A nitric oxide-mediated mechanism regulates lipolysis in human adipose tissue in vivo

Possible nitric oxide (NO)-mediated effects on lipolysis were investigated in vivo in human subcutaneous adipose tissue using microdialysis, as well as in vitro on isolated fat cells of non-obese, healthy volunteers. NO donors were added to the ingoing dialysate solvents. Changes in lipolysis and local blood flow were investigated by measuring glycerol levels and ethanol ratios, respectively, in the microdialysates. It was shown that the NO synthase inhibitor, NG-monomethyl L-arginine (L-NMMA), but not the biologically inactive enantiomer NG-monomethyl D-arginine (D-NMMA), increased glycerol levels in the microdialysates without causing a change of local blood flow. In addition, L-NMMA increased glycerol levels in the microdialysate when local blood flow was stimulated with hydralazine. Nitric oxide gas as well as the NO donor, nitroglycerine, reduced glycerol release from isolated adipocytes in vitro. Expression of inducible nitric oxide synthase (iNOS) in human adipose tissue was shown by Western blot analysis. Biologically active NOS was demonstrated by measuring total enzymatic activity. In conclusion, the data demonstrate that inhibition of NO release in subcutaneous adipose tissue results in an increased lipolysis in vivo. These effects, which were also observed in vitro, are independent of local blood flow changes. Furthermore, the demonstration of enzymatic NOS activity and the expression of inducible nitric oxide synthase (iNOS) in adipose tissue indicate that locally synthesized NO may play a role in the physiological control of lipolysis in human adipose tissue. Keywords: Adipose tissue, nitric oxide synthase, glycerol, microdialysis, isolated fat cells Introduction Nitric oxide (NO) is formed in cells from L-arginine, which in the presence of oxygen, is converted to L-citrulline by NO synthases (NOS). Three different isoforms of NOS have been cloned and sequenced (reviewed in Michel & Feron, 1997). Two of the isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), are constitutive, and produce small amounts of NOS following stimuli that raise intracellular Ca2+ concentrations. The third isoform, inducible NOS (iNOS), is expressed after induction by such agents as cytokines and bacterial lipopolysaccharide. The action of NO is difficult to study since it reacts readily with oxygen, superoxide radicals or hydrogen peroxide to produce NO2, peroxynitrite and nitrate/nitrite, respectively (Henry et al., 1991). However, the effects of NO can be studied indirectly by adding NO donors, using scavengers, or by reduction of NO synthesis using NOS inhibitors such as the L-arginine analogue, NG-monomethyl L-arginine (L-NMMA). NOS isoforms are expressed in several different cell types and NO is believed to be involved in the regulation of many events, including inflammation, vascular tone and metabolism (reviewed in Christopherson & Bredt, 1997; Michel & Feron, 1997). However, it is not known if NO also regulates lipid metabolism. Adipose tissue is the largest reservoir of lipids in the body. These are mobilized by lipolysis in fat cells. Lipolysis in adipose tissue is under hormonal regulation, the most well known being insulin and catecholamine effects on antilipolytic and lipolytic mechanisms. Recently, we suggested a dual cholinergic mechanism that also regulates lipolysis in vivo (Andersson & Arner, 1995). In theory, the latter regulatory system could utilize NO as a mediator, since NO plays a role in the parasympathetic modulation of myocardial contractility (Hare et al., 1998). Nitric oxide synthase has been demonstrated in white adipose tissue of the rat (Ribiere et al., 1996) indicating this tissue as a potential source of NO production. To date, no evidence for an involvement of NO in lipolysis has been presented. The major aim of this study was therefore to investigate whether NO has effects on lipolysis in vivo. We used microdialysis to investigate the effects of L-NMMA on local lipolysis in situ in human abdominal subcutaneous adipose tissue (Lafontan & Arner, 1996). We also studied total NOS activity and expression of NOS in this tissue, as well as the effect of NO on lipolysis in isolated fat cells in vitro.