprabasal epidermal layers. The compatibility of ATGL deficiency with normal epidermal function indicated that CGI-58 may stimulate an epidermal triglyceride lipase beyond ATGL required for the adequate provision of fatty acids as a substrate for ω-O-acylceramide synthesis. Pharmacological inhibition of ATGL enzyme activity similarly reduced triglyceride-hydrolytic activities in wild-type and CGI-58 overexpressing epidermis implicating that CGI-58 participates in ω-O-acylceramide biogenesis independent of its role as a coactivator of epidermal triglyceride catabolism.

The heart predominantly utilizes fatty acids (FAs) as energy substrate. FAs that enter cardiomyocytes can be activated and directly oxidized within mitochondria (and peroxisomes) or they can be esterified and intracellularly deposited as triacylglycerol (TAG) often simply referred to as fat. An increase in cardiac TAG can be a signature of the diseased heart and may implicate a minor role of TAG synthesis and breakdown in normal cardiac energy metabolism. Often overlooked, the heart has an extremely high TAG turnover and the transient deposition of FAs within the cardiac TAG pool critically determines the availability of FAs as energy substrate and signaling molecules. We herein review the recent literature regarding the enzymes and co-regulators involved in cardiomyocyte TAG synthesis and catabolism and discuss the interconnection of these metabolic pathways in the normal and diseased heart. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.

Nonalcoholic fatty liver disease (NAFLD) is characterized by triglyceride (TG) accumulation and endoplasmic reticulum (ER) stress. Because fatty acids (FAs) may trigger ER stress, we hypothesized that the absence of adipose triglyceride lipase (ATGL/PNPLA2)–the main enzyme for intracellular lipolysis, releasing FAs, and closest homolog to adiponutrin (PNPLA3) recently implicated in the pathogenesis of NAFLD–protects against hepatic ER stress. Wild-type (WT) and ATGL knockout (KO) mice were challenged with tunicamycin (TM) to induce ER stress. Serum biochemistry, hepatic TG and FA profiles, liver histology, and gene expression for markers of hepatic lipid metabolism, ER stress, and inflammation were explored. Moreover, cell-culture experiments were performed in Hepa1.6 cells after the knockdown of ATGL before FA and TM treatment. TM increased hepatic TG accumulation in ATGL KO, but not in WT, mice. Lipogenesis and β-oxidation were repressed at the gene-expression level (sterol regulatory element-binding transcription factor 1c, fatty acid synthase, acetyl coenzyme A carboxylase 2, and carnitine palmitoyltransferase 1 alpha) in both WT and ATGL KO mice. Genes for very-low-density lipoprotein (VLDL) synthesis (microsomal triglyceride transfer protein and apolipoprotein B) were down-regulated by TM in WT and even more in ATGL KO mice, which displayed strongly reduced serum VLDL cholesterol levels. Notably, ER stress markers glucose-regulated protein, C/EBP homolog protein, spliced X-box-binding protein, endoplasmic-reticulum–localized DnaJ homolog 4, and inflammatory markers Tnfα and iNos were induced exclusively in TM-treated WT, but not ATGL KO, mice. Total hepatic FA profiling revealed a higher palmitic acid/oleic acid (PA/OA) ratio in WT mice, compared to ATGL KO mice, at baseline. Phosphoinositide-3-kinase inhibitor–known to be involved in FA-derived ER stress and blocked by OA–was increased in TM-treated WT mice only. In line with this, in vitro OA protected hepatocytes from TM-induced ER stress. Conclusions: Lack of ATGL may protect from hepatic ER stress through alterations in FA composition. ATGL could constitute a new therapeutic strategy to target ER stress in NAFLD. (HEPATOLOGY 2012;56:270–280 )

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.

BACKGROUND AND PURPOSE Adipose triglyceride lipase (ATGL) has been identified as a rate-limiting enzyme of mammalian triglyceride catabolism. Deletion of the ATGL gene in mice results in severe lipid accumulation in a variety of tissues including the heart. In the present study we investigated cardiac function in ATGL-deficient mice and the potential therapeutic effects of the PPARα and γ agonists Wy14,643 and rosiglitazone, respectively. EXPERIMENTAL APPROACH Hearts isolated from wild-type (WT) mice and ATGL(-/-) mice treated with Wy14,643 (PPARα agonist), rosiglitazone (PPARγ agonist) or vehicle were perfused at a constant flow using the Langendorff technique. Left ventricular (LV) pressure–volume relationships were established, and the response to adrenergic stimulation was determined with noradrenaline (NA). KEY RESULTS Hearts from ATGL(-/-) mice generated higher LV end-diastolic pressure and lower LV developed pressure as a function of intracardiac balloon volume compared to those from WT mice. Likewise, passive wall stress was increased and active wall stress decreased in ATGL(-/-) hearts. Contractile and microvascular responses to NA were substantially reduced in ATGL(-/-) hearts. Cardiac contractility was improved by treating ATGL(-/-) mice with the PPARα agonist Wy14,643 but not with the PPARγ agonist rosiglitazone. CONCLUSIONS AND IMPLICATIONS Our results indicate that lipid accumulation in mouse hearts caused by ATGL gene deletion severely affects systolic and diastolic function, as well as the response to adrenergic stimulation. The beneficial effects of Wy14,643 suggest that the cardiac phenotype of these mice is partially due to impaired PPARα signalling.

Lipolysis is defined as the catabolism of triacylglycerols stored in cellular lipid droplets. Recent discoveries of essential lipolytic enzymes and characterization of numerous regulatory proteins and mechanisms have fundamentally changed our perception of lipolysis and its impact on cellular metabolism. New findings that lipolytic products and intermediates participate in cellular signaling processes and that "lipolytic signaling" is particularly important in many nonadipose tissues unveil a previously underappreciated aspect of lipolysis, which may be relevant for human disease.

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.

Growth hormone (GH)-activated signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid (GC)-responsive glucocorticoid receptor (GR) are important signal integrators in the liver during metabolic and physiologic stress. Their deregulation has been implicated in the development of metabolic liver diseases, such as steatosis and progression to fibrosis. Using liver-specific STAT5 and GR knockout mice, we addressed their role in metabolism and liver cancer onset. STAT5 single and STAT5/GR double mutants developed steatosis, but only double-mutant mice progressed to liver cancer. Mechanistically, STAT5 deficiency led to the up-regulation of prolipogenic sterol regulatory element binding protein 1 (SREBP-1) and peroxisome proliferator activated receptor gamma (PPAR-γ) signaling. Combined loss of STAT5/GR resulted in GH resistance and hypercortisolism. The combination of both induced expression of adipose tissue lipases, adipose tissue lipid mobilization, and lipid flux to the liver, thereby aggravating STAT5-dependent steatosis. The metabolic dysfunctions in STAT5/GR compound knockout animals led to the development of hepatic dysplasia at 9 months of age. At 12 months, 35% of STAT5/GR-deficient livers harbored dysplastic nodules and ∼60% hepatocellular carcinomas (HCCs). HCC development was associated with GH and insulin resistance, enhanced tumor necrosis factor alpha (TNF-α) expression, high reactive oxygen species levels, and augmented liver and DNA damage parameters. Moreover, activation of the c-Jun N-terminal kinase 1 (JNK1) and STAT3 was prominent. Conclusion Hepatic STAT5/GR signaling is crucial for the maintenance of systemic lipid homeostasis. Impairment of both signaling cascades causes severe metabolic liver disease and promotes spontaneous hepatic tumorigenesis. (hepatology 2011;54:1398–1409)

Objective— The consequences of macrophage triglyceride (TG) accumulation on atherosclerosis have not been studied in detail so far. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for the initial step in TG hydrolysis. Because ATGL knockout (KO) mice exhibit massive TG accumulation in macrophages, we used ATGL KO mice to study the effects of macrophage TG accumulation on atherogenesis. Methods and Results— Low-density lipoprotein receptor (LDLr) KO mice were transplanted with bone marrow from ATGL KO (ATGL KO→LDLr KO) or wild-type (WT→LDLr KO) mice and challenged with a Western-type diet for 9 weeks. Despite TG accumulation in ATGL KO macrophages, atherosclerosis in ATGL KO→LDLr KO mice was 43% reduced associated with decreased plasma monocyte chemoattractant protein-1 (MCP-1) and macrophage interleukin-6 concentrations. This coincided with a reduced amount of macrophages, possibly because of a 39% increase in intraplaque apoptosis and a decreased migratory capacity of ATGL KO macrophages. The reduced number of white blood cells might be due to a 36% decreased Lin − Sca-1 + cKit + hematopoietic stem cell population. Conclusion— We conclude that the attenuation of atherogenesis in ATGL KO→LDLr KO mice is due to decreased infiltration of less inflammatory macrophages into the arterial wall and increased macrophage apoptosis.

Obesity elicits an immune response characterized by myeloid cell recruitment to key metabolic organs, including adipose tissue. However, the response of immune cells to nonpathologic metabolic stimuli has been less well studied, and the factors that regulate the metabolic-dependent accumulation of immune cells are incompletely understood. Here we characterized the response of adipose tissue macrophages (ATMs) to weight loss and fasting in mice and identified a role for lipolysis in ATM recruitment and accumulation. We found that the immune response to weight loss was dynamic; caloric restriction of high-fat diet–fed mice led to an initial increase in ATM recruitment, whereas ATM content decreased following an extended period of weight loss. The peak in ATM number coincided with the peak in the circulating concentrations of FFA and adipose tissue lipolysis, suggesting that lipolysis drives ATM accumulation. Indeed, fasting or pharmacologically induced lipolysis rapidly increased ATM accumulation, adipose tissue chemoattractant activity, and lipid uptake by ATMs. Conversely, dietary and genetic manipulations that reduced lipolysis decreased ATM accumulation. Depletion of macrophages in adipose tissue cultures increased expression of adipose triglyceride lipase and genes regulated by FFA, and increased lipolysis. These data suggest that local lipid fluxes are central regulators of ATM recruitment and that once recruited, ATMs form lipid-laden macrophages that can buffer local increases in lipid concentration.

Macrophage phagocytosis is an essential biological process in host defense and requires large amounts of energy. To date, glucose is believed to represent the prime substrate for ATP production in macrophages. To investigate the relative contribution of free fatty acids (FFAs) in this process, we determined the phagocytosis rates in normal mouse macrophages and macrophages of adipose triglyceride lipase (ATGL)-deficient mice. ATGL was shown to be the rate-limiting enzyme for the hydrolysis of lipid droplet-associated triacylglycerol (TG) in many tissues. Here, we demonstrate that Atgl(-/-) macrophages fail to efficiently hydrolyze cellular TG stores leading to decreased cellular FFA concentrations and concomitant accumulation of lipid droplets, even in the absence of exogenous lipid loading. The reduced availability of FFAs results in decreased cellular ATP concentrations and impaired phagocytosis suggesting that fatty acids must first go through a cycle of esterification and re-hydrolysis before they are available as energy substrate. Exogenously added glucose cannot fully compensate for the phagocytotic defect in Atgl(-/-) macrophages. Hence, phagocytosis was also decreased in vivo when Atgl(-/-) mice were challenged with bacterial particles. These findings imply that phagocytosis in macrophages depends on the availability of FFAs and that ATGL is required for their hydrolytic release from cellular TG stores. This novel mechanism links ATGL-mediated lipolysis to macrophage function in host defense and opens the way to explore possible roles of ATGL in immune response, inflammation, and atherosclerosis.

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.