Translocation Of Hormone-sensitive Lipase Research Articles

DECR1 directly activates HSL to promote lipolysis in cervical cancer cells

Fatty acids have a high turnover rate in cancer cells to supply energy for tumor growth and proliferation. Lipolysis is particularly important for the regulation of fatty acid homeostasis and in the maintenance of cancer cells. In the current study, we explored how 2,4-Dienoyl-CoA reductase (DECR1), a short-chain dehydrogenase/reductase associated with mitochondrial and cytoplasmic compartments, promotes cancer cell growth. We report that DECR1 overexpression significantly reduced the triglyceride (TAG) content in HeLa cells; conversely, DECR1 silencing increased intracellular TAG content. Subsequently, our experiments demonstrate that DECR1 promotes lipolysis via effects on hormone sensitive lipase (HSL). The direct interaction of DECR1 with HSL increases HSL phosphorylation and activity, facilitating the translocation of HSL to lipid droplets. The ensuing enhancement of lipolysis thus increases the release of free fatty acids. Downstream effects include the promotion of cervical cancer cell migration and growth, associated with the enhanced levels of p62 protein. In summary, high levels of DECR1 serves to enhance lipolysis and the release of fatty acid energy stores to support cervical cancer cell growth.

Trafficking of cholesterol from lipid droplets to mitochondria in bovine luteal cells: Acute control of progesterone synthesis.

The corpus luteum is a transient endocrine gland that synthesizes and secretes the steroid hormone, progesterone, which is vital for establishment and maintenance of pregnancy. Luteinizing hormone (LH) via activation of protein kinase A (PKA) acutely stimulates luteal progesterone synthesis via a complex process, converting cholesterol via a series of enzymatic reactions, into progesterone. Lipid droplets in steroidogenic luteal cells store cholesterol in the form of cholesterol esters, which are postulated to provide substrate for steroidogenesis. Early enzymatic studies showed that hormone sensitive lipase (HSL) hydrolyzes luteal cholesterol esters. In this study, we tested whether HSL is a critical mediator of the acute actions of LH on luteal progesterone production. Using LH-responsive bovine small luteal cells our results reveal that LH, forskolin, and 8-Br cAMP-induced PKA-dependent phosphorylation of HSL at Ser563 and Ser660, events known to promote HSL activity. Small molecule inhibition of HSL activity and siRNA-mediated knock down of HSL abrogated LH-induced progesterone production. Moreover, western blotting and confocal microscopy revealed that LH stimulates phosphorylation and translocation of HSL to lipid droplets. Furthermore, LH increased trafficking of cholesterol from the lipid droplets to the mitochondria, which was dependent on both PKA and HSL activation. Taken together, these findings identify a PKA/HSL signaling pathway in luteal cells in response to LH and demonstrate the dynamic relationship between PKA, HSL, and lipid droplets in luteal progesterone synthesis.

Cytological and functional characteristics of fascia adipocytes in rats: A unique population of adipocytes

The dermal adipocytes, superficial fascia and subcutaneous adipose tissue (SAT) exist in the interspaces between the dermis and muscular fascia. They are adjacent to each other and traditionally recognized as one SAT. Recently, the dermal adipocyte was redefined as a unique population independent from the SAT. Also, we identified a novel type of adipogenic progenitors in rat superficial fascia. This study aimed to examine cytological and functional characteristics of fascial adipocytes in rats. Superficial fascia had no adipocytes in neonatal rats but gradually appeared numbers of adipocytes in growing rats. Adipogenic progenitors were found to reside in fascia and had strong ability in spontaneous and induced adipogenic differentiation in vitro. Differentiated fascial adipocytes versus subcutaneous or visceral adipocytes expressed increased adipose triglyceride lipase but decreased beta-adrenoreceptor, perilipin-1 and hormone-sensitive lipase (HSL), thus having high basal lipolysis but low lipolysis response to catecholamines. Phosphorylation of perilipin-1 and HSL and translocation of HSL to lipid droplets were attenuated in response to catecholamines rather than post-adrenoreceptoral lipolytic stimulators. The results suggested that superficial fascia was an origin of adipocytes with distinct developmental, cytological and functional characteristics. We proposed that fascial adipocytes could be considered as a unique population of adipocytes in the body. The fascia origin of adipocytes as an adipogenic model might logically explain fat neogenesis occurred at anatomical locations where originally exist no adipose tissues and thereby no adipose-derived stromal precursors. Also, the special histoanatomical relations and overlaps between the dermis, superficial fascia, SAT, and their adipocytes were discussed.

Hormone-sensitive lipase preferentially redistributes to lipid droplets associated with perilipin-5 in human skeletal muscle during moderate-intensity exercise.

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) are the key enzymes involved in intramuscular triglyceride (IMTG) lipolysis. In isolated rat skeletal muscle, HSL translocates to IMTG-containing lipid droplets (LDs) following electrical stimulation, but whether HSL translocation occurs in human skeletal muscle during moderate-intensity exercise is currently unknown. Perilipin-2 (PLIN2) and perilipin-5 (PLIN5) proteins have been implicated in regulating IMTG lipolysis by interacting with HSL and ATGL in cell culture and rat skeletal muscle studies. This study investigated the hypothesis that HSL (but not ATGL) redistributes to LDs during moderate-intensity exercise in human skeletal muscle, and whether the localisation of these lipases with LDs was affected by the presence of PLIN proteins on the LDs. HSL preferentially redistributed to PLIN5-associated LDs whereas ATGL distribution was not altered with exercise; this is the first study to illustrate the pivotal step of HSL redistribution to PLIN5-associated LDs following moderate-intensity exercise in human skeletal muscle. Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) control skeletal muscle lipolysis. ATGL is present on the surface of lipid droplets (LDs) containing intramuscular triglyceride (IMTG) in both the basal state and during exercise. HSL translocates to LD in ex vivo electrically stimulated rat skeletal muscle. Perilipin-2- and perilipin-5-associated lipid droplets (PLIN2+ and PLIN5+ LDs) are preferentially depleted during exercise in humans, indicating that these PLINs may control muscle lipolysis. We aimed to test the hypothesis that in human skeletal muscle in vivo HSL (but not ATGL) is redistributed to PLIN2+ and PLIN5+ LDs during moderate-intensity exercise. Muscle biopsies from 8 lean trained males (age 21±1years, BMI 22.6±1.2kgm-2 and V̇O2 peak 48.2±5.0mlmin-1 kg-1 ) were obtained before and immediately following 60min of cycling exercise at ∼59% V̇O2 peak . Cryosections were stained using antibodies targeting ATGL, HSL, PLIN2 and PLIN5. LDs were stained using BODIPY 493/503. Images were obtained using confocal immunofluorescence microscopy and object-based colocalisation analyses were performed. Following exercise, HSL colocalisation to LDs increased (P<0.05), and was significantly greater to PLIN5+ LDs (+53%) than to PLIN5- LDs (+34%) (P<0.05), while the increases in HSL colocalisation to PLIN2+ LDs (+16%) and PLIN2- LDs (+28%) were not significantly different. Following exercise, the fraction of LDs colocalised with ATGL (0.53±0.04) did not significantly change (P<0.05) and was not affected by PLIN association to the LDs. This study presents the first evidence of exercise-induced HSL redistribution to LDs in human skeletal muscle and identifies PLIN5 as a facilitator of this mechanism.

Adipose triglyceride lipase protein abundance and translocation to the lipid droplet increase during leptin-induced lipolysis in bovine adipocytes

Proper regulation of lipid metabolism is critical for preventing the development of metabolic diseases. It is clear that leptin plays a critical role in the regulation of energy homeostasis by regulating energy intake. However, leptin can also regulate energy homeostasis by inducing lipolysis in adipocytes, but it is unclear how the major lipases are involved in leptin-stimulated lipolysis. Therefore, the objectives of this study were to determine if (1) leptin acts directly to induce lipolysis in bovine adipocytes, (2) the potential lipases involved in leptin-induced lipolysis in bovine adipocytes, and (3) increases translocation of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) during leptin-stimulated lipolysis in bovine stromal vascular cell–derived adipocytes. As hypothesized, leptin induced a lipolytic response (P = 0.02) in isolated adipocytes which was accompanied by an increase in phosphorylation of signal transducer and activator of transcription (STAT)3 (P = 0.03), a well-documented secondary messenger of leptin, and ATGL protein abundance (P < 0.01). Protein abundance of STAT3, perilipin, HSL, and phosphorylation of HSL by PKA and AMPK were not altered during leptin-stimulated lipolysis (P > 0.05). Immunostaining techniques were employed to determine the location of HSL and ATGL. Both lipases translocated to the lipid droplet after 2 h of exposure to isoproterenol (P < 0.02). However, only ATGL was translocated to the lipid droplet during leptin-stimulated lipolysis (P = 0.04), indicating ATGL may be the active lipase in leptin-stimulated lipolysis. In summary, leptin stimulates lipolysis in bovine adipocytes. The lack of phosphorylated HSL and translocation of HSL to the lipid droplet during leptin-stimulated lipolysis suggest minimal activity by PKA. Interestingly, leptin-stimulated lipolysis is accompanied by an increase in ATGL protein abundance and translocation to the lipid droplet, indicating its involvement in leptin-stimulated lipolysis either due to an increase in protein abundance or through a novel lipolytic cascade.

Habitual exercise training acts as a physiological stimulator for constant activation of lipolytic enzymes in rat primary white adipocytes

It is widely accepted that lipolysis in adipocytes are regulated through the enzymatic activation of both hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) via their phosphorylation events. Accumulated evidence shows that habitual exercise training (HE) enhances the lipolytic response in primary white adipocytes with changes in the subcellular localization of lipolytic molecules. However, no study has focused on the effect that HE exerts on the phosphorylation of both HSL and ATGL in primary white adipocytes. It has been shown that the translocation of HSL from the cytosol to lipid droplet surfaces requires its phosphorylation at Ser-563. In primary white adipocytes obtained from HE rats, the level of HSL and ATGL proteins was higher than that in primary white adipocytes obtained from sedentary control (SC) rats. In HE rats, the level of phosphorylated ATGL and HSL was also significantly elevated compared with that in SC rats. These differences were confirmed by Phos-tag SDS-PAGE, a technique used to measure the amount of total phosphorylated proteins. Our results suggest that HE can consistently increase the activity of both lipases, thereby enhancing the lipolysis in white fat cells. Thus, HE helps in the prevention and treatment of obesity-related diseases by enhancing the lipolytic capacity.

Ablation of Perilipin 1 Alters Whole Body Glucose Homeostasis

Lipid droplets are cellular organelles that enclose neutral lipid depots and play dynamic roles in lipid metabolism by lipid droplet proteins. Perilipin 1 is one of the major lipid droplet binding proteins in adipocytes. Perilipin1 protein is phosphorylated by protein kinase A and its phosphorylation is necessary for translocation of hormone-sensitive lipase to the lipid droplets to facilitate lipolysis. As well known, depletion of perilipin 1 gene in mice is resistance to diet-induced obesity as well as increases basal lipolysis and reduces stimulated lipolysis. However, it is largely unknown whether insulin signaling is changed in perilipin 1 knockout (Plin1 KO) mice compared with wild type (WT) mice. To investigate potential roles for perilipin 1 in glucose homeostasis, we performed glucose tolerance test in Plin1 KO mice and WT mice. We discovered that the glucose responsiveness was more sensitive in Plin1 KO mice compared with WT mice under normal chow diet condition. Moreover, insulin downstream signalings were altered in metabolic tissues of Plin1 KO mice. Together, these data suggest that perilipin 1 would be involved in systemic glucose metabolism, at least in part, through insulin signaling. This work was supported by National Creative Research Initiative Program (2011-0018312) funded by the ministry of Education, Science, and Technology (MEST). J. H. Sohn and J. S. Han were supported by the BK21 program, Korea.

QRFP-43 inhibits lipolysis by preventing ligand-induced complex formation between perilipin A, caveolin-1, the catalytic subunit of protein kinase and hormone-sensitive lipase in 3T3-L1 adipocytes

QRFP (RFamide) peptides are neuropeptides involved in food intake and adiposity regulation in rodents. We have previously shown that QRFP-43 (43RFa) and QRFP-26 (26RFa) inhibited isoproterenol (ISO)-induced lipolysis in adipocytes. However, the antilipolytic signaling pathways activated by QRFP peptides have not been investigated. In the present study, 3T3-L1 adipocytes were used to identify the main pathways involved in QRFP-43 decreasing ISO-induced lipolysis. Our results show that QRFP-43 reduced ISO-induced phosphorylation of perilipin A (PLIN) and hormone-sensitive lipase (HSL) on Ser660 by 43 and 25%, respectively, but increased Akt phosphorylation by 44%. However, the inhibition of phosphodiesterase 3B (PDE3B), a regulator of lipolysis activated by Akt, did not reverse the antilipolytic effect of QRFP-43. PDE3B inhibition reversed the decrease of Ser660 HSL phosphorylation associated with QRFP-43 antilipolytic effect. QRFP-43 also prevented PKC activation and ISO-induced Src kinases activation leading to the inhibition of the caveolin-1 (CAV-1) translocation on lipid droplets. Indeed, QRFP-43 attenuated phorbol 12-myristate 13-acetate-induced lipolysis and ISO-induced extracellular signal-regulated and Src kinases by 28, 37 and 48%, respectively. The attenuation of ISO-induced lipolysis by QRFP-43 was associated with a decrease of phosphorylated Ser660 HSL, PKA-catalytic (PKA-c) subunit and CAV-1 translocation on lipid droplets by 37, 50 and 46%, respectively. The decrease in ISO-induced CAV-1 and PKA-c translocation was associated with a reduction of PLIN phosphorylation by 44% in QRFP-43-treated adipocytes. These results suggest that QRFP-43 attenuated ISO-induced lipolysis by preventing the formation of an active complex on lipid droplets and the activation of Src kinases and PKC.

Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis

Long-term cytokine-mediated inflammation is a risk factor for obesity and type 2 diabetes mellitus (T2DM). Our previous studies reveal significant associations between promoter single nucleotide polymorphisms (SNPs) of interleukin (IL)-4 and T2DM, as well as between SNPs in genes encoding IL-4/IL-4 receptor and high density lipoproteins. Our animal study reveals that IL-4 regulates glucose/lipid metabolism by promoting glucose tolerance and inhibiting lipid deposits. The above results strongly suggest the involvement of IL-4 in energy homeostasis. In the present study, we focus on examining the regulatory mechanism of IL-4 to lipid metabolism. Our results show that IL-4 inhibits adipogenesis by downregulating the expression of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein-α. Additionally, IL-4 promotes lipolysis by enhancing the activity and translocation of hormone sensitive lipase (HSL) in mature adipocytes, which suggests that IL-4 plays a pro-lipolytic role in lipid metabolism by boosting HSL activity. Our results demonstrate that IL-4 harbors pro-lipolysis capacity by inhibiting adipocyte differentiation and lipid accumulation as well as by promoting lipolysis in mature adipocytes to decrease lipid deposits. The above findings uncover the novel roles of IL-4 in lipid metabolism and provide new insights into the interactions among cytokine/immune responses, insulin sensitivity, and metabolism.

Lipolytic actions of secretin in mouse adipocytes

Secretin (Sct), a classical gut hormone, is now known to play pleiotropic functions in the body including osmoregulation, digestion, and feeding control. As Sct has long been implicated to regulate metabolism, in this report, we have investigated a potential lipolytic action of Sct. In our preliminary studies, both Sct levels in circulation and Sct receptor (SctR) transcripts in adipose tissue were upregulated during fasting, suggesting a potential physiological relevance of Sct in regulating lipolysis. Using SctR knockout and Sct knockout mice as controls, we show that Sct is able to stimulate lipolysis in vitro in isolated adipocytes dose- and time-dependently, as well as acute lipolysis in vivo. H-89, a protein kinase A (PKA) inhibitor, was found to attenuate lipolytic effects of 1 μM Sct in vitro, while a significant increase in PKA activity upon Sct injection was observed in the adipose tissue in vivo. Sct was also found to stimulate phosphorylation at 660ser of hormone sensitive lipase (HSL) and to bring about the translocation of HSL from cytosol to the lipid droplet. In summary, our data demonstrate for the first time the in vivo and in vitro lipolytic effects of Sct, and that this function is mediated by PKA and HSL.

Curcumin attenuates lipolysis stimulated by tumor necrosis factor-α or isoproterenol in 3T3-L1 adipocytes

Background and aimCurcumin, an active component derived from dietary spice turmeric (Curcuma longa), has been demonstrated antihyperglycemic, antiinflammatory and hypocholesterolemic activities in obesity and diabetes. These effects are associated with decreased level of circulating free fatty acids (FFA), however the mechanism has not yet been elucidated. The flux of FFA and glycerol from adipose tissue to the blood stream primarily depends on the lipolysis of triacylglycerols in the adipocytes. Adipocyte lipolysis is physiologically stimulated by catecholamine hormones. Tumor necrosis factor-α (TNFα) stimulates chronic lipolysis in obesity and type 2 diabetes. In this study, we examined the role of curcumin in inhibiting lipolytic action upon various stimulations in 3T3-L1 adipocytes. MethodsGlycerol release from TNFα or isoproterenol-stimulated 3T3-L1 adipocytes in the absence or presence of curcumin was determined using a colorimetric assay (GPO-Trinder). Western blotting was used to investigate the TNFα-induced phosphorylation of MAPK and perilipin expression. Fatcake and cytosolic fractions were prepared to examine the isoproterenol-stimulated hormone-sensitive lipase translocation. ResultsTreatment with curcumin attenuated TNFα-mediated lipolysis by suppressing phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2) and reversing the downregulation of perilipin protein in TNFα-stimulated adipocytes (p<0.05). The acute lipolytic response to adrenergic stimulation of isoproterenol was also restricted by curcumin (10–20μM, p<0.05), which was compatible with reduced perilipin phosphorylation(29%, p<0.05) and hormone-sensitive lipase translocation(20%, p<0.05). ConclusionsThis study provides evidence that curcumin acts on adipocytes to suppress the lipolysis response to TNFα and catecholamines. The antilipolytic effect could be a cellular basis for curcumin decreasing plasma FFA levels and improving insulin sensitivity.

Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes

In obesity, there is an increase in reactive oxygen species (ROS) within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL) is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies.

Opposite and redundant roles of the two Drosophila perilipins in lipid mobilization

Lipid droplets are the main lipid storage sites in cells. Lipid droplet homeostasis is regulated by the surface accessibility of lipases. Mammalian adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are two key lipases for basal and stimulated lipolysis, respectively. Perilipins, the best known lipid droplet surface proteins, can either recruit lipases or prevent the access of lipases to lipid droplets. Mammals have five perilipin proteins, which often exhibit redundant functions, precluding the analysis of the exact role of individual perilipins in vivo. Drosophila have only two perilipins, PLIN1/LSD-1 and PLIN2/LSD-2. Previous studies revealed that PLIN2 is important for protecting lipid droplets from lipolysis mediated by Brummer (BMM), the Drosophila homolog of ATGL. In this study, we report the functional analysis of PLIN1 and Drosophila HSL. Loss-of-function and overexpression studies reveal that unlike PLIN2, PLIN1 probably facilitates lipid mobilization. HSL is recruited from the cytosol to the surface of lipid droplets under starved conditions and PLIN1 is necessary for the starved induced lipid droplet localization of HSL. Moreover, phenotypic analysis of plin1;plin2 double mutants revealed that PLIN1 and PLIN2 might have redundant functions in protecting lipid droplets from lipolysis. Therefore, the two Drosophila perilipins have both opposite and redundant roles. Domain swapping and deletion analyses indicate that the C-terminal region of PLIN1 confers functional specificity to PLIN1. Our study highlights the complex roles of Drosophila perilipin proteins and the evolutionarily conserved regulation of HSL translocation by perilipins.

Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.

Ursolic acid stimulates lipolysis in primary‐cultured rat adipocytes

Ursolic acid (UA) is a pentacyclic triterpenic acid with many biological functions naturally existing in many kinds of food. To investigate whether UA can accelerate lipolysis, primary-cultured rat adipocytes were treated with UA, and glycerol release in the culture medium was measured. UA stimulated lipolysis significantly. Furthermore, the lipolytic effect of UA was inhibited by the protein kinase A (PKA) specific inhibitor H89, suggesting that UA exerted its lipolytic function through the cAMP-dependent PKA pathway. Downstream targets of the PKA pathway, hormone-sensitive lipase (HSL) and perilipin A were checked, UA enhanced lipolysis by promoting the translocation of HSL from the cytosol to the lipid droplets and inhibiting the expression of perilipin A. Additionally, adipose triglyceride lipase (ATGL), a novel rate-limiting lipase in the lipolytic catabolism, was upregulated by UA. UA-induced expression of ATGL could not be blocked by H89, suggesting that ATGL upregulation is not regulated by the PKA pathway. These findings suggest that UA significantly stimulates lipolysis by translocating HSL, decreasing perilipin A expression by the PKA pathway, and up-regulating ATGL in primary cultured adipocytes. Thus, UA is a promising candidate for the treatment of obesity.

Vimentin Is a Functional Partner of Hormone Sensitive Lipase And Facilitates Lipolysis

Lipolysis involves a number of components including signaling pathways, droplet-associated proteins, and lipases such as hormone-sensitive lipase (HSL). We used surface enhanced laser desorption/ionization time-of-flight mass spectroscopy to identify cellular proteins that might interact with HSL and potentially influence lipolysis. Using recombinant HSL as bait on protein chips, clusters of proteins of 14.7-18.9, 25.8-26.8, 36.1, 44.3-49.1, and 53.7 kDa were identified that interact with HSL, particularly when lysates were examined from beta-agonist treated mouse adipocytes. The ability to detect these interacting proteins was markedly diminished when the adipocytes were treated with insulin. A very similar pattern of proteins was identified when anti-HSL IgG was used as the bait. Following immunocapture, the identification of the prominent 53.7 kDa protein was carried out by tryptic digestion and MS analysis and determined to be vimentin. The interaction of HSL with vimentin, and its hormonal dependence, was confirmed by coimmunoprecipitation. beta-Agonist stimulated lipolysis and the rate of HSL translocation were impaired in vimentin null adipocytes, even though normal amounts of lipases and droplet-associated proteins are expressed. The current studies provide evidence that vimentin participates in lipolysis through direct, hormonally regulated interactions with HSL.

Biguanides and thiazolidinediones inhibit stimulated lipolysis in human adipocytes through activation of AMP-activated protein kinase

In rodent adipocytes, activated AMP-activated protein kinase reduces the lipolytic rate. As the hypoglycaemic drugs metformin and thiazolidinediones activate this enzyme in rodents, we tested the hypothesis that in addition to their known actions they could have an anti-lipolytic effect in human adipocytes. Adipose tissue was obtained from individuals undergoing plastic surgery. Adipocytes were isolated and incubated with lipolytic agents (isoprenaline, atrial natriuretic peptide) and biguanides or thiazolidinediones. Lipolysis was quantified by the glycerol released in the medium. AMP-activated protein kinase activity and phosphorylation state were determined using standard procedures. In human adipocytes, isoprenaline and atrial natriuretic peptide stimulated the lipolytic rate three- to fourfold. Biguanides and thiazolidinediones activated AMP-activated protein kinase and inhibited lipolysis by 30-40%, at least in part by inhibiting hormone-sensitive lipase translocation to the lipid droplet. Inhibition of AMP-activated protein kinase by compound C precluded this inhibitory effect on lipolysis. Stimulation of lipolysis also induced an activation of AMP-activated protein kinase concomitant with a drop in ATP concentration. We show for the first time in human adipocytes that biguanides and thiazolidinediones activate AMP-activated protein kinase, thus counteracting lipolysis induced by lipolytic agents. In addition, beta-agonist- or ANP-stimulated lipolysis increases AMP-activated protein kinase activity. This is because of an increase in the AMP/ATP ratio, linked to activation of some of the released fatty acids into acyl-CoA. AMP-activated protein kinase activation could represent a physiological means of avoiding a deleterious drain of energy during lipolysis but could be used to restrain pharmacological release of fatty acids.

Functional interaction of hormone-sensitive lipase and perilipin in lipolysis

Adipocyte lipolysis is controlled by complex interactions of lipases, cofactors, and structural proteins associated with lipid droplets. Perilipin (Plin) A is a major droplet-associated protein that functions as a scaffold, both suppressing basal and facilitating cAMP-dependent protein kinase (PKA)-stimulated lipolysis. Plin is required for the translocation of hormone-sensitive lipase (HSL) from the cytosol to lipid droplets upon stimulation. In these studies, we provide direct evidence for a physical interaction of HSL with Plin. By coexpressing HSL with truncation mutations of Plin, we demonstrate using coimmunoprecipitation that HSL can interact with an N-terminal region located between amino acids 141 and 200 of Plin A as well as with a C-terminal region located between amino acids 406 and 480. The N-terminal construct, Plin 1-200, which does not associate with lipid droplets but interacts with HSL, can function as a dominant negative for PKA-stimulated lipolysis. Using confocal microscopy of Plin truncations, we demonstrate that sequences between amino acids 463 and 517 may be important for or participate in lipid targeting. The results suggest the translocation of HSL to the lipid droplet occurs by virtue of Plin localization to the surface of lipid droplets and a physical interaction of HSL occurring with sequences within the N-terminal region of Plin.

Direct Effect of Glucocorticoids on Lipolysis in Adipocytes

Hypercortisolemia and glucocorticoid treatment cause elevated level of circulating free fatty acids (FFAs). The basis of this phenomenon has long been linked to the effect of glucocorticoids permitting and enhancing the adipose lipolysis response to various hormones. In this study, we demonstrate that glucocorticoids directly stimulate lipolysis in rat primary adipocytes in a dose- and time-responsive manner; this lipolytic action was attenuated by treatment with the glucocorticoid antagonist RU486. Dexamethasone down-regulates mRNA and protein levels of cyclic-nucleotide phosphodiesterase 3B, thereby elevating cellular cAMP production and activating protein kinase A (PKA). On inhibition of PKA but not other kinases, the lipolysis response ceases. Furthermore, dexamethasone induces phosphorylation and down-regulation of perilipin, a lipid droplet-associating protein that modulates lipolysis; this effect is restored by RU486 or PKA inhibitor H89. Dexamethasone up-regulates mRNA and protein levels of hormone-sensitive lipase (HSL) and adipose triglyceride lipase; these effects, parallel to increased lipolysis, are attenuated by RU486 or actinomycin D. Phosphorylation at Ser-563 and Ser-660 residues of HSL and activity of cellular lipases are elevated on dexamethasone stimulation but abrogated by the coaddition of H89. However, dexamethasone does not induce HSL translocation to the lipid droplet surface in differentiated adipocytes. We show that elevated FFA concentration in plasma is associated with increased lipase activity and lipolysis in vivo in adipose tissues of dexamethasone-treated rats. Therefore, the lipolytic action of glucocorticoids liberates FFA efflux from adipocytes to the bloodstream, which could be a cellular basis of systemic FFA elevation in response to glucocorticoid challenge.

Contribution of Adipose Triglyceride Lipase and Hormone-sensitive Lipase to Lipolysis in hMADS Adipocytes

Lipolysis is the catabolic pathway by which triglycerides are hydrolyzed into fatty acids. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) have the capacity to hydrolyze in vitro the first ester bond of triglycerides, but their respective contributions to whole cell lipolysis in human adipocytes is unclear. Here, we have investigated the roles of HSL, ATGL, and its coactivator CGI-58 in basal and forskolin-stimulated lipolysis in a human white adipocyte model, the hMADS cells. The hMADS adipocytes express the various components of fatty acid metabolism and show lipolytic capacity similar to primary cultured adipocytes. We show that lipolysis and fatty acid esterification are tightly coupled except in conditions of stimulated lipolysis. Immunocytochemistry experiments revealed that acute forskolin treatment promotes HSL translocation from the cytosol to small lipid droplets and redistribution of ATGL from the cytosol and large lipid droplets to small lipid droplets, resulting in enriched colocalization of the two lipases. HSL or ATGL overexpression resulted in increased triglyceride-specific hydrolase capacity, but only ATGL overexpression increased whole cell lipolysis. HSL silencing had no effect on basal lipolysis and only partially reduced forskolin-stimulated lipolysis. Conversely, silencing of ATGL or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Altogether, these results suggest that ATGL/CGI-58 acts independently of HSL and precedes its action in the sequential hydrolysis of triglycerides in human hMADS adipocytes.