MTORC1 inhibition via rapamycin promotes triacylglycerol lipolysis and release of free fatty acids in 3T3-L1 adipocytes.
ABSTRACT: Signaling by mTOR complex 1 (mTORC1) promotes anabolic cellular processes in response to growth factors, nutrients, and hormonal cues. Numerous clinical trials employing the mTORC1 inhibitor rapamycin (aka sirolimus) to immuno-suppress patients following organ transplantation have documented the development of hypertriglyceridemia and elevated serum free fatty acids (FFA). We therefore investigated the cellular role of mTORC1 in control of triacylglycerol (TAG) metabolism using cultured murine 3T3-L1 adipocytes. We found that treatment of adipocytes with rapamycin reduced insulin-stimulated TAG storage ~50%. To determine whether rapamycin reduces TAG storage by upregulating lipolytic rate, we treated adipocytes in the absence and presence of rapamycin and isoproterenol, a β2-adrenergic agonist that activates the cAMP/protein kinase A (PKA) pathway to promote lipolysis. We found that rapamycin augmented isoproterenol-induced lipolysis without altering cAMP levels. Rapamycin enhanced the isoproterenol-stimulated phosphorylation of hormone sensitive lipase (HSL) on Ser-563 (a PKA site), but had no effect on the phosphorylation of HSL S565 (an AMPK site). Additionally, rapamycin did not affect the isoproterenol-mediated phosphorylation of perilipin, a protein that coats the lipid droplet to initiate lipolysis upon phosphorylation by PKA. These data demonstrate that inhibition of mTORC1 signaling synergizes with the β-adrenergic-cAMP/PKA pathway to augment phosphorylation of HSL to promote hormone-induced lipolysis. Moreover, they reveal a novel metabolic function for mTORC1; mTORC1 signaling suppresses lipolysis, thus augmenting TAG storage.
Project description:Chemical stimulation of white adipose tissue (WAT) causes adipose afferent reflex (AAR) and sympathetic activation. This study is to investigate the effects of AAR on lipolysis and the mechanisms of attenuated lipolysis response to enhanced AAR in obesity. Obesity was caused by high-fat diet for 12 weeks in rats. AAR was induced by injection of capsaicin into inguinal WAT or electrical stimulation of epididymal WAT afferent nerve. AAR caused sympathetic activation, which was enhanced in obesity rats. AAR increased cAMP levels and PKA activity, promoted hormone sensitive lipase (HSL) and perilipin phosphorylation, and increased lipolysis in WAT, which were attenuated in obesity rats. PKA activity, cAMP, perilipin and ?-adrenoceptor levels were reduced, while HSL was upregulated in adipocytes from obesity rats. In primary adipocytes, isoproterenol increased cAMP levels and PKA activity, promoted HSL and perilipin phosphorylation, and increased lipolysis, which were attenuated in obesity rats. The attenuated effects of isoproterenol in adipocytes from obesity rats were prevented by a cAMP analogue dbcAMP. The results indicate that reduced lipolysis response to enhanced AAR in obesity is attributed to the impaired activation of ?-adrenoceptor-cAMP-PKA-HSL pathway. Increased cAMP level in adipocytes rectifies the attenuated lipolysis in obesity.
Project description:In liver steatosis (i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the ?-adrenergic (?-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to ?-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the ?-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). ?-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this ?-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in ?-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that ?-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.
Project description:Beiging of white adipose tissue has potential antiobesity and antidiabetes effects, yet the underlying signaling mechanisms remain to be fully elucidated. Here we show that adipose-specific knockout of Rheb, an upstream activator of mechanistic target of rapamycin complex 1 (mTORC1), protects mice from high-fat diet-induced obesity and insulin resistance. On the one hand, Rheb deficiency in adipose tissue reduced mTORC1 signaling, increased lipolysis, and promoted beiging and energy expenditure. On the other hand, overexpression of Rheb in primary adipocytes significantly inhibited CREB phosphorylation and uncoupling protein 1 (UCP1) expression. Mechanistically, fat-specific knockout of Rheb increased cAMP levels, cAMP-dependent protein kinase (PKA) activity, and UCP1 expression in subcutaneous white adipose tissue. Interestingly, treating primary adipocytes with rapamycin only partially alleviated the suppressing effect of Rheb on UCP1 expression, suggesting the presence of a novel mechanism underlying the inhibitory effect of Rheb on thermogenic gene expression. Consistent with this notion, overexpression of Rheb stabilizes the expression of cAMP-specific phosphodiesterase 4D5 (PDE4D5) in adipocytes, whereas knockout of Rheb greatly reduced cellular levels of PDE4D5 concurrently with increased cAMP levels, PKA activation, and UCP1 expression. Taken together, our findings reveal Rheb as an important negative regulator of beige fat development and thermogenesis. In addition, Rheb is able to suppress the beiging effect through an mTORC1-independent mechanism.
Project description:<h4>Purpose</h4>The aim of this study was to investigate the effect of phosphotyrosine interaction domain containing 1 (PID1) on the insulin-induced activation of the AKT (protein kinase B)/protein kinase A (PKA)/hormone-sensitive lipase (HSL) pathway and lipolysis.<h4>Methods</h4>Sprague-Dawley rats were fed either chow or a high-fat diet (HFD). The levels of insulin, glycerol, free fatty acids (FFAs) and PID1 mRNA expression were measured in the 2 groups. Furthermore, we examined the role of PID1 in the regulation of the AKT/PKA/HSL cascade and lipolysis in the 3T3-L1 cell line.<h4>Results</h4>Adipose tissue from HFD rats exhibited elevated PID1 expression, which showed a positive correlation with insulin levels and lipolysis. In 3T3-L1 adipocytes, we found that the antilipolytic effect of insulin is mediated by AKT and that phosphorylated AKT results in the promotion of PDE3B expression, the dephosphorylation of PKA and HSL and the suppression of glycerol release. However, overexpression of PID1 and treatment with 1 ?M isoproterenol and 100 nM insulin for 24 h resulted in an increased release of glycerol and a noticeable inhibition of AKT phosphorylation, PDE3B expression and the phosphorylation of PKA/HSL in 3T3-L1 cells. In contrast, knockdown of PID1 and treatment with the above reagents inhibited lipolysis and activated the phosphorylation of AKT, which resulted in the dephosphorylation of PKA and HSL.<h4>Conclusions</h4>Our findings indicate that PID1 in adipose tissue increases lipolysis by altering the antilipolytic action of insulin. This suggests that PID1 may represent a new therapeutic target to ameliorate adipocyte lipolysis and hence improve insulin sensitivity.
Project description:CD36 has been linked to the etiology of insulin resistance and inflammation. We explored its function in regulating adipose tissue lipolysis, which influences fat accumulation by liver and muscle and overall metabolism. Knockdown of CD36 in differentiated 3T3-L1 adipocytes decreased lipolysis in response to 10 ?M of the ?-adrenergic agonist isoproterenol (by 42%), 10 ?M of the adenyl cyclase activator forskolin (by 32%), and 500 ?M of the phosphodiesterase (PDE) inhibitor isobutylmethylxanthine (by 33%). All three treatments in the knockdown adipocytes were associated with significant decreases of cAMP levels and of the hormone-sensitive lipase (HSL) and perilipin phosphorylation. An important role for PDE was supported by the lack of inhibition of the lipolysis induced by the poorly hydrolyzable dibutyryl cAMP analog. An additional contributory mechanism was diminished activation of the Src-ERK1/2 pathway. Regulation of lipolysis and lipolytic signaling by CD36 was reproduced with adipose tissue from CD36(-/-) mice. The importance of surface CD36 in this regulation was suggested by the finding that the plasma membrane-impermeable CD36 inhibitor sulfo-N-succinimidyl oleate (20 ?M) decreased lipolysis. Interestingly, isoproterenol induced CD36 internalization, and this process was blocked by HSL inhibition, suggesting feedback regulation of adipocyte lipolysis via CD36 trafficking.
Project description:Cardiotrophin-1 (CT-1) is a cytokine with antiobesity properties and with a role in lipid metabolism regulation and adipose tissue function. The aim of this study was to analyze the molecular mechanisms involved in the lipolytic actions of CT-1 in adipocytes. Recombinant CT-1 (rCT-1) effects on the main proteins and signaling pathways involved in the regulation of lipolysis were evaluated in 3T3-L1 adipocytes and in mice. rCT-1 treatment stimulated basal glycerol release in a concentration- and time-dependent manner in 3T3-L1 adipocytes. rCT-1 (20 ng/ml for 24 h) raised cAMP levels, and in parallel increased protein kinase (PK)A-mediated phosphorylation of perilipin and hormone sensitive lipase (HSL) at Ser660. siRNA knock-down of HSL or PKA, as well as pretreatment with the PKA inhibitor H89, blunted the CT-1-induced lipolysis, suggesting that the lipolytic action of CT-1 in adipocytes is mainly mediated by activation of HSL through the PKA pathway. In ob/ob mice, acute rCT-1 treatment also promoted PKA-mediated phosphorylation of perilipin and HSL at Ser660 and Ser563, and increased adipose triglyceride lipase (desnutrin) content in adipose tissue. These results showed that the ability of CT-1 to regulate the activity of the main lipases underlies the lipolytic action of this cytokine in vitro and in vivo, and could contribute to CT-1 antiobesity effects.
Project description:(1) Background: Acute administration of the cannabinoid receptor 1 (CB1R) inverse agonist Rimonabant (SR141716A) to fed Wistar rats was shown to elicit a rapid and short-lasting elevation of serum free fatty acids. (2) Methods: The effect of Rimonabant on lipolysis in isolated primary rat adipocytes was studied to raise the possibility for direct mechanisms not involving the (hypothalamic) CB1R. (3) Results: Incubation of these cells with Rimonabant-stimulated lipolysis to up to 25% of the maximal isoproterenol effect, which was based on both CB1R-dependent and independent mechanisms. The CB1R-dependent one was already effective at Rimonabant concentrations of less than 1 µM and after short-term incubation, partially additive to ?-adrenergic agonists and blocked by insulin and, in part, by adenosine deaminase, but not by propranolol. It was accompanied by protein kinase A (PKA)-mediated association of hormone-sensitive lipase (HSL) with lipid droplets (LD) and dissociation of perilipin-1 from LD. The CB1R-independent stimulation of lipolysis was observed only at Rimonabant concentrations above 1 µM and after long-term incubation and was not affected by insulin. It was recapitulated by a cell-free system reconstituted with rat adipocyte LD and HSL. Rimonabant-induced cell-free lipolysis was not affected by PKA-mediated phosphorylation of LD and HSL, but abrogated by phospholipase digestion or emulsification of the LD. Furthermore, LD isolated from adipocytes and then treated with Rimonabant (>1 µM) were more efficient substrates for exogenously added HSL compared to control LD. The CB1R-independent lipolysis was also demonstrated in primary adipocytes from fed rats which had been treated with a single dose of Rimonabant (30 mg/kg). (4) Conclusions: These data argue for interaction of Rimonabant (at high concentrations) with both the LD surface and the CB1R of primary rat adipocytes, each leading to increased access of HSL to LD in phosphorylation-independent and dependent fashion, respectively. Both mechanisms may lead to direct and acute stimulation of lipolysis at peripheral tissues upon Rimonabant administration and represent targets for future obesity therapy which do not encompass the hypothalamic CB1R.
Project description:The release of free fatty acids (FFAs) from adipocytes (i.e. lipolysis) is increased in obesity and is a contributory factor to the development of insulin resistance. A recently identified adipokine, apelin, is up-regulated in states of obesity. Although apelin is secreted by adipocytes, its functions in them remain largely unknown. To determine whether apelin affects lipolysis, FFA, glycerol, and leptin levels, as well as abdominal adiposity, were measured at baseline and after reintroduction of exogenous apelin in apelin-null mice. To examine apelin's effects in vitro, isoproterenol-induced FFA/glycerol release, and hormone-sensitive lipase (HSL) and acetyl CoA carboxylase phosphorylation were investigated in 3T3-L1 cells and isolated wild-type adipocytes. Serum FFA, glycerol, and leptin concentrations, as well as abdominal adiposity, were significantly increased in apelin-null vs. wild-type mice; these changes were ameliorated in response to exogenous apelin. Apelin also reduced isoproterenol-induced FFA release in adipocytes isolated from wild-type but not APJ-null mice. In 3T3-L1 cells and isolated adipocytes, apelin attenuated isoproterenol-induced FFA/glycerol release. Apelin's inhibition was reversed by pertussis toxin, the G(q) inhibitor glycoprotein antagonist 2A, and the AMP-activated protein kinase inhibitors compound C and dorsomorphin. Apelin increased HSL phosphorylation at Ser-565 and also abrogated isoproterenol-induced HSL phosphorylation at Ser-563. Notably, apelin increased acetyl CoA carboxylase phosphorylation, suggesting AMPK activation. In conclusion, apelin negatively regulates lipolysis. Its actions may be mediated by pathways involving G(q), G(i), and AMP-activated protein kinase.
Project description:In metazoans, target of rapamycin complex 1 (TORC1) plays the key role in nutrient- and hormone-dependent control of metabolism. However, the role of TORC1 in regulation of triglyceride storage and metabolism remains largely unknown.In this study, we analyzed the effect of activation and inhibition of the mammalian TORC1 (mTORC1) signaling pathway on the expression of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), lipolysis, lipogenesis, and lipid storage in different mammalian cells.Activation of mTORC1 signaling in 3T3-L1 adipocytes by ectopic expression of Rheb inhibits expression of ATGL and HSL at the level of transcription, suppresses lipolysis, increases de novo lipogenesis, and promotes intracellular accumulation of triglycerides. Inhibition of mTORC1 signaling by rapamycin or by knockdown of raptor stimulates lipolysis primarily via activation of ATGL expression. Analogous results have been obtained in C2C12 myoblasts and mouse embryonic fibroblasts with genetic ablation of tuberous sclerosis 2 (TSC2) gene. Overexpression of ATGL in these cells antagonized the lipogenic effect of TSC2 knockout.Our findings demonstrate that mTORC1 promotes fat storage in mammalian cells by suppression of lipolysis and stimulation of de novo lipogenesis.
Project description:Lipoic acid (LA) is a naturally occurring compound with beneficial effects on obesity. The aim of this study was to evaluate its effects on lipolysis in 3T3-L1 adipocytes and the mechanisms involved. Our results revealed that LA induced a dose- and time-dependent lipolytic action, which was reversed by pretreatment with the c-Jun N-terminal kinase inhibitor SP600125, the PKA inhibitor H89, and the AMP-activated protein kinase activator AICAR. In contrast, the PI3K/Akt inhibitor LY294002 and the PDE3B antagonist cilostamide enhanced LA-induced lipolysis. LA treatment for 1 h did not modify total protein content of hormone-sensitive lipase (HSL) but significantly increased the phosphorylation of HSL at Ser(563) and at Ser(660), which was reversed by H89. LA treatment also induced a marked increase in PKA-mediated perilipin phosphorylation. LA did not significantly modify the protein levels of adipose triglyceride lipase or its activator comparative gene identification 58 (CGI-58) and inhibitor G(0)/G(1) switch gene 2 (G0S2). Furthermore, LA caused a significant inhibition of adipose-specific phospholipase A2 (AdPLA) protein and mRNA levels in parallel with a decrease in the amount of prostaglandin E(2) released and an increase in cAMP content. Together, these data suggest that the lipolytic actions of LA are mainly mediated by phosphorylation of HSL through cAMP-mediated activation of protein kinase A probably through the inhibition of AdPLA and prostaglandin E(2).