Activation of hormone-sensitive lipase from adipose tissue by cyclic AMP-dependent protein kinase

Lipase activation requiring cyclic-3′,5′-adenosine monophosphate and ATP was demonstrated in crude fractions of human adipose tissue homogenates. Activation was totally blocked by addition of the specific protein kinase inhibitor. Levels of endogenous protein kinase were adequate to support clear-cut activation but in partially purified preparations addition of exogenous (rabbit muscle) kinase further enhanced activation. When tissue was treated with epinephrine prior to homogenization the degree of activation in partially purified fractions was distinctly reduced. The mechanism of activation of hormone-sensitive lipase in human adipose tissue is thus shown, like that in rat adipose tissue, to be linked to a cyclic AMP-dependent protein kinase.     

Studies on the hormone-sensitive lipase of adipose tissue

Sucrose gradient centrifugation has been used to examine the triglyceride lipases present in extracts of rat epididymal adipose tissue. The aqueous infranatant recovered between the pellet and fat cake of tissue homogenates which had been centrifuged at 40,000 g was shown to contain two types of triglyceride lipase activity. One of these appears in the 15s region and has been identified as the active form of the "hormone-sensitive lipase" believed to be responsible for initiating the hydrolysis of tissue triglyceride stores in response to lipolytic stimuli. The activity of this enzyme was selectively increased in extracts prepared from tissue exposed to epinephrine and decreased in extracts of insulin-treated tissue. The increased lipolytic activity of extracts of tissue from fasted or fasted-refed rats was also found largely in this region. When the tissue was incubated with orthophosphate-(32)P, radioactivity was incorporated into a protein migrating at 15s. A second peak of triglyceride lipase activity appeared in the 6s region coincident with the location of the monoglyceride and diglyceride lipase activities. The amount of 6s triglyceride lipase activity did not correlate with changes in the lipolytic activity of the tissue from which the extracts were prepared, and its physiological function remains to be elucidated. The lipoprotein lipase and the short-chain triglyceride lipase ("tributyrinase") each moved more slowly in the gradient than the 6s triglyceride lipase. Both the 6s and 15s enzymes were shown to be present in washed adipocytes isolated from the tissue by collagenase digestion.     

Rapid assay for hormone-sensitive lipase activity of adipose tissue

A highly specific and rapid assay for hormone-sensitive lipase activity of rat adipose tissue is described. The method employs emulsified 2,3-di-O-oleyl-[9,10-(3)H(2)]oleoyl glycerol as a substrate; it is very sensitive and is suitable for serial sampling.     

Role of hormone-sensitive lipase in beta-adrenergic remodeling of white adipose tissue

Free fatty acids (FFA) are important extracellular and intracellular signaling molecules and are thought to be involved in beta-adrenergic-induced remodeling of adipose tissue, which involves a transient inflammatory response followed by mitochondrial biogenesis and increased oxidative capacity. This work examined the role of hormone-sensitive lipase (HSL), a key enzyme of acylglycerol metabolism, in white adipose tissue (WAT) remodeling using genetic inactivation or pharmacological inhibition. Acute treatment with the beta(3)-adrenergic agonist CL-316,243 (CL) induced expression of inflammatory markers and caused extravasation of myeloid cells in WAT of wild-type (WT) mice. HSL-knockout (KO) mice had elevated inflammatory gene expression in the absence of stimulation, and acute injection of CL did not further recruit myeloid cells, nor did it further elevate inflammatory gene expression. Acute pharmacological inhibition of HSL with BAY 59-9435 (BAY) had no effect on inflammatory gene expression in WAT or in cultured 3T3-L1 adipocytes. However, BAY prevented induction of inflammatory cytokines by beta-adrenergic stimulation in WAT in vivo and in cultured 3T3-L1 adipocytes. Chronic CL treatment stimulated mitochondrial biogenesis, expanded oxidative capacity, and increased lipid droplet fragmentation in WT mice, and these effects were significantly impaired in HSL-KO mice. In contrast to HSL-KO mice, mice with defective signaling of Toll-like receptor 4, a putative FFA receptor, showed normal beta-adrenergic-induced remodeling of adipose tissue. Overall, results reveal the importance of HSL activity in WAT metabolic plasticity and inflammation.     

p-nitrophenyl butyrate hydrolyzing activity of hormone-sensitive lipase from bovine adipose tissue

The "esterase" activity of hormone-sensitive lipase (HSL) was studied using water-soluble p-nitrophenyl butyrate (PNPB) as a substrate. Bovine adipose tissue HSL was purified to near homogeneity by precipitation at pH 5.0, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, and high performance ion-exchange columns on Mono Q and Mono S. The purified preparation hydrolyzed emulsified triolein and cholesteryl oleate (CO), and water-soluble PNPB. In the two last steps of purification, the elution profile of the CO-hydrolyzing activity coincided with that of PNPB-hydrolyzing activity. The HSL was adsorbed to heparin-Sepharose and the CO- and PNPB-hydrolyzing activities were eluted together in the same peak. Diisopropylfluorophosphate (DFP) strongly inhibited the HSL activities and the inhibition profiles of the triolein-; CO-, and PNPB-hydrolyzing activities were essentially identical. Only one polypeptide of Mr 84,000 in partial purified HSL fraction was labeled by affinity labeling with [3H]DFP. On digestion of the enzyme with trypsin, the triolein- and CO-hydrolyzing activities were lost more rapidly than the PNPB-hydrolyzing activity. Phosphorylation increased the triolein-hydrolyzing activity to 40% more than that of the control, but did not affect the CO- and PNPB-hydrolyzing activities.     

Reversible protein kinase activation of hormone-sensitive lipase from chicken adipose tissue

Hormone-sensitive lipase partially purified from adipose tissue of laying hens was markedly activated by cyclic AMP-dependent protein kinase. Activation was approximately 4-fold (ranging up to as great as 10-fold) compared with the much lower degree of activation obtained with analogous preparations from rat and human adipose tissues (59 and 86%, respectively). The partially purified preparations contained adequate endogenous protein kinase activity to effect complete activation with addition of cyclic AMP, ATP, and Mg(2+). Activation was blocked by protein kinase inhibitor (from rabbit skeletal muscle) but could be restored fully by addition of excess exogenous protein kinase (from bovine skeletal muscle). The fully activated lipase was slowly deactivated by dialysis at 4 degrees C and then rapidly and almost fully reactivated by addition of cyclic AMP and ATP-Mg(2+). Reactivation was blocked by protein kinase inhibitor. This deactivation-reactivation cycle was rapid at 23 degrees C with dialysis against charcoal and could be demonstrated repeatedly using a single preparation. The reversible deactivation of protein kinase-activated enzyme is presumed to reflect the action of a lipase phosphatase. Lipase prepared from tissue previously exposed to glucagon yielded a much smaller degree of activation than lipase prepared from tissue not exposed to the lipolytic hormone, indicating that the physiological hormone-induced activation is probably similar to or identical with the protein kinase activation demonstrated in the cell-free preparations. Under the conditions of assay used, the partially purified lipase fraction contained diglyceride, monoglyceride, and lipoprotein lipase activities. However, treatment with cyclic AMP-dependent protein kinase had virtually no effect on these lipase activities.     

Stimulation of the hormone-sensitive triacylglycerol lipase from adipose tissue by phosphatidylethanolamine

The activity of a pigeon adipose tissue hormone-sensitive triacylglycerol lipase preparation was increased from 2- to 5-fold by the presence of phosphatidylethanolamine in assays with three different methods of preparing triolein substrates. Phosphatidylethanolamine from egg yolk produced the greatest stimulation of lipase activity; the stimulation was concentration-dependent but was not time-dependent. A comparable increase in triacylglycerol lipase activity due to phosphatidylethanolamine was also observed with enzyme preparations from chicken and rat adipose tissue. Phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, Triton X-100 and sodium dodecyl sulfate all inhibited enzyme activity. Phosphatidylethanolamine had no effect on acid lipase activity in the pigeon adipose tissue preparation. Preincubation of the pigeon adipose tissue lipase with ATP, cyclic AMP and protein kinase resulted in a 2.15-fold activation of hydrolase activity determined in the absence of phosphatidylethanolamine. In contrast, non-activated and protein kinase-activated forms of the lipase were characterized as having very nearly the same activity in assays with substrate preparations containing phosphatidylethanolamine. The phosphatidylethanolamine-dependent stimulation of lipase activity was characterized kinetically as being due to an increase in maximal velocity. The modulation of the adipose tissue hormone-sensitive lipase activity by phospholipids could be involved in the hormonal regulation of lipolysis.     

Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism

The mobilization of free fatty acids from adipose triacylglycerol (TG) stores requires the activities of triacylglycerol lipases. In this study, we demonstrate that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes contributing to TG breakdown in in vitro assays and in organ cultures of murine white adipose tissue (WAT). To differentiate between ATGL- and HSL-specific activities in cytosolic preparations of WAT and to determine the relative contribution of these TG hydrolases to the lipolytic catabolism of fat, mutant mouse models lacking ATGL or HSL and a mono-specific, small molecule inhibitor for HSL (76-0079) were used. We show that 76-0079 had no effect on TG catabolism in HSL-deficient WAT but, in contrast, essentially abolished free fatty acid mobilization in ATGL-deficient fat. CGI-58, a recently identified coactivator of ATGL, stimulates TG hydrolase activity in wild-type and HSL-deficient WAT but not in ATGL-deficient WAT, suggesting that ATGL is the sole target for CGI-58-mediated activation of adipose lipolysis. Together, ATGL and HSL are responsible for more than 95% of the TG hydrolase activity present in murine WAT. Additional known or unknown lipases appear to play only a quantitatively minor role in fat cell lipolysis.     

Human adipose tissue hormone-sensitive lipase: identification and comparison with other species

The mRNA for human hormone-sensitive lipase (HSL) was identified using Northern blot analysis and a cDNA-probe for rat HSL. As in the rat, human adipose tissue expresses a single mRNA species of 3.3 kb. Using Western blotting with a polyclonal rabbit antibody towards rat adipose tissue HSL, the corresponding enzyme in human adipose tissue was identified with an apparent 88 kDa polypeptide, thus slightly larger than the rat and bovine 84 kDa, and the mouse and guinea-pig 82 kDa species. Additional evidence for the identification was provided by the inhibition of HSL diacylglycerol lipase activity by the anti-rat HSL antibody, and by NaF, DFP and Hg2+, known inhibitors of HSL. The concentration of the enzyme, as reflected by its activity per g tissue and the specific activity was about two thirds of that in the rat adipose tissue (200 g rats). The identification of the human enzyme protein made it possible to directly demonstrate its phosphorylation by cAMP-dependent protein kinase, thus extending the previous report regarding activation of the lipase with this kinase and ATP-Mg2+ in human adipose tissue extracts (Khoo, J.C., Aquino, A.A. and Steinberg, D. (1974) J. Clin. Invest. 53, 1124-1131).     

Role of phosphoprotein phosphatases in reversible deactivation of chicken adipose tissue hormone-sensitive lipase.

The reversible deactivation of chicken adipose tissue hormone-sensitive lipase alpha(previously activated with Mg2+ ATP and adenosine 3':5'-monophosphate) required Mg2+ and was inhibited by phosphate. These results are consistent with the assumption that deactivation of the protein kinase-activated enzyme is catalyzed by a lipase phosphatase. Cholesterol ester is catalyzed by a lipase phosphatase. Cholesterol ester hydrolase similarly was activated and reversibly deactivated. The activity of endogenous lipase phosphatase in pH 5.2 precipitate fractions was reduced, and in some cases eliminated, by incubation at 50 degrees for 20 min in buffer containing 20% glycerol. Heating at 50 degrees greatly increased the apparent percentage activation of triglyceride and cholesterol ester hydrolases but this was due to a selective decrease in basal (nonactivated) hydrolase activities. Essentially all endogenous lipase phosphatase could be removed by treatment of the pH 5.2 precipitate fraction with ATP-Sepharose affinity gel. The addition of a partially purified preparation of rat liver phosphorylase phosphatase deactivated triglyceride and cholesterol ester hydrolases. The deactivation process was concentration, 5 mM) and was inhibited by 5 mM phosphate and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipase alpha was also observed with crude prepa- and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipas alpha was also observed with crude preparations of phosphoprotein phosphatases from rat and turkey hearts, and from rat epididymal fat pads. Thus, hormone-sensitive lipase is deactivated by a variety of phosphoprotein phosphatases from different tissues and different species, implying a low degree of specificity for the deactivating system.     

Inflammatory response in white adipose tissue in the non-obese hormone-sensitive lipase null mouse model

In the present study, a local inflammatory response in white adipose tissue from the nonobese HSL-null mouse model is demonstrated. The protein levels of several well-known markers of inflammation, like TNFalpha and ferritin HC, were highly increased and accompanied by an activation of NFkappaB. A number of macrophage proteins, i.e., gal-3, Capg, and MCP-4, were expressed at increased levels and immunohistochemical analyses revealed an increased infiltration of F4/80+ cells.     

Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue

A polyclonal rabbit antibody was used to detect hormone-sensitive lipase in rat organs other than white adipose tissue. Inhibition of tissue diacylglycerol lipase activity by the anti-hormone-sensitive lipase, and by NaF, Hg2+ and diisopropyl fluorophosphate, known inhibitors of the hormone-sensitive lipase, demonstrated its presence in the adrenals, ovaries, testes, heart and skeletal muscle, but not in the liver and kidneys. After enrichment by immunoprecipitation an immunoreactive protein, corresponding to the adipose tissue hormone-sensitive lipase 84 kDa subunit, and some additional, higher Mrapp proteins, were detected by Western blotting in the same tissues. The adipose tissue contained greater than 80% of the total hormone-sensitive lipase, with 5-10- and 50-100-fold lower specific activity in the steroid-producing and the muscle tissues, respectively.     

Direct evidence for protein phosphatase-catalyzed dephosphorylation/deactivation of hormone-sensitive lipase from adipose tissue

Incubation of purified hormone-sensitive lipase, 32P-phosphorylated with the catalytic subunit of cyclic AMP-dependent protein kinase and [gamma-32P]ATP-Mg2+, with partially purified protein phosphatase from the same tissue caused a rapid decrease of the 32P content of the enzyme protein. Deactivation of the lipase towards emulsified trioleoylglycerol was temporally related to the dephosphorylation with approx. 80% decrease of both phosphorylation and activity within 30 min. Addition of ATP-Mg and cyclic AMP-dependent protein kinase to the dephosphorylated lipase was shown to rephosphorylate and reactivate the enzyme. These findings are the first direct demonstration of reversible protein phosphatase-catalyzed dephosphorylation/deactivation of hormone-sensitive lipase.