Gudrun Fredrikson

Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of adipocyte triacylglycerol

The respective roles of monoacylglycerol lipase and hormone-sensitive lipase in the sequential hydrolysis of adipose tissue triacylglycerols have been examined. An adipose tissue preparation, containing both lipases in approximately the same proportion as in the intact tissue, hydrolyzed emulsified tri- or dioleoylglycerol to fatty acids and glycerol, with little accumulation of di- or monooleoylglycerol. Selective removal of the monoacylglycerol lipase by immunoprecipitation markedly reduced the glycerol release. Isolated hormone-sensitive lipase hydrolyzed acylglycerols with a marked accumulation of monoacylglycerol in accordance with the positional specificity of this enzyme (Fredrikson, G. and Belfrage, P. (1983) J. Biol. Chem. 258, 14253-14256). Addition of increasing amounts of isolated monoacylglycerol lipase led to a corresponding increase in glycerol release, due to hydrolysis of the monoacylglycerols formed. The reaction proceeded to completion when the relative proportion of the two lipases was similar to that in the intact tissue. These findings indicate that hormone-sensitive lipase catalyzes the hydrolysis of triacylglycerol in the rate-limiting step of adipose tissues lipolysis, and of the resulting diacylglycerol, whereas the action of monoacylglycerol lipase is required in the final hydrolysis of the 2-monoacylglycerols produced.

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.

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes. Studies using the selective inhibitor wortmannin.

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC50≈ 100 nM), the insulin‐induced activation and phosphorylation of cGMP‐inhibited cAMP phosphodiesterase (cGI‐PDE) as well as the activation of the insulin‐stimulated cGI‐PDE kinase (IC50≈ 10–30 nM). No direct effects of the inhibitor on the insulin‐stimulated cGI‐PDE kinase, the cGI‐PDE and the hormone‐sensitive lipase were observed. These data suggest that activation of PI 3‐kinase upstream of the insulin‐stimulated cGI‐PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin‐induced cGI‐PDE activation/ phosphorylation and anti‐lipolysis.

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.

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-

Localization of hormone-sensitive lipase to rat Sertoli cells and its expression in developing and degenerating testes

Using in situ hybridization, hormone‐sensitive lipase was found to be expressed in a stage‐dependent manner in Sertoli cells of rat testis. No expression was found in Leydig cells but expression in spermatids could not be excluded. These results suggest a role for hormone‐sensitive lipase in the metabolism of lipid droplets in Sertoli cells, in contrast to its previously proposed function in steroid biosynthesis. The expression of testicular hormone‐sensitive lipase mRNA and protein, both larger in size compared to other tissues, coincided with the onset of spermatogenesis and was dependent on scrotal localization of the testis, suggesting a temperature‐dependent, pretranslational regulation of expression.

Hormone-sensitive lipase in brown adipose tissue: Identification and effect of cold exposure

Hormone-sensitive lipase (HSL) in brown adipose tissue from mice was identified through immunoprecipitation with a polyclonal antibody (anti-HSL) towards rat white fat HSL and Western blotting. An 82 kDa polypeptide, slightly smaller than the rat white fat HSL 84 kDa subunit, was detected and its identity as HSL verified by inhibition properties. The HSL concentration per g tissue was several-fold higher in the mouse brown adipose tissue than in the rat white adipose tissue, but the specific activities per mg protein were similar. Cold-exposure (4°C of the mice for 24 h approximately doubled the HSL concentration but this increase parallelled the overall protein increase and did not reflect a specific effect on the HSL.

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).

Incorporation of Hormone-Sensitive Lipase into Phosphatidylcholine Vesicles

Enzymatically active, detergent-solubilized purified hormone-sensitive lipase (HSL) was incorporated into phosphatidylcholine (PC) vesicles, using a detergent-dialysis procedure with small PC vesicles, obtained by sonication, as phospholipid source and CHAPS, a zwitterionic bile-salt derivative, as detergent. Association of enzyme protein with the PC vesicles was verified by floatation in a discontinuous dextran gradient and by gel chromatography. An average of 35% of added HSL was incorporated into the vesicles. The vesicles were shown, by quasi-elastic light scattering and electron microscopy, to have a diameter of approximately 160 nm. The vesicleassociated HSL could be phosphorylated by cyclic AMP-dependent protein kinase. The vesicles were stable, both with regard to enzyme activity and size, for at least 4 days when stored at 4°C. The preparation of detergent-free, vesicle-associated and stable HSL provides new possibilities to study some of its properties, and supports and extends the previous report (Holm, C., Fredrikson, G., and Belfrage, P.,J. Biol. Chem. 261, 15659–15661, 1986) which demonstrated the amphiphilic character of HSL.

Expression of biologically active hormone-sensitive lipase in mammalian (COS) cells

cDNAs encoding rat adipose tissue hormone‐sensitive lipase were expressed in COS cells, under the control of the SV40 promoter to half the level in rat adipocytes, the richest native source of the enzyme. A cDNA lacking most of the long 5′‐untranslated region of the full‐length rat hormone‐sensitive lipase cDNA was, with regard to the lipase activity, on the average 70% more efficiently expressed than the full‐length cDNA. The recombinant protein was almost identical to hormone‐sensitive lipase of rat adipose tissue with respect to specific activity, susceptibility to inhibitors, molecular size, phosphorylation and activation by cyclic AMP‐dependent protein kinase. The described eukaryotic expression system will allow analysis of effects of amino acid substitutions introduced into the lipase molecule by site‐directed mutagenesis.