Knockdown Of Adipose Triglyceride Lipase Research Articles

The Combined Inhibition of Autophagy and Diacylglycerol Acyltransferase-Mediated Lipid Droplet Biogenesis Induces Cancer Cell Death during Acute Amino Acid Starvation.

Lipid droplets (LDs) are dynamic organelles involved in the management of fatty acid trafficking and metabolism. Recent studies suggest that autophagy and LDs serve complementary roles in the protection against nutrient stress, but the autophagy-LD interplay in cancer cells is not well understood. Here, we examined the relationship between autophagy and LDs in starving HeLa cervical cancer- and MDA-MB-231 breast cancer cells. We found that acute amino acid depletion induces autophagy and promotes diacylglycerol acyltransferase 1 (DGAT1)-mediated LD accumulation in HeLa cells. Inhibition of autophagy via late-stage autophagy inhibitors, or by knocking down autophagy-related 5 (ATG5), reduced LD accumulation in amino acid-starved cancer cells, suggesting that autophagy contributes to LD biogenesis. On the contrary, knockdown of adipose triglyceride lipase (ATGL) increased LD accumulation, suggesting that LD breakdown is mediated by lipolysis under these conditions. Concurrent inhibition of autophagy by silencing ATG5 and of LD biogenesis using DGAT inhibitors was effective in killing starving HeLa cells, whereas cell survival was not compromised by suppression of ATGL-mediated lipolysis. Autophagy-dependent LD biogenesis was also observed in the aggressive triple-negative MDA-MB-231 breast cancer cells deprived of amino acids, but these cells were not sensitized to starvation by the combined inhibition of LD biogenesis and autophagy. These findings reveal that while targeting autophagy-driven and DGAT-mediated LD biogenesis reduces the resilience of HeLa cervical cancer cells to amino acid deprivation, this strategy may not be successful in other cancer cell types.

Hepatocyte-specific deletion of adipose triglyceride lipase (adipose triglyceride lipase/patatin-like phospholipase domain containing 2) ameliorates dietary induced steatohepatitis in mice.

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. 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. 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.

A Role for Lipid Metabolism in Tyrosine Kinase Inhibitor (TKI) Resistance of Chronic Myeloid Leukemia (CML)

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are remarkably effective therapies in chronic myeloid leukemia (CML). Despite success, TKIs do not target the CML leukemic stem cell (LSC), meaning patients must be treated for life at a high economic burden and often with significant side effects. Our previous work demonstrated a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML, which is profoundly downregulated in TKI resistance (>3-fold, p<0.02) (Gonzalez MA, et al. Cancer Res 2020 #648). Low G0S2 expression not only correlated with TKI resistance, but also with a shorter overall survival in chronic phase (p=0.036), and transformation from the chronic to the blast phase of disease (p<0.05). G0S2 is known to regulate apoptosis, quiescence, lipid metabolism, oxidative phosphorylation, and also acts as an inhibitor of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in intracellular lipolysis. We hypothesized that G0S2-mediated ATGL inhibition underlies its tumor suppressor activity in CML and TKI resistance.To address this hypothesis, we first assessed ATGL protein expression in CML cell lines (K562 and KU812) in the presence and absence of the BCR-ABL TKI, imatinib. Importantly, ATGL protein expression remained unchanged in graded doses of imatinib, suggesting that ATGL expression occurs in a BCR-ABL1-independent manner. To assess the functional role of ATGL in CML and TKI resistance, we performed shRNA-mediated ATGL knockdown (shATGL) in both cell lines. Although ectopic G0S2 impaired survival of CML cell lines and patient samples, we observed a different phenotype upon ATGL knockdown. Rather, shATGL alone increased colony formation by ~30% in both cell lines in the absence but not presence of imatinib (K562, p=0.04; KU812, p=0.0024). Knockdown of ATGL had no effect on colony formation of normal cord blood CD34 + progenitors (p=0.742). Interestingly, when we ectopically expressed G0S2 into K562 cells with simultaneous ATGL knockdown, the phenotype in colony formation assays mimicked ectopic G0S2 expression, reducing survival by ~50% (p=0.05). These data suggest that the tumor suppressor role of G0S2 in CML is independent of ATGL. However, while ectopic G0S2 expression alone had no effect on apoptosis of CML cell lines, when combined with ATGL knockdown, imatinib-mediated apoptosis was markedly increased. This was similar to data observed in blast phase CML patient samples upon ectopic G0S2 expression. Consistently, RNA sequencing (RNAseq) data for CML patients revealed that ATGL mRNA is highly downregulated in blast phase (n=14) compared with patients in the chronic (n=52) or accelerated (n=11) phases of the disease (p<0.0001). These data suggest that ATGL abrogates G0S2-mediated apoptosis in the presence of imatinib. We next performed colony formation assays of K562 cells with G0S2 knockdown (shG0S2) versus a non-targeting control (shNT) in the absence and presence of an ATGL inhibitor (Atglistatin, 50 µM) and a fatty acid oxidation (FAO) inhibitor (TMZ, 10 µM). Atglistatin had no effect on colony formation in the shNT-expressing cells, but reduced survival by ~40% upon G0S2 knockdown. Similarly, TMZ reduced survival of shNT cells by ~30%, while it reduced survival by ~90% upon G0S2 knockdown. These data suggest that the phenotypic effects of G0S2 knockdown in TKI resistance in part requires FAO.The effect of G0S2 on lipid metabolism was further confirmed with RNAseq and metabolomics/lipidomics analyses. RNAseq revealed no overlapping pathways between K562 cells expressing ectopic G0S2 versus shATGL. Lipidomics analyses revealed that G0S2 knockdown reduced expression of tri- and di-glycerides, whereas ectopic G0S2 promoted triglyceride accumulation in K562 cells. G0S2 knockdown also resulted in substantial changes of phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Finally, metabolomics data implicated a role for G0S2 as a negative regulator of the mitochondrial electron transport chain. Altogether, these findings suggest that G0S2 and lipid metabolism play a role in regulating leukemic stem and progenitor cell survival as well as TKI response in vitro. Therefore, restoring G0S2 expression and inhibiting FAO, combined with BCR-ABL1 inhibition, may be a novel clinical strategy to induce treatment-free remission in CML patients and eradicate the CML LSC. DisclosuresNo relevant conflicts of interest to declare.

Adipose triglyceride lipase promotes the proliferation of colorectal cancer cells via enhancing the lipolytic pathway.

Abnormal lipid metabolism is the sign of tumour cells. Previous researches have revealed that the lipolytic pathway may contribute to the progression of colorectal cancer (CRC). However, adipose triglyceride lipase (ATGL) role in CRC cells remains unclear. Here, we find that elevated ATGL positively correlates with CRC clinical stages and negatively associates with overall survival. Overexpression of ATGL significantly promotes CRC cell proliferation, while knockdown of ATGL inhibits the proliferation and promotes the apoptosis of CRC cells in vitro. Moreover, in vivo experiments, ATGL promotes the growth of CRC cells. Mechanistically, ATGL enhances the carcinogenic function of CRC cells via promoting sphingolipid metabolism and CoA biosynthesis pathway‐related gene levels by degrading triglycerides, which provides adequate nutrition for the progression of CRC. Our researches clarify for the first time that ATGL is a novel oncogene in CRC and may provide an important prognostic factor and therapeutic target for CRC.

ATGL activity regulates GLUT1-mediated glucose uptake and lactate production via TXNIP stability in adipocytes

Traditionally, lipolysis has been regarded as an enzymatic activity that liberates fatty acids as metabolic fuel. However, recent work has shown that novel substrates, including a variety of lipid compounds such as fatty acids and their derivatives, release lipolysis products that act as signaling molecules and transcriptional modulators. While these studies have expanded the role of lipolysis, the mechanisms underpinning lipolysis signaling are not fully defined. Here, we uncover a new mechanism regulating glucose uptake, whereby activation of lipolysis, in response to elevated cAMP, leads to the stimulation of thioredoxin-interacting protein (TXNIP) degradation. This, in turn, selectively induces glucose transporter 1 surface localization and glucose uptake in 3T3-L1 adipocytes and increases lactate production. Interestingly, cAMP-induced glucose uptake via degradation of TXNIP is largely dependent upon adipose triglyceride lipase (ATGL) and not hormone-sensitive lipase or monoacylglycerol lipase. Pharmacological inhibition or knockdown of ATGL alone prevents cAMP-dependent TXNIP degradation and thus significantly decreases glucose uptake and lactate secretion. Conversely, overexpression of ATGL amplifies the cAMP response, yielding increased glucose uptake and lactate production. Similarly, knockdown of TXNIP elicits enhanced basal glucose uptake and lactate secretion, and increased cAMP further amplifies this phenotype. Overexpression of TXNIP reduces basal and cAMP-stimulated glucose uptake and lactate secretion. As a proof of concept, we replicated these findings in human primary adipocytes and observed TXNIP degradation and increased glucose uptake and lactate secretion upon elevated cAMP signaling. Taken together, our results suggest a crosstalk between ATGL-mediated lipolysis and glucose uptake.

Adipose triglyceride lipase activity regulates cancer cell proliferation via AMP-kinase and mTOR signaling

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.

Sequential lipolysis and lipophagy pathways orchestrate lipid droplet breakdown in hepatocytes

Neutral lipids are a critical source for cellular energy and are stored within specialized organelles known as lipid droplets (LD). Degradation of LDs for energy utilization is accomplished by two principal pathways: lipolysis that is activated by cAMP to recruit adipose triglyceride lipase (ATGL) to the LD surface, and lipophagy that utilizes the autophagic machinery to target and traffic LDs for degradation by lysosomal acid lipase (LAL). However, the mechanisms underlying synergy between these seemingly distinct pathways are not well understood. Previous studies show that autpohagic membrane cannot effectively engulf large organelles, but is size‐restricted to small cargo. Based on this, we HYPOTHESIZE that lipolysis and lipophagy are sequential pathways whereby lipolysis first reduces the size of large LDs to allow for subsequent autophagic engulfment of small LDs for lipophagy.MethodsUsing light and electron microscopy combined with biochemical approaches, we explored LD size and the sequential nature of lipolysis and lipophagy in primary rat and AML12 hepatocytes. Live‐cell microscopy was used for the first time to monitor LDs transitioning from lipolysis in the cytoplasm to lipophagy within endolysosomal vesicles.ResultsConfocal microscopy of endogenous ATGL revealed a preferential association with large LDs with an average size of 4.0 μm2, seven‐fold larger than LDs not associated with ATGL. In striking comparison, LDs associated with components of the autophagic machinery (LC3, Lamp3) were 50% smaller in size (0.39 μm2). Consistent with this observation, siRNA knockdown of ATGL resulted in the persistence of large LDs (2.8 μm2), whereas knockdown of LAL left cells filled with numerous small LDs (1.5 μm2). These observations were mimicked by inhibitors of ATGL (atglistatin) and LAL (lalistat). In support of these findings, differential centrifugation of AML12 homogenates allowed for separation of two distinct populations of LDs: a buoyant fraction of large LDs enriched in the cytosolic lipase ATGL, and a second collection of smaller, less buoyant LDs enriched in lysosomal enzymes such as Lamp2A, the autophagosome marker LC3‐II, and the endosomal marker Tsg101. Most interesting are time lapse movies of live cells showing engulfment of small LDs by a Lamp3‐positive late endosomal compartment, suggesting that these organelles play an important role in the direct targeting and engulfment of LDs for lipophagy.ConclusionHepatocytes appear to catabolize LDs in a tandem stepwise process that first utilizes cytosolic lipases to substantially reduce LD size, thereby allowing for subsequent engulfment of small LDs by autophagosomes as well as late endosomes and lysosomes.Support or Funding InformationSupported by NIAAA RO1AA020735 to MAM; NIDDK award T32DK007352 to MBS and RJS, and the Mayo Clinic Kogod Aging Fellowship to MBS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipid Droplet Size Determines Cooperation of Lipolysis and Lipophagy in Hepatocytes.

Fatty liver disease affects 80–100 million Americans and is a precursor to irreversible and sometimes fatal liver damage. The initial stages of fatty liver disease are marked by the hepatocellular accumulation of fat‐storage organelles known as lipid droplets (LDs) that are degraded by two pathways: lipolysis that involves the recruitment of cytosolic lipases (ATGL, HSL) to the LD surface, and lipophagy that relies on autophagic membranes to target and traffic LDs for lysosomal degradation. Importantly, the mechanisms underlying a cooperation between these seemingly distinct pathways are not well understood. Previous studies have shown that the autophagic engulfment of cytosolic cargo is size‐restricted. Because of this, we HYPOTHESIZE that lipolysis and lipophagy occur as sequential processes initiated by cytosolic lipases to reduce LD size and allow for subsequent engulfment by autophagic membranes.MethodsIn AML12 hepatocytes, immunofluorescence microscopy and biochemical approaches were used to determine the enrichment of lipolysis vs lipophagy machinery on large vs. small LDs. Live‐cell microscopy was used for the first time to monitor LDs transitioning from lipolysis to lipophagic engulfment.ResultsConfocal microscopy of AML12 cells immunolabeled for adipose triglyceride lipase (ATGL) revealed a preferential association with large LDs with an average area of 4.0 μm2, seven‐fold larger than LDs not associated with ATGL. In striking comparison, LDs associated with components of the autophagic machinery (LC3) were two‐fold smaller in area (0.39 μm2). Consistent with this observation was the finding that siRNA knockdown of ATGL resulted in the persistence of large LDs (2.8 μm2), whereas knockdown of lysosomal acid lipase (LAL) left cells filled with many LDs of a smaller size (1.5 μm2). These observations were mimicked by inhibitors of ATGL (atglistatin) and lysosomal acidification (chloroquine). In support of these findings, differential centrifugation of AML12 homogenates allowed for separation of two distinct populations of LDs: a buoyant fraction of large LDs enriched in the cytosolic lipase ATGL, and a second collection of smaller, less buoyant LDs enriched in lysosomal enzymes such as Lamp2A and the autophagosome marker LC3‐II. Most interesting are time lapse movies of live cells showing engulfment of small LDs by a LAMP3‐positive late endosomal/lysosomal compartment, suggesting that these organelles play an important role in the direct targeting and engulfment of LDs for lipophagy.ConclusionHepatocytes appear to catabolize LDs in a tandem stepwise process that first utilizes cytosolic lipases to substantially reduce LD size, thereby allowing for subsequent engulfment by autophagosomes as well as late endosomes and lysosomes.Support or Funding InformationThis study was generously supported by funding from NIAAA R01AA020735, NIDDK T32DK007352, and the Mayo Clinic Robert and Arlene Kogod Center on Aging.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduced expression of adipose triglyceride lipase decreases arachidonic acid release and prostacyclin secretion in human aortic endothelial cells

Background: Vascular endothelial cells represent an important source of arachidonic acid (AA)-derived mediators involved in the generation of anti- or proatherogenic environments. Evidence emerged (in mast cells), that in addition to phospholipases, neutral lipid hydrolases as adipose triglyceride lipase (ATGL) also participate in this process.Objective: To examine the impact of ATGL on AA-release from cellular phospholipids (PL) and on prostacyclin secretion in human aortic endothelial cells (HAEC).Methods and results: siRNA-mediated silencing of ATGL promoted lipid droplet formation and TG accumulation in HAEC (nile red stain). ATGL knockdown decreased the basal and A23187 (calcium ionophore)-induced release of 14C-AA from (14C-AA-labeled) HAEC. In A23187-stimulated ATGL silenced cells, this was accompanied by a decreased content of 14C-AA in cellular PL and a decreased secretion of prostacyclin (determined by 6-keto PGF1α EIA).Conclusions: In vascular endothelial cells, the efficiency of stimulus-induced AA release and prostacyclin secretion is dependent on ATGL.

Hepatic ATGL manipulation regulates lipid metabolism through altered autophagy

Adipose triglyceride lipase (ATGL) catalyzes the rate‐limiting step of triacylglycerol (TAG) hydrolysis in multiple tissues. Our laboratory has previously shown that hepatic ATGL preferentially channels hydrolyzed fatty acids (FAs) to β‐oxidation with a concomitant induction of peroxisome proliferator activated receptor‐α (PPAR‐α), independent of a FA carrier. Additionally, our work describes a novel signaling axis involving cAMP/PKA, ATGL‐catalyzed lipolysis and sirtuin1 (SIRT1) activation that governs transcriptional regulation of oxidative metabolism and mitochondrial biogenesis. Recent studies have also shown SIRT1 promotes autophagy, which also contributes to lipid droplet catabolism (lipophagy). Thus, the objective of these studies is to determine the effects of ATGL on autophagy in the liver. Hepatic ATGL overexpression promotes the expression of autophagy genes, whereas ATGL knockdown reduces the expression of the same genes. ATGL regulates autophagosome biogenesis and lysosomal levels through alterations in the microtubule‐associated protein 1A/1B‐light chain 3 (LC3), sequestome1 (p62) and lysosomal‐associated membrane protein 1 (LAMP1). Use of a chemical inhibitor of ATGL, ATGListatin (Astat) in hepatocytes shows a marked suppression of autophagy, similar to the effects observed with ATGL knockdown in vivo. Finally, ATGL requires SIRT1 to govern its effects on autophagy as evidenced by studies showing that ATGL overexpression in liver‐specific SIRT1 KO mice is unable to induce autophagy. Thus, our findings identify ATGL‐mediated lipolysis as an important and potent regulator of autophagy that could have important implications for NAFLD etiology and its comorbidities.Support or Funding InformationFunding sources include the NIDDK (DK085008 to DGM) and the Minnesota Obesity Center (DK050456).

Contribution of lipase deficiency to mitochondrial dysfunction and insulin resistance in hMADS adipocytes.

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular lipid catabolism. We have previously shown decreased expression and activity of these lipases in adipose tissue of obese insulin resistant individuals. Here we hypothesized that lipase deficiency might impact on insulin sensitivity and metabolic homeostasis in adipocytes not just by enhancing lipid accumulation, but also by altering lipid and carbohydrate catabolism in a peroxisome proliferator-activated nuclear receptor (PPAR)-dependent manner. To address our hypothesis, we performed a series of in vitro experiments in a human white adipocyte model, the human multipotent adipose-derived stem (hMADS) cells, using genetic (siRNA) and pharmacological knockdown of ATGL and/or HSL. We show that ATGL and HSL knockdown in hMADS adipocytes disrupted mitochondrial respiration, which was accompanied by a decreased oxidative phosphorylation (OxPhos) protein content. This lead to a reduced exogenous and endogenous palmitate oxidation following ATGL knockdown, but not in HSL deficient adipocytes. ATGL deficiency was followed by excessive triacylglycerol accumulation, and HSL deficiency further increased diacylglycerol accumulation. Both single and double lipase knockdown reduced insulin-stimulated glucose uptake, which was attributable to impaired insulin signaling. These effects were accompanied by impaired activation of the nuclear receptor PPARα, and restored on PPARα agonist treatment. The present study indicates that lipase deficiency in human white adipocytes contributes to mitochondrial dysfunction and insulin resistance, in a PPARα-dependent manner. Therefore, modulation of adipose tissue lipases may provide a promising strategy to reverse insulin resistance in obese and type 2 diabetic patients.

Inhibition of adipose triglyceride lipase (ATGL) by the putative tumor suppressor G0S2 or a small molecule inhibitor attenuates the growth of cancer cells.

The G0/G1 switch gene 2 (G0S2) is methylated and silenced in a wide range of human cancers. The protein encoded by G0S2 is an endogenous inhibitor of lipid catabolism that directly binds adipose triglyceride lipase (ATGL). ATGL is the rate-limiting step in triglyceride metabolism. Although the G0S2 gene is silenced in cancer, the impact of ATGL in the growth and survival of cancer cells has never been addressed. Here we show that ectopic expression of G0S2 in non-small cell lung carcinomas (NSCL) inhibits triglyceride catabolism and results in lower cell growth. Similarly, knockdown of ATGL increased triglyceride levels, attenuated cell growth and promoted apoptosis. Conversely, knockdown of endogenous G0S2 enhanced the growth and invasiveness of cancer cells. G0S2 is strongly induced in acute promyelocytic leukemia (APL) cells in response to all trans retinoic acid (ATRA) and we show that inhibition of ATGL in these cells by G0S2 is required for efficacy of ATRA treatment. Our data uncover a novel tumor suppressor mechanism by which G0S2 directly inhibits activity of a key intracellular lipase. Our results suggest that elevated ATGL activity may be a general property of many cancer types and potentially represents a novel target for chemotherapy.

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.

The autophagic inhibitor 3‐methyladenine potently stimulates PKA‐dependent lipolysis in adipocytes

BACKGROUND AND PURPOSE The class III PI3K inhibitor, 3-methyladenine (3-MA), is commonly used to selectively block autophagy. Recent findings suggest a strong relationship between autophagy and lipid turnover. Here, we explore the effect of 3-MA on adipocyte lipolysis. EXPERIMENTAL APPROACH Assays were performed in 3T3-L1 cells. Cells were treated with 3-MA and wortmannin, a pan PI3K and autophagy inhibitor. Pharmacological and genetic manipulation of endogenous autophagic and lipolytic pathways was used to ascertain the contribution of 3-MA to the observed effects on lipolysis. KEY RESULTS 3T3-L1 cells that were exposed to 3-MA showed a consistent increase in lipolysis, approximately 50% over basal levels. The effect of 3-MA was not secondary to autophagic inhibition as treatment of 3T3-L1 cells with wortmannin yielded no such changes. Dosing and time course experiments showed that 3-MA's ability to activate lipolysis was more sensitive than its inhibitory effect on autophagy. Knockdown of adipose triglyceride lipase (ATGL) negated the stimulatory effect of 3-MA by >90%, indicating that 3-MA enhanced ATGL-dependent hydrolysis of triacylglycerols. Additionally, the lipolytic effect of 3-MA was dependent on the activation of PKA and 3-MA induced a rapid and potent elevation of intracellular cAMP levels in adipocytes. CONCLUSIONS AND IMPLICATIONS Cumulatively, we show that 3-MA potently modulated a cellular mechanism and its underlying signalling pathways not associated with autophagy. Furthermore, we describe a novel stimulatory effect on a major signalling pathway. Our findings provide valuable information to studies employing 3-MA as a specific inhibitor for PI3K and autophagy.

Hepatic ATGL knockdown uncouples glucose intolerance from liver TAG accumulation

Adipose triglyceride lipase (ATGL) is the predominant triacylglycerol (TAG) hydrolase in mammals; however, the tissue-specific effects of ATGL outside of adipose tissue have not been well characterized. Hence, we tested the contribution of hepatic ATGL on mediating glucose tolerance and insulin action. Glucose or insulin tolerance tests and insulin signaling were performed in C57BL/6 mice administered control (nongene specific shRNA) or Atgl shRNA adenoviruses. Glucose and lipid metabolism assays were conducted in primary hepatocytes isolated from mice transduced with control or Atgl shRNA adenoviruses. Knocking down hepatic ATGL completely abrogated the increase in serum insulin following either 1 or 12 wk of feeding a high-fat (HF) diet despite higher hepatic TAG content. Glucose tolerance tests demonstrated that ATGL knockdown normalized glucose tolerance in HF-diet-fed mice. The observed improvements in glucose tolerance were present despite unaltered hepatic insulin signaling and increased liver TAG. Mice with suppressed hepatic ATGL had reduced hepatic glucose production in vivo, and hepatocytes isolated from Atgl shRNA-treated mice displayed a 26% decrease in glucose production and a 38% increase in glucose oxidation compared to control cells. Taken together, these data suggest that hepatic ATGL knockdown enhances glucose tolerance by increasing hepatic glucose utilization and uncouples impairments in insulin action from hepatic TAG accumulation.

Lipolytic Products Activate Peroxisome Proliferator-activated Receptor (PPAR) α and δ in Brown Adipocytes to Match Fatty Acid Oxidation with Supply

β-Adrenergic receptors (β-ARs) promote brown adipose tissue (BAT) thermogenesis by mobilizing fatty acids and inducing the expression of oxidative genes. β-AR activation increases the expression of oxidative genes by elevating cAMP, but whether lipolytic products can modulate gene expression is not known. This study examined the role that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) plays in the induction of gene expression. Activation of brown adipocytes by β-AR agonism or 8-bromo-cyclic AMP increased the expression of PGC1α, PDK4, PPARα, uncoupling protein 1 (UCP1), and neuron-derived orphan receptor-1 (NOR-1), and concurrent inhibition of HSL reduced the induction of PGC1α, PDK4, PPARα, and UCP1 but not NOR-1. Similar results were observed in the BAT of mice following pharmacological or genetic inhibition of HSL and in brown adipocytes with stable knockdown of ATGL. Conversely, treatments that increase endogenous fatty acids elevated the expression of oxidative genes. Pharmacological antagonism and siRNA knockdown indicate that PPARα and PPARδ modulate the induction of oxidative genes by β-AR agonism. Using a live cell fluorescent reporter assay of PPAR activation, we demonstrated that ligands for PPARα and -δ, but not PPARγ, were rapidly generated at the lipid droplet surface and could transcriptionally activate PPARα and -δ. Knockdown of ATGL reduced cAMP-mediated induction of genes involved in fatty acid oxidation and oxidative phosphorylation. Consequently, ATGL knockdown reduced maximal oxidation of fatty acids, but not pyruvate, in response to cAMP stimulation. Overall, the results indicate that lipolytic products can activate PPARα and PPARδ in brown adipocytes, thereby expanding the oxidative capacity to match enhanced fatty acid supply.

Reduced Expression of Adipose Triglyceride Lipase Enhances Tumor Necrosis Factor α-induced Intercellular Adhesion Molecule-1 Expression in Human Aortic Endothelial Cells via Protein Kinase C-dependent Activation of Nuclear Factor-κB

We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNFα-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFκB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IκBα were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNFα-induced ICAM-1 expression via activation of NFκB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state.

Intracellular fatty acids suppress β-adrenergic induction of PKA-targeted gene expression in white adipocytes

β-Adrenergic receptor (β-AR) activation elevates cAMP levels in fat cells and triggers both metabolic and transcriptional responses; however, the potential interactions between these pathways are poorly understood. This study investigated whether lipolysis affects β-AR-mediated gene expression in adipocytes. Acute β(3)-adrenergic receptor (β(3)-AR) stimulation with CL 316,243 (CL) increased expression of PKA-targeted genes PCG-1α, UCP1, and NOR-1 in mouse white fat. Limiting lipolysis via inhibition of hormone-sensitive lipase (HSL), a direct target of PKA, sharply potentiated CL induction of PCG-1α, UCP1, and NOR-1. CL also induced greater expression of PKA-targeted genes in white fat of HSL-null mice compared with wild-type littermates, further indicating that HSL activity limits PKA-mediated gene expression. Inhibiting HSL in 3T3-L1 adipocytes also potentiated the induction of PGC-1α, UCP1, and NOR-1 by β-AR activation, as did siRNA knockdown of adipose triglyceride lipase, the rate-limiting enzyme for lipolysis. Conversely, treatments that promote intracellular fatty acid accumulation suppressed induction of PGC-1α and UCP1 through β-AR stimulation. Analysis of β-adrenergic signaling indicated that excessive intracellular fatty acid production inhibits adenylyl cyclase activity and thereby reduces PKA signaling to the nucleus. Lastly, partially limiting lipolysis by inhibition of HSL increased the induction of oxidative gene expression and mitochondrial electron transport chain activity in white adipose tissue and facilitated fat loss in mice treated for 5 days with CL. Overall, our results demonstrate that fatty acids limit the upregulation of β-AR-responsive genes in white adipocytes and suggest that limiting lipolysis may be a novel means of enhancing β-AR signaling.

Adipose triglyceride lipase is a major hepatic lipase that regulates triacylglycerol turnover and fatty acid signaling and partitioning

Despite advances in our understanding of the ways in which nutrient oversupply and triacylglycerol (TAG) anabolism contribute to hepatic steatosis, little is known about the lipases responsible for regulating hepatic TAG turnover. Recent studies have identified adipose triglyceride lipase (ATGL) as a major lipase in adipose tissue, although its role in the liver is largely unknown. Thus, we tested the contribution of ATGL to hepatic lipid metabolism and signaling. Adenovirus-mediated knockdown of hepatic ATGL resulted in steatosis in mice and decreased hydrolysis of TAG in primary hepatocyte cultures and in vitro assays. In addition to altering TAG hydrolysis, ATGL was shown to play a significant role in partitioning hydrolyzed fatty acids between metabolic pathways. Although ATGL gain and loss of function did not alter hepatic TAG secretion, fatty acid oxidation was increased by ATGL overexpression and decreased by ATGL knockdown. The effects on fatty acid oxidation coincided with decreased expression of peroxisome proliferator-activated receptor α (PPAR-α) and its target genes in mice with suppressed hepatic ATGL expression. However, PPAR-α agonism was unable to normalize the effects of ATGL knockdown on PPAR-α target gene expression, and this suggests that ATGL influences PPAR-α activity independently of ligand-induced activation. Taken together, these data show that ATGL is a major hepatic TAG lipase that plays an integral role in fatty acid partitioning and signaling to control energy metabolism.

Hepatic Adipose Triglyceride Lipase (ATGL) mediates hepatic triglyceride turnover, fatty acid channeling and PPAR‐alpha activity

Adipose triglyceride lipase (ATGL) catalyzes the first step in triglyceride (TAG) hydrolysis. Because most studies have focused on ATGL in adipose tissue, its role and function in other tissues is relatively unknown. To elucidate the metabolic functions of hepatic ATGL, we employed gain and loss‐of‐function studies in primary hepatocyte cultures and adenovirus‐mediated knockdown in vivo. ATGL overexpression in primary hepatocyte cultures exhibited resistance to fatty acid‐induced steatosis, increased catabolism of hydrolyzed fatty acids and increased PPAR‐α activity. Treament of hepatocytes with siRNA against ATGL resulted in decreased rates of TAG hydrolysis and increased TAG accumulation along with suppressed fatty acid oxidation and PPAR‐α activity. However, the large changes in TAG turnover and fatty acid oxidation did not affect media TAG suggesting that rates of VLDL synthesis were unaltered. Interestingly, ATGL shRNA‐treated mice exhibited 150% more hepatic TAG and increased liver weight. Serum TAG levels of the mice did not change in response to ATGL knockdown while the expression of PPAR‐α and its target genes decreased, thus supporting the previous data from in vitro studies. Taken together, these data show that ATGL is a major TAG lipase in the liver that acts, perhaps in part through PPAR‐α, to channel hydrolyzed fatty acids towards oxidative pathways as opposed to VLDL secretory pathways.