Effects of hormones on the rate of the triacylglycerol/fatty acid substrate cycle in adipocytes and epididymal fat pads

Abstract

Brian Brooks, Jonathan R.S. Arch + and Eric A. Newsholme* Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU and + Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey KT18 5XQ, England Received 12 July 1982 Hormone Triacylglycerol/ fatty acid substrate cycle Substrate cycling Noradrenaline Glucagon Triacylglycerolsynthesis 1. INTRODUCTION It has been suggested by some workers that sub- strate ('futile') cycles are either of little of no sig- nificance in metabolism [1] whereas others suggest that they are of considerable importance in in- creasing sensitivity in metabolic control [2-4]. One prediction of the hypothesis that cycling increases sensitivity is that the rate will vary from one con- dition to another and that some hormones may specifically increase the cycling rate. Evidence for the existence of the triacylglycerol/fatty acid cycle in adipose tissue was presented by Steinberg [5] who compared the rates of fatty acid and glycerol production by isolated epididymal fat pads. This method has been used to investigate the effect of some hormones on the rate of the triacylglycerol/ fatty acid cycle in isolated adipocytes of the rat. In addition, a radiochemical method that depends upon the incorporation of tritium from tritiated water into the glycerol and fatty acid moieties of triacylglycerol has been developed for the mea- surement of the rate of this cycle in epididymal fat pads of the rat. The effects of some hormones have been investigated using both techniques and the results are reported and discussed here. 2. MATERIALS AND METHODS Male Wistar rats were obtained from OLAC (1976) Ltd. (Blackthorn, Bicester, Oxon OX60TP). * To whom correspondence should be addressed Chemicals, biochemicals and enzymes were ob- tained from sources given in [6] except that crys- talline glucagon, ACTH and TSH were obtained from Sigma Chemical Co. (Poole, Dorset) and radiochemicals from The Radiochemical Centre (Amersham HP7 9LL). Rats were killed by cervical dislocation and the epididymal fat pads were removed. Only the thin- ner distal portion of the pad was used for incuba- tion and up to 200 mg fresh tissue was incubated in 1.5 ml Krebs-Ringer bicarbonate buffer con- taining 1 mM glucose and 4% (w/v) defatted al- bumin [6]. Fat cells were prepared as in [7] and in- cubated in 1.3 ml Krebs-Ringer buffer as in [6]. The incubation was terminated by addition of 300/tl 2 M H2SO4 (which does not precipitate al- bumin); 200 ttl was taken for assay of fatty acid and 1 ml for assay of glycerol. For the latter, the cells and albumin were precipitated by addition of trichloroacetic acid (200/d, 40% (w/v)). The pre- cipitate was removed by centrifugation and univer- sal indicator (15/~1) was added to the supernatant and neutralised by addition of 7 M KOH from a microsyringe. The concentrations of glycerol and fatty acids were determined as in [8,9]. The rate of cycling was calculated as follows: cycling -- [(3 x rate of glycerol production)