Analysis of lipid metabolism in adipocytes using a fluorescent fatty acid derivative. I. Insulin stimulation of lipogenesis.
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.
Müller
G
G
Hoechst-Marion-Roussel, Pharmaceutical Research Site, DG Metabolism, Frankfurt am Main, Germany.
Jordan
H
H
Petry
S
S
Wetekam
E M
EM
Schindler
P
P
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D005227:Fatty Acids; D005456:Fluorescent Dyes; D007328:Insulin / Q000494:pharmacology*; D008055:Lipids / Q000096:biosynthesis*; D008297:Male; D051381:Rats; D017208:Rats, Wistar
D005227:Fatty Acids; D005456:Fluorescent Dyes; D007328:Insulin; D008055:Lipids