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.     

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.     

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.     

Hormone-sensitive lipase of adipose tissue.

Some physiologic aspects of the mobilization and fate of free fatty acids are reviewed. The molecular mechanism of the activation of hormone-sensitive lipase in adipose tissue is then discussed. Recent evidence established that hormone-sensitive lipase, concerned with fat mobilization, is both functionally and immunochemically distinct from lipoprotein lipase, concerned with uptake of plasma triglycerides. Lipoprotein lipase activity is not altered by cyclic AMP-dependent protein kinase. The latter enzyme enhances not only triglyceride hydrolase but also monoglyceride, diglyceride and cholesterol ester hydrolase activities in chicken adipose tissue. Finally, it is shown that the activation of all four acyl hydrolases is reversible, the deactivation being magnesium-dependent. Protein phosphatase fractions from heart and liver active against phosphorylase a can reversibly deactivate adipose tissue hormone-sensitive lipase, implying a low degree of substrate specificity for lipase phosphatase.     

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.     

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.     

Hormone-sensitive lipase from swine adipose tissue: identification and some properties

Swine adipose tissue hormone-sensitive lipase, purified 475-fold to 10% protein purity, has been identified as a polypeptide of Mr = 84,000. The enzyme has high specific activity against tri-, di- and monoacylglycerols, as well as cholesterol esters, and is inhibited by millimolar NaF, and micromolar HgCl2 and DFP. The enzyme polypeptide serves as a substrate for cyclic AMP-dependent protein kinase. The characteristics of the hormone-sensitive lipase from swine adipose tissue are similar to those reported previously for the enzyme from rat. They differ from those reported for the lipase from chicken adipose tissue, and possible reasons for these differences are discussed.     

Changes in the expression of uncoupling proteins and lipases in porcine adipose tissue and skeletal muscle during feed deprivation*(1)

The hormone-sensitive and lipoprotein lipases are critical determinants of the metabolic adaptation to starvation. Additionally, the uncoupling proteins have emerged with potential roles in the metabolic adaptations required by energy deficiency. The objective of this study was to evaluate the expression (mRNA abundance) of uncoupling proteins 2 and 3 and that of hormone-sensitive and lipoprotein lipase in the adipose tissue and skeletal muscle of the pig in relationship to feed deprivation. Thirty-two male castrates (87 kg +/- 5%) were assigned at random to fed and feed-deprived treatment groups. After 96 hr, the pigs were euthanized and adipose and skeletal muscle tissue obtained for total RNA extraction and nuclease protection assays. Feed deprivation increased uncoupling protein 3 mRNA abundance 103-237% (P < 0.01) in longissimus and red and white semitendinosus muscle. In contrast, the increase in uncoupling protein 3 mRNA in adipose tissue was only 23% (P < 0.06), and adipose uncoupling protein 2 mRNA was not influenced (P > 0.66) by feed deprivation. The increased abundance of uncoupling protein 2 mRNA in the longissimus muscle of feed-deprived pigs was small (22%), but significant (P < 0.04). The expression of hormone-sensitive lipase was increased 46% and 64% (P < 0.04) in adipose tissue and longissimus muscle, respectively, by feed deprivation, whereas adipose lipoprotein lipase expression was reduced (P < 0.01) to 20% of that of the fed group. Longissimus lipoprotein lipase expression in the feed-deprived group was 37% of that of the fed group (P < 0.01), and similar reductions were detected in red and white semitendinosus muscle. Overall, these findings indicate that uncoupling protein 3 expression in skeletal muscle is quite sensitive to starvation in the pig, whereas uncoupling protein 2 changes are minimal. Furthermore, we conclude that hormone-sensitive lipase is upregulated at the mRNA level with prolonged feed deprivation, whereas lipoprotein lipase is downregulated.     

Cholesterol esterase in rat adipose tissue and its activation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

A high level of cholesterol esterase activity, comparable to that of hormone-sensitive triglyceridase, has been demonstrated in rad adipose tissue. Essentially all of the activity was in the isolated adipocytes, primarily in the 100,000 times g supernatant fraction of the adipocytes. Cholesterol esterase activity in the 100,000 times g supernatant fraction was increased 40 plus or minus 16% by incubation with ATP (0.5 mM), Mg-2+ (1.25 mM), and cyclic adenosine 3':5'-monophosphate (cyclic AMP) (10 muM), conditions which also activated hormone-sensitive triglyceridase. Protein kinase inhibitor (rabbit skeletal muscle) blocked activation, and activation was restored by the addition of excess protein kinase (bovine skeletal muscle). In extracts prepared from adipocytes first incubated for 5 min with 10 muM epinephrine and 1 mM theophylline, there was no cyclic AMP-dependent cholesterol esterase activation, implying that the enzyme had been activated by a similar mechanism in the intact cell. The physiological role of this high level of cholesterol esterase activity in adipose tissue is unclear. Its relationship to hormone-sensitive triglyceride lipase, with which it extensively co-fractionates, and its possible involvement in fat mobilization remain to be determined.     

Activation of chicken adipose tissue diglyceride lipase by cyclic AMP-dependent protein kinase and its deactivation by purified protein phosphatase.

Diglyceride lipase of chicken adipose tissue was found to be activated by cyclic AMP-dependent protein kinase to the same extent as hormone-sensitive triglyceride lipase (3-to 10-fold) when lipase assays were carried out in buffers of low ionic strength. Sodium phosphate (50 mM) or sodium chloride (100 mM) preferentially enhanced the basal (nonactivated) form of diglyceride lipase, which minimized the apparent activation by protein kinase. The activated diglyceride lipase was readily deactivated by a pure protein phosphatase from bovine heart (MW 35,000) and the deactivated enzyme was then reactivated by protein kinase.     

Inhibitors of protein phosphatase-1. Inhibitor-1 of bovine adipose tissue and a dopamine- and cAMP-regulated phosphoprotein of bovine brain are identical

Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP-dependent protein kinase on a threonyl residue; this phosphorylation was necessary for inhibition of protein phosphatase-1. Bovine adipose tissue inhibitor-1 was compared directly with rabbit skeletal muscle inhibitor-1 and with a 32000-Mr, dopamine- and cAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of protein phosphatase-1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor-1 was found to be very similar and possibly identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer electrophoresis/chromatography; elution on reverse-phase HPLC; chymotryptic peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct from that of skeletal muscle (inhibitor-1).     

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).     

Enzymes catalyzing the hydrolysis of long-chain monoacyglycerols in rat adipose tissue

Acetone-ether preparations of epididymal fat pads from fasted or fed rats contained two enzymes catalyzing the hydrolysis of long-chain monoacylglycerols. The enzymes were identified as monoacylglycerol lipase (Tornqvist, H. and Belfrage, P., (1976) J. Biol Chem. 251, 813--819) and lipoprotein lipase by their apparent pI values after electrofocusing in non-ionic detergent, selective inhibition properties, substrate specificity and positional specificity. It was estimated that monoacylglycerol lipase accounted for about 90% of the total monoacylglycerol-hydrolyzing activity in acetone-ether preparations from fasted and 70% from fed rats. Its enzyme activity did not change with the nutritional state in contrast to that of lipoprotein lipase. The latter enzyme hydrolyzed 2-monoacylglycerols at a much lower rate than the 1(3)-isomers. Monoacylglycerol lipase was located almost entirely in the adipocytes, thus most of the enzyme activity towards monoacylglycerols in the adipose tissue was found in this site. Fractionated sucrose homogenates of rat epididymal fat pads also contained a third enzyme with monoacylglycerol-hydrolyzing activity, identified as hormone-sensitive lipase by its pI, selective inhibition properties and substrate specificity. It was estimated that hormone-sensitive lipase accounted for less than 20% of the total activity against monoacylglycerols in these tissue preparations from fasted rats. Over-all quantitative estimations emphasized the dominant role of monoacylglycerol lipase over the other two enzymes in the hydrolysis of monoacylglycerols.     

The in vitro effect of growth hormone on adipose tissue lipoprotein lipase in rats

The $\text{in vitro}$ effect of growth hormone on adipose tissue lipoprotein lipase was studied in rats. Epididymal adipose tissue was incubated with human growth hormone in the presence of heparin. Growth hormone at a concentration of 0.1 μg per ml decreased by approximately 20% (p<0.005) the heparin-releasable lipoprotein lipase in rat adipose tissue. Discussion was focussed on the reciprocal changes caused by growth hormone of the activities of lipoprotein lipase and hormone-sensitive lipase in the rat adipose tissue.     

Hormone-sensitive lipase of rat adipose tissue: correlation of activity with a protein of molecular weight 84 000

Hormone-sensitive lipase of rat adipose tissue was partially purified. The enzyme retained its capacity to be activated by cyclic AMP-dependent protein kinase throughout purification. When the partially purified 32P-labeled preparation was subjected to two-dimensional gel electrophoresis, the enzyme activity was found to be associated with a 32P-labeled protein of molecular weight 84 000. The result suggests that this 32P-labeled protein represents hormone-sensitive lipase or the catalytic subunit of the enzyme.     

Growth hormone-mediated breakdown of body fat: effects of GH on lipases in adipose tissue and skeletal muscle of old rats fed different diets.

Lipid storage and breakdown is mainly controlled by lipoprotein lipase and hormone-sensitive lipase. The aim of this work was to elucidate whether growth hormone mediated loss of adipose tissue involves a concerted action on tissue lipases, and to what degree such events are modulated by dietary regimen. Twelve-month-old rats fed first a high-fat diet or a low-fat diet for 14 weeks were injected with saline or growth hormone (4 mg/kg/d) for four days or three weeks in different combinations with either high- or low-fat diets. In adipose tissue, growth hormone generally inhibited lipoprotein lipase and also attenuated the inhibiting effect of insulin on hormone-sensitive lipase activity. Growth hormone treatment combined with restricted high-fat feeding reduced the activity of both lipases in adipose tissue and stimulated hormone-sensitive lipase in muscle. Generally, plasma levels of free fatty acids, glycerol and cholesterol were reduced by growth hormone, and in combination with restricted high-fat feeding, triglyceride levels improved too. We conclude that growth hormone inhibits lipid storage in adipose tissue by reducing both lipoprotein lipase activity and insulin's inhibitory action on hormone-sensitive lipase. We also propose that growth hormone's effects on tissue lipases and blood lipids are modulated by dietary regimen.     

Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. I. Phosphorylase and phosphorylase phosphatase.

Glycogen phosphorylase from swine adipose tissue was purified nearly 700-fold using ethanol precipitation, DEAE-cellulose adsorption, AMP-agarose affinity chromatography, and agarose gel filtration. The purified enzyme migrated as one major and several minor components during polyacrylamide gel electrophoresis. Activity was associated with the major component and at least one of the minor components. The molecular weight of the disaggregated, reduced, and alkylated enzyme, estimated by polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate, was 90,000. Stability of the purified enzyme was considerably increased in the presence of AMP. The isoelectric pH of the enzyme in crude homogenates was 6.3. The sedimentation coefficient of the purified enzyme (7.9 S) and that in crude homogenates (7.3 S) was determined by sucrose density gradient sedimentation. Optimal pH for activity was between pH 6.5 and 7.1. Apparent Km values for glycogen and inorganic phosphate were 0.9 mg/ml and 6.6 mM, respectively. The Ka for AMP was 0.21 mM. Enzyme activity was increased by K2SO4, KF, KCl, and MgCl2 and decreased by NaCl, Na2SO4, D-glucose, and ATP. Inhibition by glucose was noncompetitive with the activator AMP; inhibition by ATP was partially competitive with AMP. The purified enzyme was activated by incubation with skeletal muscle phosphorylase kinase. Enzyme in crude homogenates was activated by the addition of MgCl2 and ATP; activation was not blocked by addition of protein kinase inhibitor, suggesting that phosphorylase kinase in homogenates of swine adipose tissue is present largely in an activated form. Deactivation of phosphorylase a by phosphorylase phosphatase was studied using enzyme purified approximately 200-fold from swine adipose tissue by ethanol precipitation, DEAE-cellulose chromatography, and gel filtration. The Km of the adipose tissue phosphatase for skeletal muscle phosphorylase a was 6 muM. The purified swine adipose tissue phosphorylase, labeled with 32-P, was inactivated and dephosphorylated by the adipose tissue phosphatase. Dephosphorylation of both skeletal muscle and adipose tissue substrates was inhibited by AMP and glucose reversed this inhibition. Several lines of evidence suggest that AMP inhibition was due to an action on the substrate rather than on the enzyme. We have previously reported that the system for phosphorylase activation in rat fat cells differs in some important characteristics from that in skeletal muscle. However, both swine fat phosphorylase and phosphorylase phosphatase have major properties very similar to those described for the enzymes from skeletal muscle.     

Triglyceride, diglyceride, monoglyceride, and cholesterol ester hydrolases in chicken adipose tissue activated by adenosine 3':5'-Monophosphate-dependent protein kinase. Chromatographic resolution and immunochemical differentiation from lipoprotein lipase.

Hormone-sensitive lipase and cholesterol ester hydrolase of chicken adipose tissue were markedly activated by adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (on the average, 235 to 275%; occasionally as much as 1000%). Diglyceride and monoglyceride hydrolases were also activated, but to a lesser extent (60 to 87%). The activation of all four hydrolases was inhibited by protein kinase inhibitor and reversed by the addition of exogenous protein kinase. Following activation by cAMP-dependent protein kinase, all four hydrolases were deactivated in a Mg2+-dependent reaction and then reactivated to or near initial levels on incubation with cAMP and Mg2+-ATP. The reversible deactivation is assumed to reflect activity of one or more protein phosphatases. The maximum activation obtainable for the four hydrolases decreased when the tissue had been previously exposed to glucagon, indicating that the glucagon-induced activation was probably similar to or identical with the activation demonstrated in cell-free preparations. The pH optima for the four hydrolase activities were similar (7.13 to 7.38). Although the absolute activities and relative degrees of kinase activation differed according to the particular emulsified substrates used, the results do not rule out the possibility that all four hydrolase activities are referable to a single hormone-sensitive hydrolase. Hormone-sensitive acyl hydrolases were separated from lipoprotein lipase by heparin-Sepharose affinity chromatography. Lipoprotein lipase was active against triolein, diolein, and monoolein, but not cholesterol oleate. Incubation of lipoprotein lipase with exogenous protein kinase, cAMP, and Mg2+ATP had no effect on any of the three hydrolase activities. Lipoprotein lipase was further purified to homogeneity and used to prepare antiserum in rabbits. The immunoglobin G fraction from these antisera completely inhibited lipoprotein lipase eluted from heparin-Sepharose columns. However, the hormone-sensitive hydrolase activities (not retained on heparin-Sepharose affinity chromatography) were not inhibited by anti-lipoprotein lipase immunoglobin G, and anti-lopoprotein lipase immunoglobin G did not affect the activation process in crude fractions. Thus, hormone-sensitive lipase and lipoprotein lipase, functionally distinct enzymes, have been physically resolved and immunochemically distinguished. Apparently lipoprotein lipase activity is not regulated, at least directly, by cAMP-dependent protein kinase.     

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.     

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.