Peter Strålfors

Regulation of adipose tissue lipolysis: effects of noradrenaline and insulin on phosphorylation of hormone-sensitive lipase and on lipolysis in intact rat adipocytes

In [l] we have demonstrated that hormone-sensitive lipase is phosphorylated in intact rat adipocytes, but the effects of hormones on the extent of this phosphorylation was not studied. The [32P]phosphorylated enzyme protein migrated in a small [“PIphosphopeptide band (1.5% of total) when proteins from adipocytes, incubated with 32Pi, were separated with SDS-PAGE. During the isolation of the [32P] phosphorylated lipase we obtained no indication of heterogeneity of this 84 000 dalton [32P]phosphopeptide band, which suggested that it mainly consisted of [32P]hormone-sensitive lipase [I]. Based on these observations we have derived a method for quantitation of [32P]hormone-sensitive lipase in adipocyte protein extracts. We had developed a technique for continuous monitoring of FFA release from adipocytes by pH-stat titration as a measure of hormone-sensitive lipase activity [2]. In combination, these techniques have enabled us to correlate the time-course of effects of noradrenaline and insulin on phosphorylation and activity of the enzyme, in intact adipocytes. The results suggest that these hormones can regulate lipolysis in the intact cell by altering the extent of phosphorylation of hormonesensitive lipase.

Hormone-sensitive lipase of rat adipose tissue: Identification and some properties of the enzyme protein

The cyclic AMP-dependent stimulation of hormonesensitive lipase activity in adipose tissue preparations, originally described by Bizack [ 1 ] and confirmed by others [2,3] has been shown to be mediated via a protein kinase-catalyzed phosphorylation [4,5]. However, direct evidence for the molecular events taking place upon stimulation is lacking since the enzyme protein has not been isolated. We have recently purified monoacylglycerol lipase from rat adipose tissue by solubilization and fractionation in non-ionic detergent [6]. Using the same technique we have been able to separate solubilized hormonesensitive lipase from other lipolytic enzymes of rat adipose tissue, to identify the enzyme protein and to demonstrate some of its properties, notably a protein kinase-catalyzed phosphorylation.

6 Hormone-Sensitive Lipase

Publisher Summary Hormone-sensitive lipase (HSL) has a major role in fatty acid mobilization from adipose tissue by catalyzing the rate-limiting step in the lipolysis of stored triacylglycerols. From previous work with adipose tissue preparations, fast-acting lipolytic hormones were generally thought to control HSL through cyclic AMP-mediated phosphorylation of the enzyme. This chapter summarizes the molecular mechanisms for short-term hormonal control of the enzyme in adipose tissue and other organs and emphasizes the current state of knowledge and the progress as the result of identification, purification, and characterization of HSL. HSL is inhibited by micromolar concentrations of diisopropyl fluorophosphate (DFP) and the inhibition is parallelled by [3H]DFP incorporation into the enzyme, indicating that a reactive serine residue is involved in its catalytic function. HSL has broad substrate specificity, hydrolyzing long-chain tri-, di-, and monoacylglycerols, as well as cholesterol esters. In several aspects, HSL is similar to other tissue lipases, such as lipoprotein lipase and hepatic lipase. However, the relatively higher diacylglycerol lipase activity and, in particular, the ability to hydrolyze cholesterol esters distinguish HSL from these lipases. Cyclic adenosine monophosphate (AMP)-dependent protein kinase phosphorylates HSL at a single serine residue in a site termed as “the “regulatory phosphorylation site,” which can be found in a proteolytic peptide of about ten amino acid residues.

Regulation of adipose tissue lipolysis: phosphorylation of hormone-sensitive lipase in intact rat adipocytes

The rate-limiting step in the hydrolysis of adipose tissue triacylglycerols is catalyzed by the hormonesensitive lipase [ 11. It has been suggested that the activity of this enzyme is regulated by its phosphorylation, mediated by hormonal alterations of the intracellular concentration of cyclic AMP [2]. Although likely to be correct, this hypothesis has been based on rather indirect experimental evidence. It is, however, supported by our recent results: the identification of the hormone-sensitive lipase protein and the demonstration that the enzyme can be phosphorylated, and activated, in vitro with a cyclic AMP-dependent protein kinase [3,4]. These findings are not necessarily relevant for conditions in the living cell. To assert the physiological relevance of the phosphorylation of the isolated enzyme its phosphorylation must be demonstrated also in the intact adipocyte, i.e., under in vivo conditions. We are studying mechanisms for the hormonal regulation of hormone-sensitive lipase, at the cellular and at the molecular level. Here we have incubated intact rat adipocytes with 3zPi under conditions giving maximal rate of lipolysis (noradrenaline stimulation). Using a recently developed procedure for extensive purification of hormone-sensitive lipase at a preparative scale [5] we have isolated the enzyme from the adipocytes and shown that it is phosphorylated. In [6] we describe how exposure of the adipocytes to hor-

Electrophoretic elution of proteins from polyacrylamide gel slices

A method for electrophoretic elution of proteins from polyacrylamide gel slices is described. Eluted proteins were retained by a discontinuous conductivity gradient (M. Otto and M. Snejdárková, Anal. Biochem. 111, 111-114 (1981)). The method has been adapted to slices from slab gels and gels that have been stained and destained. Proteins were eluted as their sodium dodecyl sulfate complexes. Minute amounts of proteins (0.1 microgram) were recovered in high yield (85-95%) in 2 h in less than 0.1 ml volume.

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6-8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (less than 5 micrograms/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent Km of 60 microM and Vmax of 20 mumol X min-1 X mg-1, corresponding values with mixed histones were 12 microM and 1.2 mumol X min-1 X mg-1. With both protein substrates the apparent Km for ATP was 11 microM. Concentrations of Mg2+ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7-2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated 32Pi from [gamma-32P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.

Phosphorylation of hormone-sensitive lipase by cyclic GMP-dependent protein kinase

In intact rat adipocytes hormone‐sensitive lipase has been shown to be phosphorylated on serine residues in two different phosphorylation sites: a regulatory site phosphorylated by cyclic AMP‐dependent protein kinase and a basal site, which does not directly affect the enzyme activity, phosphorylated by cyclic AMP‐independent protein kinase(s) [(1984) Proc. Natl. Acad. Sci USA 81, 3317‐3321]. Cyclic GMP‐dependent protein kinase catalyzed the phosphorylation of the same two phosphorylation sites on the isolated enzyme, at serine residues. Both sites were phosphorylated at about the same rate, with the hormone‐sensitive lipase activity concomitantly enhanced.

Phosphorylation of the basal site of hormone-sensitive lipase by glycogen synthase kinase-4

In rat adipocytes hormone‐sensitive lipase is phosphorylated at two sites termed ‘regulatory’ and ‘basal’, in the former case by cyclic AMP‐dependent protein kinase causing an activation of the lipase [(1984) Proc. Natl. Acad. Sci. USA 81, 3317‐3321]. Here, the basal phosphorylation site was found to be phosphorylated by glycogen synthase kinase‐4 without any effects on lipase activity, or on the extent of its activation subsequent to phosphorylation of the regulatory site. Glycogen synthase kinase‐3, casein kinase‐I, and casein kinase‐II did not phosphorylate the lipase. Phosphorylase kinase phosphorylated it to a very low extent at a third phosphorylation site not phosphorylated in the fat cell.

Autolysis of isolated adipocytes by endogenously produced fatty acids.

Isolated rat adipocytes, in which lipolysis was maximally stimulated by isoproterenol, continued to produce fatty acids in excess of the high‐affinity binding capacity of the fatty acid acceptor bovine serum albumin in the incubation medium. At an average of 17 mol fatty acids/mol serum albumin, there was a burst of lactate dehydrogenase activity recovered in the incubation medium, indicating cell lysis. It is concluded that endogenously produced fatty acids will cause autolysis of adipocytes in vitro. Actively lipolysing adipocytes were more fragile than resting cells, since increasing amounts of lactate dehydrogenase activity was recovered in the medium during active lipolysis.

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.