Translocation of Hormone-sensitive Lipase and Perilipin upon Lipolytic Stimulation of Rat Adipocytes

Abstract

Adipocyte lipolysis was compared with hormone-sensitive lipase (HSL)/perilipin subcellular distribution and perilipin phosphorylation using Western blot analysis. Under basal conditions, HSL resided predominantly in the cytosol and unphosphorylated perilipin upon the lipid droplet. Upon lipolytic stimulation of adipocytes isolated from young rats with the ␤-adrenergic agonist, isoproterenol, HSL translocated from the cytosol to the lipid droplet, but there was no movement of perilipin from the droplet to the cytosol; however, perilipin phosphorylation was observed. By contrast, upon lipolytic stimulation and perilipin phosphorylation in cells from more mature rats, there was no HSL translocation but a significant movement of perilipin away from the lipid droplet. Adipocytes from younger rats had markedly greater rates of lipolysis than those from the older rats. Thus high rates of lipolysis require translocation of HSL to the lipid droplet and translocation of HSL and perilipin can occur independently of each other. A loss of the ability to translocate HSL to the lipid droplet probably contributes to the diminished lipolytic response to catecholamines with age.The molecular basis of the acute hormonal regulation of lipolysis in adipocytes remains unclear. Although the ratelimiting step of lipolysis appears to be catalyzed by hormonesensitive lipase (HSL), 1 an enzyme that is acutely regulated by hormones that elevate cAMP and activate cAMP-dependent protein kinase (reviewed in Refs. 1 and 2), the activation of purified HSL by phosphorylation in vitro (3) is insufficient to account for the stimulation of lipolysis in vivo (4), suggesting the involvement of additional mechanisms.It has now been shown that upon lipolytic stimulation of the fat cell, HSL protein translocates from a cytoplasmic compartment to the lipid droplet (5), and subsequent immunofluorescence studies using anti-HSL antibodies in 3T3-L1 adipocytes have demonstrated that HSL is distributed throughout the cytoplasm of unstimulated cells, but moves to the surface of lipid droplets in these cells upon lipolytic stimulation (6). In addition to the well established mechanism of cAMP-dependent activation of HSL, Okuda et al. (7,8) proposed the so-called "hormone-sensitive substrate theory" of lipolysis in which the hormone did not act on the lipase, but on the endogenous lipid substrate. Wise and Jungas (9) went on to propose a dual mechanism of lipolytic activation by catecholamines involving "substrate activation," suggesting that some factor at the surface of intact lipid droplets may be necessary for the hormonal stimulation of lipolysis. More recent work would predict that this factor would be expected to facilitate the translocation of HSL, presumably by some alteration in the lipid droplet surface, and may involve the formation of smaller lipid droplets observed in 3T3-L1 adipocytes and Leydig cells (10,11).Recent studies have demonstrated that differences in patterns of lipid droplet protein expression are partly responsible for the differing l...

A computational model of adipose tissue metabolism: Evidence for intracellular compartmentation and differential activation of lipases

“…The localized metabolic subdomain is consistent with a small cytosolic volume due to large lipid droplet in adipocyte (Moore et al, 2005;Denton et al, 1966). Furthermore, the volume fraction of this metabolic subdomain estimated by the model corresponds to the volume fraction of intracellular water space (1~4% of total tissue volume) measured from in vitro studies of adipose fat pad (Denton et al, 1966;Crofford and Renold, 1965) Recent in vitro studies of adipocyte lipid mobilization showed that major lipolytic enzymes and proteins are co-localized in a subcellular domain during beta-adrenergic stimulation (Granneman et al, 2007;Moore et al, 2005;Clifford et al, 2000). The localization of enzyme complexes reduces the transit time of metabolites which allows faster cellular dynamics (Welch and Easterby, 1994).…”

“…The reaction rate coefficients are related by a thermodynamic constraint (or Haldane relationship): (7) In the blood compartment, the breakdown of TG to FAs and glycerol is the only reaction that is catalyzed by LPL. Since some LPL is carried by blood (Karpe et al, 1998), the activity of LPL reaction depends on adipose blood flow: (8) where K m,LPL and K m,Q are phenomenological M-M constants for the LPL reaction.…”

Carboxylesterase 3 (EC 3.1.1.1) Is a Major Adipocyte Lipase

“…In vivo, lipolysis is determined both by intrinsic lipase capacity and by access of lipase(s) to TAGs of the lipid droplet. This has been elegantly demonstrated for HSL, which displaces from cytosol to the lipid droplet surface following ␤-adrenergic stimulation, a process that involves protein kinase A-mediated HSL phosphorylation (21). A reciprocal displacement is observed for perilipin A, a protein that coats the lipid droplet surface and that may form a barrier to access by lipases to their TAG substrate (22).…”

Integrative physiology of human adipose tissue

“…19 Phosphorylation of HSL by protein kinase A is accompanied by translocation of HSL from the adipocyte cytosol to the surface of the lipid droplet 20 and also by phosphorylation of perilipin, a protein that appears to coat the lipid droplet and to move away upon stimulation, to allow HSL access. 20 Acute activation of lipolysis, via perilipin and HSL phosphorylation, may be brought about by catecholamines acting through b-adrenergic receptors, although in the situation of overnight fasting, when lipolysis increases steadily, b-adrenergic stimulation appears not to be involved; 21 progressive removal of insulin inhibition may be more important. Overnight secretion of growth hormone 22 and the morning rise in cortisol 23 play additional modulatory roles.…”