The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of hormone-sensitive lipase to the surfaces of lipid storage droplets

Abstract

Hormone-sensitive lipase catalyzes the rate-limiting step in the release of fatty acids from triacylglycerol-rich lipid storage droplets of adipocytes, which contain the body’s major energy reserves. Hormonal stimulation of cAMP formation and the activation of cAMP-dependent protein kinase leads to the phosphorylation of hormone-sensitive lipase and a large increase in lipolysis in adipocytes. By contrast, phosphorylation of hormone-sensitive lipase by the kinase in vitro results in a comparatively minor increase in catalytic activity. In this study, we investigate the basis for this discrepancy by using immunofluorescence microscopy to locate hormone-sensitive lipase in lipolytically stimulated and unstimulated 3T3-L1 adipocytes. In unstimulated cells, hormone-sensitive lipase is diffusely distributed throughout the cytosol. Upon stimulation of cells with the β-adrenergic receptor agonist, isoproterenol, hormone-sensitive lipase translocates from the cytosol to the surfaces of intracellular lipid droplets concomitant with the onset of lipolysis, as measured by the release of glycerol to the culture medium. Both hormone-sensitive lipase translocation and lipolysis are reversed by the incubation of cells with the β-adrenergic receptor antagonist, propranolol. The treatment of cells with cycloheximide fails to inhibit lipase translocation or lipolysis, indicating that the synthesis of nascent proteins is not required. Cytochalasin D and nocodazole used singly and in combination also failed to have a major effect, thus suggesting that the polymerization of microfilaments and microtubules and the formation of intermediate filament networks is unnecessary. Hormone-sensitive lipase translocation and lipolysis were inhibited by N-ethylmaleimide and a combination of deoxyglucose and sodium azide. We propose that the major consequence of the phosphorylation of hormone-sensitive lipase following the lipolytic stimulation of adipocytes is the translocation of the lipase from the cytosol to the surfaces of lipid storage droplets.

Introduction

The largest energy reserves in mammals are the triacylglycerols housed within intracellular lipid storage droplets in adipocytes. Energy is released from these cells as fatty acids in response to catecholamine stimulation of β-adrenergic receptors. The resulting elevation of intracellular cAMP activates cAMP-dependent protein kinase (PKA) which, in turn, phosphorylates hormone-sensitive lipase (HSL) [1], the rate-limiting enzyme catalyzing lipolysis. The activation of PKA and phosphorylation of HSL produces very large increases (>50-fold) in lipolysis in suspensions of primary adipocytes (for review, see [2], [3], [4]). By contrast, PKA-mediated phosphorylation of HSL in vitro results in an approximate two-fold increase in enzyme activity as assessed by measuring the hydrolysis of emulsified radiolabeled triacylglycerols [5]. One hypothesis to explain the large increase in lipolysis in intact adipocytes is that the phosphorylation of HSL promotes the translocation of the enzyme from a cytosolic location to the surface of the lipid droplet. Previous studies have demonstrated that lipolytic stimulation of primary rat adipocytes leads to a shift in the location of HSL from primarily supernatant fractions to the floating fat cake fraction of subsequently homogenized cells [6]. Also, stimulation of cultured 3T3-L1 adipocytes leads to a loss of lipolytic activity from soluble fractions of cell homogenates [7]; the latter study did not test for lipolytic activity in the floating fat cake. Thus, support for the translocation hypothesis derives from studies with fractionated cells, which cannot distinguish unequivocally between a physiological association of HSL with lipid droplets in intact cells and association that may be secondary to the disruption and fractionation of cells.

In the present study, we used immunofluorescence microscopy to determine the location of HSL in both resting and lipolytically-stimulated 3T3-L1 adipocytes. The data demonstrate that HSL translocates from the cytosol to the surfaces of lipid droplets upon stimulation of cells with isoproterenol, an agonist of β-adrenergic receptors. Additionally, we demonstrate that intact cytoskeletal elements including microfilaments, microtubules and intermediate filaments are not required for HSL translocation or the subsequent lipid hydrolysis. ATP is required for the process by promoting either the phosphorylation of the lipase or the subsequent translocation.