The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.
For facilitation of the experimental analysis of the mechanism and regulation of mobilization of fatty acids from adipose triacylglycerol (TAG) stores, which also represents important targets for pharmacological intervention with the pathogenesis of diabetes and obesity, we developed a convenient and reliable non-radioactive cell-based assay. Isolated rat adipocytes are incubated with the fluorescent fatty acid derivative, 12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid (NBD-FA), in the presence of insulin. The resulting NBD-FA-labeled TAG is efficiently cleaved by hormone-sensitive lipase (HSL) in vitro. After removal of insulin and excess of free NBD-FA, lipolysis is initiated by addition of isoproterenol and/or adenosine deaminase. The amount of NBD-FA generated in total or released into the incubation medium in the presence of modulatory hormones or compounds is then monitored by thin layer chromatography and fluorescence imaging. Release of NBD-FA, glycerol and [3H]oleic acid from TAG follows similar kinetics and concentration dependence in response to various lipolytic and anti-lipolytic stimuli as well as inhibitors of HSL. Release of NBD-FA from adipocytes correlates well to translocation of HSL from the cytosol to TAG droplets. In addition, we found that a cell-free system consisting of NBD-FA-labeled TAG droplets with endogenous associated HSL closely reflects the lipolytic state of the adipocytes used for its preparation. In conclusion, release of NBD-FA from TAG in vivo and in vitro can be used as accurate index for (regulation of) lipolysis in primary and cultured adipocytes.
Title: Analysis of lipid metabolism in adipocytes using a fluorescent fatty acid derivative. I. Insulin stimulation of lipogenesis Muller G, Jordan H, Petry S, Wetekam EM, Schindler P Ref: Biochimica & Biophysica Acta, 1347:23, 1997 : PubMed
Stimulation of lipid synthesis (lipogenesis) is one of the most pronounced metabolic actions of insulin. Here we demonstrate insulin-stimulated lipogenesis in isolated rat adipocytes using a fatty acid derivative which carries a fluorophore. Three major fluorescent lipid products (lipids 1, 2, 3) are generated as revealed by TLC analysis and subsequent fluorescent scanning or imaging. Lipolytic digestion and labeling studies suggest monoacylglycerol-3-phosphate and diacylglycerol (-3-phosphate) structures harboring a single fluorescent fatty acyl residue each for lipids 1 and 3 (2), respectively. Fluorescent triglycerides are not generated. Assaying acylation with isolated microsomes using the purified lipids 1 and 3 indicates that incorporation of one fluorescent fatty acyl residue into glycerol(-3-phosphate) interferes with subsequent esterification. Pretreatment of the adipocytes with insulin significantly stimulates synthesis of lipids 1 and 2, only. The insulin concentration-response relationship (EC50 = 0.5 nM) and the maximal insulin response for synthesis of lipid 1 (stimulation factor = 14- to 20-fold at low glucose and 3- to 7-fold at high glucose) are comparable with those for incorporation of [3-3H]glucose into total adipocyte lipids. Thus this fluorescence-based assay may be useful for studying insulin action and lipogenesis.
