Rat Hormone-sensitive Lipase Research Articles

CB1 Receptor-Dependent and Independent Induction of Lipolysis in Primary Rat Adipocytes by the Inverse Agonist Rimonabant (SR141716A).

(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 β-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.

Hormone‐sensitive lipase (HSL) is also a retinyl ester hydrolase: evidence from mice lacking HSL

Here, we investigated the importance of hormone-sensitive lipase (HSL) as a retinyl ester hydrolase (REH). REH activity was measured in vitro using recombinant HSL and retinyl palmitate. The expression of retinoic acid (RA)-regulated genes and retinoid metabolites were measured in high-fat diet fed HSL-null mice using real-time quantitative PCR and triple-stage liquid chromatography/tandem mass spectrometry, respectively. Age- and gender-matched wild-type littermates were used as controls. The REH activity of rat HSL was found to be higher than that against the hitherto best known HSL substrate, i.e., diacylglycerols. REH activity in white adipose tissue (WAT) of HSL-null mice was completely blunted and accompanied by increased levels of retinyl esters and decreased levels of retinol, retinaldehyde and all-trans RA. Accordingly, genes known to be positively regulated by RA were down-regulated in HSL-null mice, including pRb and RIP140, key factors promoting differentiation into the white over the brown adipocyte lineage. Dietary RA supplementation partly restored WAT mass and the expression of RA-regulated genes in WAT of HSL-null mice. These findings demonstrate the importance of HSL as an REH of adipose tissue and suggest that HSL via this action provides RA and other retinoids for signaling events that are crucial for adipocyte differentiation and lineage commitment.

Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates

BackgroundHormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo.Methodology/Principal FindingsIn this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. 32P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S0.5 values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S0,5 using the TO substrate.ConclusionsOur results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region.

Identification of Neutral Cholesterol Ester Hydrolase, a Key Enzyme Removing Cholesterol from Macrophages

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.

Signaling the Signal, Cyclic AMP-dependent Protein Kinase Inhibition by Insulin-formed H2O2 and Reactivation by Thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

Chromosomal localization of the hormone sensitive lipase (LIPE) and insulin receptor (INSR) genes in pigs.

Using rat hormone sensitive lipase (LIPE) and human insulin receptor (INSR) cDNA probes, the LIPE gene was assigned to chromosome 6p11-q21 and the INSR gene to chromosome 2q11-q21 in pigs by in situ hybridization. In humans, these two genes are located on the q and p arms of chromosome 19, respectively. The present results provide the first in situ hybridization mapping data for porcine chromosome 2.

CGI-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation

A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. We previously showed that Comparative Gene Identification-58 (CGI-58), a product of the causal gene of Chanarin-Dorfman syndrome, interacts with perilipin on LDs. In this study, we investigated the function of CGI-58 using RNA interference. Notably, CGI-58 knockdown caused an abnormal accumulation of LDs in both 3T3-L1 preadipocytes and Hepa1 hepatoma cells. CGI-58 knockdown did not influence the differentiation of 3T3-L1 adipocytes but reduced the activity of both basal and cAMP-dependent protein kinase-stimulated lipolysis. In vitro studies showed that CGI-58 itself does not have lipase/esterase activity, but it enhanced the activity of adipose triglyceride lipase. Upon lipolytic stimulation, endogenous CGI-58 was rapidly dispersed from LDs into the cytosol along with small particulate structures. This shift in localization depends on the phosphorylation of perilipin, because phosphorylated perilipin lost the ability to bind CGI-58. During lipolytic activation, LDs in adipocytes vesiculate into micro-LDs. Using coherent anti-Stokes Raman scattering microscopy, we pursued the formation of micro-LDs in single cells, which seemed to occur in cytoplasmic regions distant from the large central LDs. CGI-58 is not required for this process. Thus, CGI-58 facilitates lipolysis in cooperation with perilipin and other factors, including lipases.

Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation

We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk. The observed changes indicate an active role of HSL in mammary gland lipid metabolism.

Function of hormone-sensitive lipase in diacylglycerol–protein kinase C pathway

To explore the functional effects of hormone-sensitive lipase (HSL) in diacylglycerol (DAG) metabolism, Chinese hamster ovary cells were stably transfected with rat HSL cDNA (wt-HSL), inactive mutant S423A-HSL cDNA (S423A) and pcDNA3 vector alone (Ct). [ 14 C ]Glucose-incorporation into triglyceride (TG) was 75% lower in the presence or absence of insulin in cells expressing wt-HSL compared to Ct or S423A. [ 14 C ]Glucose-incorporation into DAG was 33% lower without insulin and 51% lower with insulin in cells expressing wt-HSL compared to Ct or S423A. Insulin stimulated glucose-incorporation into DAG 2.2-fold in S423A and Ct cells, whereas only a 50% increase was observed in cells expressing wt-HSL. Phospholipase C-mediated release of DAG from membrane phospholipids was reduced 70% in cells expressing wt-HSL compared to Ct or S423A. Western blot analysis showed that membrane-bound protein kinase C (PKC)-α and -ε were decreased 40–50% in cells expressing wt-HSL grown in high glucose with insulin. These data show that HSL potentially hydrolyzes cellular DAG generated either by de novo synthesis from glucose or release from membrane phospholipids by phospholipase C, resulting in a reduction in the translocation of DAG-sensitive PKCs.

Fatty acid specificity of hormone-sensitive lipase: implication in the selective hydrolysis of triacylglycerols

The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of fatty acid mobilization, but could contribute to explain the preferential mobilization of some highly unsaturated fatty acids compared with others.

Expression, activity, and subcellular localization of testicular hormone-sensitive lipase during postnatal development in the guinea pig.

The present work reports on testicular hormone-sensitive lipase (HSL), the biological significance of which has been documented in male fertility. The HSL protein levels and enzymatic activity were measured, respectively, by densitometry of immunoreactive bands in Western blots, performed with antibodies against recombinant rat HSL, and by spectrophotometry in seminiferous tubules (STf) and interstitial tissue (ITf) enriched fractions generated from neonatal, pubertal, and adult guinea pig testes. In addition, HSL was studied in subcellular fractions obtained from STf isolated from adult testes and in epididymal spermatozoa (Spz). A 104-kDa HSL protein was detected in STf and ITf, the expression and activity of which increased with testicular development. Three immunoreactive bands of 104, 110, and 120 kDa were detected in the lysosomal subfraction, and two bands of 104 and 120 kDa were detected in Spz. The HSL activity was positively correlated with free (FC) and esterified (EC) cholesterol ratios in STf and ITf, but not with triglyceride (TG) levels, during testicular development. Immunolabeling localized HSL to elongated spermatids and Sertoli cells, where its distribution was stage-dependent, and within the cells lining the excurrent ducts of the testis. The findings of the 104- and 120-kDa HSL immunoreactive bands and of HSL activity in Spz as well, as the detection of the 104-, 110-, and 120-kDa immunoreactive bands in lysosomes, suggest that part of HSL may originate from germ cells and be imported in Sertoli cells. The HSL protein levels and enzymatic activity in ITf and STf were positively correlated with serum testosterone levels during development. To the best of our knowledge, this study is the first to contribute insights regarding the impact of HSL on FC:EC cholesterol ratios and TG levels in the interstitial tissue and tubules in relation to serum testosterone levels during postnatal development, and regarding the immunolocalization of the enzyme in regions of the male gamete consistent with spermatozoa-oocyte interaction.

A role for hormone-sensitive lipase in the selective mobilization of adipose tissue fatty acids

The mobilization of fatty acids from rat and human fat cells is selective according to molecular structure, and notably carbon atom chain length. This study aimed at examining whether the release of individual fatty acids from triacylglycerols (TAG) by hormone-sensitive lipase (HSL) plays a role in the selectivity of fatty acid mobilization. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 18 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation degree from 0 to 3 double bond(s), was measured by comparing the composition of non-esterified fatty acids (NEFA) to that of the original TAG. The relative hydrolysis (% in NEFA/% in TAG) differed between fatty acids, being about 5-fold and 3-fold higher for the most (18:1 n-7) than for the least (24:0) readily released fatty acid by recombinant rat and human HSL, respectively. Relationships were found between the chain length of fatty acids and their relative hydrolysis. Among 12–24 carbon atom saturated fatty acids, the relative hydrolysis markedly decreased (by about 5- and 3-times for recombinant rat and human HSL, respectively) with increasing chain length. We conclude that fatty acids are selectively released from TAG by HSL according to carbon atom chain length. These data provide insight on the mechanism by which fatty acids are selectively mobilized from fat cells.

Substrate-Dependent Lipolysis Induced by Isoproterenol

The relationship between isoproterenol-induced lipolysis and the phosphorylation of perilipin and hormone-sensitive lipase (HSL) was examined using cell-free systems consisting of lipid droplets isolated from rat fat cells and HSL, and/or trioleoylglycerol emulsified with gum arabic and HSL. Isoproterenol was found to stimulate lipolysis in the cell-free system with the lipid droplets without an increase in the phosphorylation of either perilipin or HSL. On the other hand, no stimulation of lipolysis was found in the cell-free system containing lipid droplets despite increases in the phosphorylation of perilipin and HSL. In the cell-free system consisting of trioleoylglycerol emulsified with gum arabic and HSL, neither isoproterenol nor increases in the phosphorylation of perilipin and HSL accelerated lipolysis. These results suggest that isoproterenol-induced lipolysis may not be mediated through the phosphorylation of perilipin and HSL, and may rather be dependent on the substrate of HSL.

Mechanism of the stimulatory action of okadaic acid on lipolysis in rat fat cells

Okadaic acid was found to induce concentration- and time-dependent lipolysis in rat fat cells in the absence of lipolytic hormones, but it did not significantly increase the total hormone-sensitive lipase (HSL) activity in these fat cells, the activity of HSL extracted from fat layer and that of HSL in the supernatant of homogenized fat cells. Western blotting of fat cell homogenate fractions with an antiserum raised against synthetic peptide derived from rat HSL showed that HSL protein shifted from the supernatant to the fat layer in response to okadaic acid, which increased the HSL protein content on the fat layer and concomitantly reduced that of the supernatant, concentration- and time-dependently. Sonication of the fat cells abolished their responsiveness to okadaic acid. The lipolytic action of okadaic acid was examined and its site was identified using a cell-free system comprising lipid droplets isolated from rat fat cells and HSL. Okadaic acid induced lipolysis in this cell-free system and sonication of the lipid droplets caused disappearance of lipolytic action of okadaic acid. Okadaic acid failed to stimulate lipolysis in a cell-free system comprising HSL and artificial lipid droplets (trioleoylglycerol emulsified with gum arabic) instead of lipid droplets isolated from rat fat cells. These results suggest that okadaic acid does not increase the catalytic activity of HSL but induces translocation of HSL to the lipid droplets isolated from rat fat cells. The site of the lipolytic action of okadaic acid in relation to the interaction between HSL and lipid droplet is discussed. —Morimoto, C., A. Kiyama, K. Kameda, H. Ninomiya, T. Tsujita, and H. Okuda. Mechanism of the stimulatory action of okadic acid on lipolysis in rat fat cells.

Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydrophobic ligands. The interaction was demonstrated in vitro by the binding of ALBP to HSL translated in vitro, to HSL in extracts of HSL overexpressing Chinese hamster ovary (CHO) cells, and to HSL in extracts of rat adipose tissue. Finally, the presence of ALBP was documented in immune complexes from rat adipose tissue immunoprecipitated with anti-HSL antibodies. The HSL-ALBP interaction was mapped to an N-terminal 300-aa region of HSL that is distinct from the C-terminal catalytic domain. These results suggest that HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic lipids.

Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages

Foam cells formed from receptor-mediated uptake of lipoprotein cholesterol by macrophages in the arterial intima are critical in the initiation, progression, and stability of atherosclerotic lesions. Macrophages accumulate cholesterol when conditions favor esterification by acyl-CoA:cholesterol acyltransferase (ACAT) over cholesterylester hydrolysis by a neutral cholesteryl-ester hydrolase, such as hormone-sensitive lipase (HSL), and subsequent cholesterol efflux mediated by extracellular acceptors. We recently made stable transfectants of a murine macrophage cell line, RAW 264.7, that overexpressed a rat HSL cDNA and had a 5-fold higher rate of cholesteryl-ester hydrolysis than control cells. The current study examined the effect of macrophage-specific HSL overexpression on susceptibility to diet-induced atherosclerosis in mice. A transgenic line overexpressing the rat HSL cDNA regulated with a macrophage-specific scavenger receptor promoter-enhancer was established by breeding with C57BL/6J mice. Transgenic peritoneal macrophages exhibited macrophage-specific 7-fold overexpression of HSL cholesterol esterase activity. Total plasma cholesterol levels in transgenic mice fed a chow diet were modestly elevated 16% compared to control littermates. After 14 weeks on a high-fat, high-cholesterol diet, total cholesterol increased 3-fold, with no difference between transgenics and controls. However, HSL overexpression resulted in thicker aortic fatty lesions that were 2.5-times larger in transgenic mice. HSL expression in the aortic lesions was shown by immunocytochemistry. Atherosclerosis was more advanced in transgenic mice exhibiting raised lesions involving the aortic wall, along with lipid accumulation in coronary arteries occurring only in transgenics. Thus, increasing cholesteryl-ester hydrolysis, without concomitantly decreasing ACAT activity or increasing cholesterol efflux, is not sufficient to protect against atherosclerosis.—Escary, J-L., H. A. Choy, K. Reue, X-P. Wang, L. W. Castellani, C. K. Glass, A. J. Lusis, and M. C. Schotz. Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages. J. Lipid Res. 1999. 40: 397–404.

Mutational analysis of structural features of rat hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triacylglycerols and cholesteryl esters. HSL activity is regulated via phosphorylation-dephosphorylation, with cyclic AMP-dependent protein kinase increasing activity following phosphorylation of a single serine and Ca2+/calmodulin-dependent protein kinase II phosphorylating another serine at a basal site. The current studies used site-directed mutagenesis to show that Ser-563 of rat HSL is phosphorylated by cyclic AMP-dependent protein kinase and that Ser-565 is phosphorylated by Ca2+/calmodulin-dependent protein kinase II. Mutation of Ser-563-->Ala eliminated HSL hydrolytic activity against cholesteryl ester, triacylglycerol, and diacylglycerol substrates to the same extent as mutation of Ser-423-->Ala, the presumed catalytic site. Mutation of Ser-565-->Ala modestly decreased HSL activity. In contrast, mutation of Ser-563-->Asp preserved HSL hydrolytic activity and even increased activity 20% above the control wild-type enzyme. Molecular modeling of the catalytic pocket of HSL suggested the involvement of Val-710. Mutation of Val-710-->Ala resulted in an 85% loss of HSL hydrolytic activity. The results of these studies illustrate the importance of the presence of a hydroxyl group or negative charge at residue 563, either for proper conformation of rat HSL or for proper stabilization of substrate to allow maintenance of hydrolytic activity, as well as the importance of the involvement of additional amino acids in the catalytic pocket of the enzyme.

Hormone-sensitive lipase overexpression increases cholesteryl ester hydrolysis in macrophage foam cells.

Atherosclerosis is a complex physiopathologic process initiated by the formation of cholesterol-rich lesions in the arterial wall. Macrophages play a crucial role in this process because they accumulate large amounts of cholesterol esters (CEs) to form the foam cells that initiate the formation of the lesion and participate actively in the development of the lesion. Therefore, prevention or reversal of CE accumulation in macrophage foam cells could result in protection from multiple pathological effects. In this report, we show that the CE hydrolysis catalyzed by neutral cholesterol ester hydrolase (nCEH) can be modulated by overexpression of hormone-sensitive lipase (HSL) in macrophage foam cells. For these studies, RAW 264.7 cells, a murine macrophage cell line, were found to be a suitable model of foam cell formation. HSL expression and nCEH activity in these cells and in peritoneal macrophages were comparable. In addition, antibody titration showed that essentially all nCEH activity in murine macrophages was accounted for by HSL. To examine the effect of HSL overexpression on foam cell formation, RAW 264.7 cells were stably transfected with a rat HSL cDNA. The resulting HSL overexpression increased hydrolysis of cellular CEs 2- to 3-fold in lipid-laden cells in the presence of an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. Furthermore, addition of cAMP produced a 5-fold higher rate of CE hydrolysis in cholesterol-laden, HSL-overexpressing cells than in control cells and resulted in nearly complete hydrolysis of cellular CEs in only 9 hours, compared with <50% hydrolysis in control cells. Thus, HSL overexpression stimulated the net hydrolysis of CEs, leading to faster hydrolysis of lipid deposits in model foam cells. These data suggest that HSL overexpression in macrophages, alone or in combination with ACAT inhibitors, may constitute a useful therapeutic approach for impeding CE accumulation in macrophages in vivo.

Overexpression of hormone-sensitive lipase in Chinese hamster ovary cells leads to abnormalities in cholesterol homeostasis

Hormone-sensitive lipase (HSL) is an intracellular enzyme that functions as both a neutral triglyceride and cholesteryl ester hydrolase. In order to explore the effects of HSL on cholesterol homeostasis, Chinese hamster ovary (CHO) cells were transfected with rat HSL and several different stable cell lines that overexpress HSL mRNA, HSL protein, and HSL activity approximately 600-fold were isolated. Cells transfected with HSL contained less cholesteryl esters and unesterified cholesterol than control cells. HSL transfectants expressed 20-60% fewer LDL receptors than control cells when grown in lipid-depleted media or in the presence of mevinolin, as assessed by binding and degradation of LDL and immunoblotting of LDL receptors. In contrast, the rate of cholesterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were increased 3- to 14-fold in HSL transfectants grown in sterol replete media. The rate of cholesterol synthesis and the activity of HMG-CoA reductase increased when cells were grown in lipid-depleted media, and remained markedly elevated compared to control cells. These results show that the regulation of LDL receptor expression and cholesterol synthesis can be dissociated through the actions of HSL and suggest multiple control mechanisms for sterol-responsive genes.

Identification of essential aspartic acid and histidine residues of hormone-sensitive lipase: apparent residues of the catalytic triad

It is expected that hormone-sensitive lipase (HSL), like most other lipases and esterases, adopts an α/β-hydrolase fold and has a catalytic triad of serine, aspartic or glutamic acid, and histidine. Recently, we have published a three-dimensional model for the C-terminal catalytic domain of HSL, having an α/β-hydrolase fold and with Ser-423 1 Residue numbers in this paper refer to the rat HSL amino acid sequence. 1 , Asp-703 and His-733 in the catalytic triad (Contreras et al. (1996) J. Biol. Chem. 271, 31426–31430). It has been shown that Ser-423, situated in the motif GXSXG, is essential for catalysis (Holm et al. (1994) FEBS Lett. 344, 234–238). The suggested aspartic acid and histidine were here probed by site-directed mutagenesis. Mutants of residues Asp-703 and His-733 are devoid of both lipase and esterase activity, which is not the case for mutants of other tested aspartic acid and histidine residues. Thus, the presented data support the three-dimensional model structure with Asp-703 and His-733 as part of the triad. © Federation of European Biochemical Societies.