Abstract
To facilitate the study of adrenoreceptor response in small needle biopsy samples of human subcutaneous adipose tissue, we developed a dual radioisotopic technique for measuring lipolysis rate. Aliquots (20-75 mg) of adipose tissue fragments were incubated in a buffered albumin medium containing [3H]palmitate and [14C]glucose, each of high specific activity. In neutral glycerides synthesized in this system, [14C]glucose is incorporated exclusively into the glyceride-glycerol moiety and 3H appears solely in the esterified fatty acid. Alpha-2 and beta-1 adrenoreceptor activation of tissue incubated in this system does not alter rates of 14C-labeled glyceride accumulation, but does produce a respective increase or decrease in the specific activity of fatty acids esterified into newly synthesized glycerides. This alteration in esterified fatty acid specific activity is reflected in the ratio of 14C:3H in newly synthesized triglycerides extracted from the incubated adipose tissue. There is a high correlation (r = 0.90) between the 14C:3H ratio in triglycerides and the rate of lipolysis as reflected in glycerol release into the incubation medium. The degree of adrenoreceptor activation by various concentrations of lipolytic and anti-lipolytic substances can be assessed by comparing this ratio in stimulated tissue to that characterizing unstimulated tissue or the incubation medium. This technique permits the study of very small, unweighed tissue biopsy fragments, the only limitation on sensitivity being the specific activity of the medium glucose and palmitate. It is, therefore, useful for serial examinations of adipose tissue adrenoreceptor dose-response characteristics under a variety of clinical circumstances.
Highlights
T o facilitate the study of adrenoreceptor response in small needle biopsy samples of human subcutaneous adipose tissue,we developeda dual radioisotopic technique for measuring lipolysis rate
We describe a radioisotopic method for the in vitro determination of lipolysis rate in very small fragments of human adipose tissue obtained by needle aspiration
The newly synthesized triglycerides of adipose tissue incubated in medium containing both 3 H 2 0 and ['4C]glucose(section F), had a 'H atom:14Cglycerideglycerol molecule ratio of 3.40 f .05 (SEM) in unstimulated tissue and 3.34 f .083 in tissue exposed to 1 X 10-6M isoproterenol
Summary
T o facilitate the study of adrenoreceptor response in small needle biopsy samples of human subcutaneous adipose tissue,we developeda dual radioisotopic technique for measuring lipolysis rate. The degree of adrenoreceptor activation by various concentrations of lipolytic and anti-lipolytic substances can be assessed by comparing this ratio in stimulated tissue to that characterizing unstimulated tissue or the incubation medium.l This technique permits the study of very small, unweighed tissue biopsy fragments, the only limitation on sensitivity being the specific activity of the medium glucose and palmitate It is, useful for serial examinations of adipose tissue adrenoreceptor dose-response characteristics under a variety of clinical circumstances.-Leibel, R. Activation of beta receptors increases lipolysisby stimulating the adenylate cyclase-cyclic AMP cascade, while alpha-2 receptor activation inhibits lipolysis by reducing levels of adenylate cyclase in the membrane [1].The physiologic balance of these opposing receptors is dependent upon anatomical site, endocrine, autonomic, and nutritional status [2] Functional studies of this receptor system in human adipose tissue have used measures of in vitro glycerol release, adenylate cyclase, and cyclic AMP accumulation, and direct ligand binding as end points [3]. The method permits a quantitative functional assessment of adrenoreceptor status in such fragments which can be obtained from any subcutaneous site [5], repeatedly, during the course of study
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