Adipose Triglyceride Lipase Knockout Mice Research Articles

Adipose Triglyceride Lipase Deficiency Aggravates Angiotensin II–Induced Atrial Fibrillation by Reducing Peroxisome Proliferator–Activated Receptor α Activation in Mice

Atrial fibrillation (AF) is a main risk factor for cerebrovascular diseases but lacks precision therapy. Adipose triglyceride lipase (ATGL) is a key enzyme involved in the intracellular degradation of triacylglycerol and plays an important role in lipid and energy metabolism. However, the role of ATGL in the regulation of AF remains unclear. In this study, AF was induced by infusion of angiotensin II (Ang II, 2000 ng/kg/min) for 3 weeks in male ATGL knockout (KO) mice and age-matched C57BL/6 wild-type mice. The atrial volume was measured by echocardiography. Atrial fibrosis, inflammatory cells, and superoxide production were detected by histologic examinations. The results showed that ATGL expression was significantly downregulated in the atrial tissue of the Ang II–infused mice. Moreover, Ang II–induced increase in the inducibility and duration of AF, atrial dilation, fibrosis, inflammation, and oxidative stress in wild-type mice were markedly accelerated in ATGL KO mice; however, these effects were dramatically reversed in the ATGL KO mice administered with peroxisome proliferator–activated receptor (PPAR)-α agonist clofibric acid. Mechanistically, Ang II downregulated ATGL expression and inhibited PPAR-α activity, activated multiple signaling pathways (inhibiting kappa B kinase α/β-nuclear factor-κB, nicotinamide adenine dinucleotide phosphate oxidase, and transforming growth factor-β1/SMAD2/3) and reducing Kv1.5, Cx40, and Cx43 expression, thereby contributing to atrial structural and electrical remodeling and subsequent AF. In summary, our results indicate that ATGL KO enhances AF inducibility, possibly through inhibiting PPAR-α activation and suggest that activating ATGL might be a new therapeutic option for treating hypertensive AF.

Abstract 11856: Characterization of Endothelial Adipose Triglyceride Lipase in a Preclinical Model of Heart Failure With Preserved Ejection Fraction

Background: Metabolic syndrome and endothelial dysfunction are identified as key drivers of Heart Failure with preserved Ejection Fraction. Endothelial cells have an important barrier function including the control of lipid transport from the coronary circulation to the myocardium. In order to determine the role of EC lipid metabolism in HFpEF, we generated mice with an EC-specific deletion of adipose triglyceride lipase and subjected these mice to a hypertensive-obese two-hit HFpEF model. Methods and Results: Cell culture experiments were performed in HUVECS with a pharmacological ATGL inhibitor NG-497. In vitro experiments showed significant reduction of lipolysis by NG-497 and marked endothelial lipid droplet accumulation. Inducible endothelial cell-specific ATGL knockout mice were generated by crossbreeding Atgl fl/fl mice with Cdh5(PAC)-CreERT2 mice. Animals were randomized in a HFpEF or control group. HFpEF mice received a high fat diet and L-NAME for 15 weeks, while animals in the control group were put on a low fat diet and water. To analyse cardiac function, conventional and speckle-tracking echocardiography were performed. After 15 weeks, cre - littermates (wt) with HFpEF developed obesity and hypertension (body weight: wt-Ctrl: 33.8±2.5g vs. wt-HFpEF: 45.1±4.4g, p=0.0001; systolic blood pressure: wt-Ctrl: 99.9±3.7mmHg vs. wt-HFpEF: 135.5±5.6mmHg, p=0.0001), which did not significantly differ in ecATGL-KO mice. In parallel, wt-HFpEF mice developed echocardiographic signs of HFpEF including diastolic dysfunction: E/é: (wt-Ctrl: -22.1±1.4 vs. wt-HFpEF: -45.5±3.8; p<0.0001), IVRT: (wt-Ctrl: 17.8±0.7ms vs. wt-HFpEF: 19.5±0.8ms; p= p<0.0001), and an impaired global longitudinal strain (GLS) (wt-Ctrl: -20.7±1.1% vs. wt-HFpEF 12.9±0.7%; p<0.0001). Parameters of diastolic dysfunction were significantly improved in ecATGLKO mice after 15weeks of HFD/ L-NAME: E/é:-29.3±2.8; p<0.0001 vs. wt-HFpEF; IVRT: 18.3±0.5; p<0.05 vs. wt-HFpEF; GLS: -19.1±1.7; p<0.0001 vs. wt-HFpEF). Conclusion: This study demonstrates that loss of ATGL in ECs may protect from HFpEF. We hypothesize that lipid droplet accumulation in coronary ATGL-deficient ECs may save the myocardium from lipid overload and lipotoxicity, as shown in Cre- HFpEF mice.

ATGL deficiency aggravates pressure overload-triggered myocardial hypertrophic remodeling associated with the proteasome-PTEN-mTOR-autophagy pathway.

Persistent myocardial hypertrophy frequently leads to heart failure (HF). Intramyocardial triacylglycerol (TAG) accumulation is closely related with cardiac remodeling and abnormal contractile function. Adipose triglyceride lipase (ATGL), a key enzyme in TAG metabolism, regulates cardiac function. However, its associated molecular pathways have not been fully defined. Here, cardiac hypertrophy and HF were induced in wild-type (WT) or ATGL knockout (KO) mice through transverse aortic constriction (TAC) for up to 4weeks. TAC in WT mice significantly reduced cardiac function and autophagy while enhancing left ventricular hypertrophy, interstitial fibrosis, inflammatory response, superoxide generation, and cardiomyocyte apoptosis, accompanied with upregulation of the proteasome activity, reduction of PTEN level and activation of AKT-mTOR signaling, and these effects were further aggravated in ATGL KO mice. Interestingly, ATGL KO-mediated cardiac dysfunction and remodeling were markedly reversed by proteasome inhibitor (epoxomicin) or autophagic activator (rapamycin), but accelerated by PTEN inhibitor (VO-OHpic) or autophagy inhibitor 3-MA. Mechanistically, ATGL KO upregulated proteasome expression and activity, which in turn mediates PTEN degradation leading to activation of AKT-mTOR signaling and inhibition of autophagy, thereby enhancing hypertrophic remodeling and HF. In conclusion, ATGL KO contributes to TAC-induced cardiac dysfunction and adverse remodeling probably associated with the proteasome-PTEN-mTOR-autophagy pathway. Therefore, modulation of this pathway may have a therapeutic effect potential for hypertrophic heart disease. TAC-induced downregulation of ATGL results in increased proteasome (β1i/β2i/β5i) activity, which in turn promotes degradation of PTEN and activation of AKT-mTOR signaling and then inhibits autophagy and ATP production, thereby leading to cardiac hypertrophic remodeling and dysfunction. Conversely, blocking proteasome activity or activating autophagy attenuates these effects.

Deletion of adipose triglyceride lipase abolishes blood flow increase after β3-adrenergic stimulation in visceral adipose tissue of mice.

Dynamic changes in adipose tissue blood flow (ATBF) with nutritional status play a role in the regulation of metabolic and endocrine functions. Activation of the sympathetic nervous system via β-adrenergic receptors (β-AR) contributes to the control of postprandial enhancement of ATBF. Herein, we sought to identify the role of each β-AR subtype in the regulation of ATBF in mice. We monitored the changes in visceral epididymal ATBF (VAT BF), induced by local infusion of dobutamine, salbutamol, and CL316,243 (a selective β1-, β2-, and β3-AR agonist, respectively) into VAT of lean CD-1 mice and global adipose triglyceride lipase (ATGL) knockout (KO) mice, using laser Doppler flowmetry. Administration of CL316,243, known to promote lipolysis in adipocytes, significantly increased VAT BF of CD-1 mice to a greater extent compared to that of the vehicle, whereas administration of dobutamine or salbutamol did not produce significant differences in VAT BF. The increase in VAT BF induced by β3-AR stimulation disappeared in ATGL KO mice as opposed to their wild-type (WT) littermates, implying a role of ATGL-mediated lipolysis in the regulation of VAT BF. Different vascular reactivities occurred despite no significant differences in vessel density and adiposity between the groups. Additionally, the expression levels of the angiogenesis-related genes were significantly higher in VAT of ATGL KO mice than in that of WT, implicating an association of ATBF responsiveness with angiogenic activity in VAT. Our findings suggest a potential role of β3-AR signaling in the regulation of VAT BF via ATGL-mediated lipolysis in mice.

Outside-in signaling by femoral cuff injury induces a distinct vascular lesion in adipose triglyceride lipase knockout mice.

Genetic deficiency of adipose triglyceride lipase (ATGL), a rate-limiting enzyme for intracellular triglyceride (TG) hydrolysis, causes TG-deposit cardiomyovasculopathy (TGCV), a recently identified rare cardiovascular disorder (ORPHA code: 565612) in humans. One of the major characteristics of TGCV is a novel type of diffuse and concentric coronary atherosclerosis with ATGL-deficient smooth muscle cells (SMCs). Patients with TGCV have intractable coronary artery disease. Therefore, it is crucial to investigate the mechanisms underlying vascular lesions in ATGL deficiency using animal models. Cuff injury is an experimental procedure to induce vascular remodeling with neointimal formation with SMCs after placing a cuff around the adventitial side of the artery without direct influence on endothelium. We report the effect of cuff injury on femoral arteries of ATGL-knockout (ATGL⁻/⁻) mice. Cuff-induced concentric neointimal formation with migrating SMCs was exacerbated in ATGL⁻/⁻ mice, mimicking atherosclerotic lesions in patients with TGCV. In the media, cell death of SMCs and loss of elastic fibers increased. Perivascular infiltrating cells expressing tumor necrosis factor-α (TNF-α) were more prominent in ATGL⁻/⁻ mice than in wild-type (WT) mice. In Boyden chamber experiments, a greater number of ATGL⁻/⁻ SMCs migrated in response to TNF-α compared to WT SMCs. These data, for the first time, demonstrated that outside-in signaling by cuff-induced neointimal formation where paracrine stimuli from adventitial infiltrating cells may lead to neointimal formation and mediolysis in ATGL-deficient conditions. Cuff injury might be a valuable model for understanding the mechanisms underlying the development of atherosclerotic lesions in patients with TGCV.

Polymethoxyselenoflavones exert anti-obesity effects through activation of lipolysis and brown adipocyte metabolism.

Polymethoxyselenoflavone (PMSF) is a compound that substitutes the oxygen atom in a flavonoid with selenium. This study aimed to investigate the effects of PMSFs on lipid metabolism in adipocytes and their anti-obesity potential. To test lipolytic and thermogenic effects of the compounds in vitro, adipocytes differentiated from immortalized pre-brown adipocyte progenitors and pre-white adipocyte cell lines were treated with 19 PMSFs. The expression levels of brown adipocyte markers and genes related to mitochondrial metabolism were analyzed by qPCR and western blot. In vivo anti-obesity effect was investigated using diet-induced obesity mouse models and adipocyte-specific ATGL knockout mice. The qPCR analysis identified 2-(3,4-dimethoxyphenyl)-4H-selenochromen-4-one (DMPSC) as the most potent brown adipogenic candidate among the 19 compounds tested in this study. DMPSC treatment significantly increased the mitochondrial content and oxidative metabolism in adipocytes in vitro. Mechanistically, DMPSC treatment increased lipolysis through activation of PKA downstream signaling. Consistently, the in vivo treatment of DMPSC increased energy consumption, reduced body weight, and improved glucose tolerance in mice fed with high-fat diets. Moreover, DMPSC treatment increased brown adipocyte marker expression and mitochondrial content in adipose tissue of mice. The anti-obesity effects were absent in adipocyte-specific ATGL knockout mice, indicating that the DMPSC effect is mediated by cytosolic lipase-dependent mechanisms. Collectively, our results indicated that DMPSC exerted anti-obesity effects partially through the PKA signaling-mediated activation of lipolysis and brown adipose tissue metabolism.

Tricaprin Rescues Myocardial Abnormality in a Mouse Model of Triglyceride Deposit Cardiomyovasculopathy.

Triglyceride deposit cardiomyovasculopathy (TGCV) is an intractable cardiovascular disease for which a specific treatment is urgently required. In TGCV, adipose triglyceride lipase (ATGL) deficiency results in the abnormal intracellular metabolism of long-chain fatty acid (LCFA) which leads to TG deposition. Medium-chain triglycerides have been used as an important functional food for various human diseases. To address the potential activities of tricaprin, a medium-chain triglyceride, on cardiac dysfunctions of TGCV, we examined the effects of tricaprin diet on Atgl knock out (KO) mice, an animal model for TGCV. Cardiac imaging tests showed that the tricaprin diet reduced TG accumulation, resulting from improvement of LCFA metabolism, and improved left ventricular function in Atgl KO mice compared to that in mice fed the control diet. In conclusion, tricaprin improved myocardial abnormality in the TGCV model, thus, it may be useful for the treatment of patients with TGCV.

Cold-Induced Thermogenesis Depends on ATGL-Mediated Lipolysis in Cardiac Muscle, but Not Brown Adipose Tissue

SummaryFatty 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.

Adipose triglyceride lipase (ATGL) deletion from adipocytes, but not skeletal myocytes, impairs acute exercise performance in mice

Fatty acids (FA) and glucose are essential energy substrates that supply exercising muscle. While the selection of substrates is highly influenced by their availability and the intensity and duration of exercise, for prolonged exercise FA are the key substrate. Adipose Triglyceride Lipase (ATGL) is the rate‐limiting enzyme mediating intracellular triacylglycerol hydrolysis and should play a key role in the mobilization of FA during exercise. However, the contribution of adipose versus skeletal muscle fatty acid mobilization to support exercise remains unclear. To gain insight, we generated adipocyte or skeletal myocyte‐specific ATGL knockout mice and determined their response to acute peak and submaximal exercise.We found that adipocyte ATGL‐mediated lipolysis was crucial to support exercise performance, likely owing to limited FA availability and reliance on a rapidly depleted carbohydrate store. Impaired skeletal muscle ATGL‐mediated lipolysis did not impact exercise tolerance and these mice exhibited improved metabolic flexibility during peak exercise.This suggests that adipocyte rather than skeletal myocyte ATGL‐mediated lipolysis plays a greater role during acute peak and submaximal endurance exercise due to limits in substrate availability and greater reliance on carbohydrate oxidation. While further investigation is needed, this suggests the role of IMCL is more of a stop‐gap substrate store to bridge carbohydrate use and FA mobilization and delivery from adipocytes to working muscle.Supported by NIH R01DK090166, HHMI Physician‐Scientist Early Career Award; NIH T32 DK007052

Downregulation of adipose triglyceride lipase promotes cardiomyocyte hypertrophy by triggering the accumulation of ceramides

Adipose triglyceride lipase (ATGL), the rate-limiting enzyme of triglyceride (TG) hydrolysis, plays an important role in TG metabolism. ATGL knockout mice suffer from TG accumulation and die from heart failure. However, the mechanisms underlying cardiac hypertrophy caused by ATGL dysfunction remain unknown. In this study, we found that ATGL expression declined in pressure overload-induced cardiac hypertrophy in vivo and phenylephrine (PE)-induced cardiomyocyte hypertrophy in vitro. ATGL knockdown led to cardiomyocyte hypertrophy, while ATGL overexpression prevented PE-induced hypertrophy. In addition, ATGL downregulation increased but ATGL overexpression reduced the contents of ceramide, which has been proved to be closely associated with cardiac hypertrophy. Moreover, the accumulation of ceramide was due to elevation of free fatty acids in ATGL-knockdown cardiomyocytes, which could be explained by the reduced activity of peroxisome proliferator-activated receptor (PPAR) α leading to imbalance of fatty acid uptake and oxidation. These observations suggest that downregulation of ATGL causes the decreased PPARα activity which results in the imbalance of FA uptake and oxidation, elevating intracellular FFA contents to promote the accumulation of ceramides, and finally inducing cardiac hypertrophy. Upregulation of ATGL could be a strategy for ameliorating lipotoxic damage in cardiac hypertrophy.

Endothelial dysfunction in adipose triglyceride lipase deficiency

Systemic knockout of adipose triglyceride lipase (ATGL), the pivotal enzyme of triglyceride lipolysis, results in a murine phenotype that is characterized by progredient cardiac steatosis and severe heart failure. Since cardiac and vascular dysfunction have been closely related in numerous studies we investigated endothelium-dependent and -independent vessel function of ATGL knockout mice. Aortic relaxation studies and Langendorff perfusion experiments of isolated hearts showed that ATGL knockout mice suffer from pronounced micro- and macrovascular endothelial dysfunction. Experiments with agonists directly targeting vascular smooth muscle cells revealed the functional integrity of the smooth muscle cell layer. Loss of vascular reactivity was restored ~50% upon treatment of ATGL knockout mice with the PPARα agonist Wy14,643, indicating that this phenomenon is partly a consequence of impaired cardiac contractility. Biochemical analysis revealed that aortic endothelial NO synthase expression and activity were significantly reduced in ATGL deficiency. Enzyme activity was fully restored in ATGL mice treated with the PPARα agonist. Biochemical analysis of perivascular adipose tissue demonstrated that ATGL knockout mice suffer from perivascular inflammatory oxidative stress which occurs independent of cardiac dysfunction and might contribute to vascular defects. Our results reveal a hitherto unrecognized link between disturbed lipid metabolism, obesity and cardiovascular disease.

Endothelial dysfunction in adipose triglyceride lipase deficiency: role of perivascular adipose tissue

Background Perivascular adipose tissue (PVAT) has been shown to be an important modulator of vascular function through release of both relaxing and contracting factors. However, the involvement of PVAT in development of endothelial dysfunction is not well understood, yet. We have recently demonstrated that mice lacking adipose triglyceride lipase (ATGL) suffer from severe endothelial dysfunction. Since vessels of these mice are coated with large amounts of PVAT, we speculated that this might potentially contribute to disturbed vascular homeostasis. Therefore, PVAT of wild type (WT) and ATGL knockout mice was characteri-zed in terms of inflammatory as well as oxidative stress. Additionally, we wanted to distinguish between

Absence of adipose triglyceride lipase protects from hepatic endoplasmic reticulum stress in mice

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. 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.

Resveratrol regulates lipolysis via adipose triglyceride lipase

Resveratrol has been reported to increase adrenaline-induced lipolysis in 3T3-L1 adipocytes. The general aim of the present work was to gain more insight concerning the effects of trans-resveratrol on lipid mobilization. The specific purpose was to assess the involvement of the two main lipases: adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in the activation of lipolysis induced by this molecule. For lipolysis experiments, 3T3-L1 and human SGBS adipocytes as well as adipose tissue from wild-type, ATGL knockout and HSL knockout mice were used. Moreover, gene and protein expressions of these lipases were analyzed. Resveratrol-induced free fatty acids release but not glycerol release in 3T3-L1 under basal and isoproterenol-stimulating conditions and under isoproterenol-stimulating conditions in SGBS adipocytes. When HSL was blocked by compound 76-0079, free fatty acid release was still induced by resveratrol. By contrast, in the presence of the compound C, an inhibitor of adenosine monophosphate-activated protein kinase, resveratrol effect was totally blunted. Resveratrol increased ATGL gene and protein expressions, an effect that was not observed for HSL. Resveratrol increased fatty acids release in epididymal adipose tissue from wild-type and HSL knockout mice but not in that adipose tissue from ATGL knockout mice. Taking as a whole, the present results provide novel evidence that resveratrol regulates lipolytic activity in human and murine adipocytes, as well as in white adipose tissue from mice, acting mainly on ATGL at transcriptional and posttranscriptional levels. Enzyme activation seems to be induced via adenosine monophosphate-activated protein kinase.

Macrophage Adipose Triglyceride Lipase Deficiency Attenuates Atherosclerotic Lesion Development in Low-Density Lipoprotein Receptor Knockout Mice

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. 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. 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.