1. The effects of insulin (2 nM and 4 nM) upon oxygen consumption ( VoJ, lipolysis rates and indirectly derived rates of fatty acid utilization, by isolated brown adipocytes from warm-acclimated (W cells) and cold-acclimated (C cells) animals, induced by noradrenaline and glucagon separately and conjointly, are reported.2. Changes in interrelationships (coupling) between the parameters under different treatment regimes were assessed using bivariate regression analyses.3. Administration of glucagon with noradrenaline increased lipolysis/fatty acid utilization coupling without concomitant increase of Vo2 suggesting that glucagon may increase re-esterification through glycogenolytic generation of glycerol 3-phosphate, trapping intracellular fatty acid in excess of the capacity of disposal mechanisms, thus conserving respiratory substrate.4. W cells were unresponsive to glucagon in terms of lipolysis and po2'; C cells responded to glucagon with parallel increases in lipolysis rate and Both cell types responded to noradrenaline alone and conjointly with glucagon; C cells-were more sensitive to these agonists than W cells.5. Lipolysis/ Vo, coupling was reduced in C cells suggesting that in cold acclimation, noradrenaline-induced lipolysis rates are in excess of the capacity of cellular oxidation/re-esterification mechanisms.6. Insulin inhibited noradrenaline and glucagon-induced lipolysis, simultaneously increasing po2, supporting the hypothesis that glucose may be a thermogenic substrate in brown adipase tissue, permitting concurrent thermogenesis and lipogenesis. C cells were more insulin-sensitive than W cells.7. The data indicate that insulin may mediate its effects (additively with noradrenaline) by activation of pyruvate dehydrogenase, generating glycolytic flux and, in the presence of noradrenaline-inhibited lipogenesis, generate additional oxaloacetate, permitting increased P-oxidation.In homeotherms, non-shivering thermogenesis is mediated primarily by brown adipose tissue (BAT) [l]. The principal mechanism of activation is by means of increased sympathetic activity with liberation of noradrenaline from the dense adrenergic terminals with which the tissue is invested, and consequent activation of P-adrenergically mediated lipolysis leading to the liberation of free fatty acids from intracellular triacylglycerols. Subsequent P-oxidation of free fatty acids, associated with increased mitochondrial proton conductance induced by increased free fatty acid concentrations [2], leads to enhanced heat production by dissipation of the mitochondrial proton gradient [3]. A second effect attributed to noradrenaline release in chronic cold exposure is increased synthesis of mitochondrial protonophore [4].While noradrenaline appears to be the prime mediator of BAT thermogenesis [5], there is evidence that non-adrenergic factors such as glucagon [6, 71 may have permissive or

Cold-acclimation is associated with development of non-shivering thermogenesis, in brown adipose tissue (BAT); adrenergically mediated lipolysis liberates free fatty acid (FFA), the thermogenic substrate, from intracellular triacylglycerol deposits (reviewed Nicholls and Locke 1984). FFA also activates the mitochondrial protonophore, thermogenin, resulting in loss of respiratory control and hence heat-dissipation {Cunningham, Weisinger and Nicholls 1986). Noradrenaline (NA) is the primary mediator of BAT thermogenesis, but pancreatic hormones have been implicated (Kuroshima, Yahata and Habara 1984). Glucose uptake and utilization by BAT is insulin-sensitive (Cooney, Caterham and Newsholme 1985), and, in vivo, glycolysis is enhanced by both NA and insulin (McCormack, Gibbins and Denton 1986). Insulin secretion is reduced in coldacclimation (Howland and Nowell 1968), and the relative insulinsensitivities of BAT from warm-and cold-acclimated animals may indicate if insulin has a functional role in the regulation of BAT. To address this question, oxygen consumption (V0 2 ) and lipolytic activity of isolated brown adipocytes from warm-and cold-acclimated rats (W-and C-cells) were measured in the presence and absence of insulin. Materials and MethodsMale Wistar albino rats (initial body weight ca. 200 g) were kept at 21±1°C (warm-acclimated) or 4±1°C (cold-acclimated), receiving food and water ad libitum and a 12 h photoperiod (0800-2000). After six weeks acclimation, animals were sacrificed, interscapular fat pads removed, BAT dissected free and held in Krebs-Henseleit bicarbonate buffered medium (KHM) containing 2.7 mM glucose (pH 7.4). Adipocytes were prepared by collagenase digestion {Levin, Comai, O'Brien and Sullivan 1982) in the presence of soybean trypsin inhibitor (0.5 mg-mi -1 ). Dispersed adipocytes were thrice washed with KHM containing 4.5 g% defatted bovine serum albumin, resuspended in KHM with albumin and counted (Neubauer haemocytometer). Viability was assessed by trypan blue exclusion and by addition of 15 mmole sodium succinate in 10 ml KHM. VO 2 was measured, polarographically for 5 minutes, 5-10 minutes after addition of 10M1 KHM, or 10 ml KHM containing 2 nM or 4 nM insulin (the plasma insulin-concentrations of cold-and warm-acclimated rats respectively (Howland and Nowell 1968)). Lipolysis was estimated by assay of glycerol, by an NAD-linked microenzymatic method, produced by 0.5-2.0 x 10 5 adipocytes incubated in 4 ml KHM (with albumin), with insulin as above, at 37°C for 30 minutes. Lipolysis was expressed Downloaded by: NYU. Copyrighted material.