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Studies on the epinephrine-sensitive lipase of adipose tissue Yamamoto, Mas

Abstract

The study of the role of adipose tissue in the maintenance of the caloric homeostasis of organisms is currently the object of widespread research. In particular, the enzymes of lipid metabolism in adipose tissue are being extensively investigated in both intact fat pads and in broken-cell preparations. Special attention is being paid to factors which control the activity of these enzymes. We have examined some properties of a lipase in epididymal fat pads of rats. The enzyme has been assayed by measuring the free fatty acids liberated when triglycerides are incubated with crude adipose tissue extracts. Quantitative measurements of free fatty acids were performed by (a) titrating the liberated acid with dilute alkali solution, and (b) reacting the free fatty acids with Cu⁺⁺ to form the chloroform-soluble copper soap of long chain fatty acids, then assaying the copper with diethyl-dithiocarbamate spectrophotometrically. It is well known that lipase activity in adipose tissue decreases during incubation in a Krebs-Ringer bicarbonate medium at 37°C. The de-activated enzyme can be activated by briefly exposing the intact tissue to epinephrine. The study of this epinephrine-sensitive lipase in adipose tissue has been the main object of this thesis. When epinephrine was added to media containing intact epididymal fat pads, the dramatic mobilization of free fatty acids from the pads into the media was observed. When epinephrine was added directly to unfractionated homogenates, little, if any, response was elicited, indicating perhaps that some activating factor was destroyed or diluted out during homogenization. When ATP, cyclic 3',5'-AMP and Mg⁺⁺ were added to unfractionated homogenates of adipose tissue, some lipase activation was observed. Similarly, when these nucleotides and Mg⁺⁺ were added to the supernatant fluid obtained from centrifuged homogenates, some activation of the lipase was observed, although the results obtained were not consistent. Other nucleotide 3',5'-cyclic phosphates generally inhibited lipase activity in the supernatant fluid. Our data indicates that epinephrine activates adipose tissue lipase only when added to the intact fat pad before homogenization. Little or no activation occurred when the amine was added to homogenates. Cyclic 3',5'-AMP had some ability to reactivate the lipase, both in unfractionated homogenates and in the supernatant fluid prepared by centrifugation. The effects, however, were not marked. It is concluded that if epinephrine-activation of adipose tissue is mediated through cyclic 3',5'-AMP, precise conditions for showing this have not yet been achieved. Additional experiments were performed on the epinephrine-sensitive lipase. Intact adipose tissue obtained from reserpinized rats was exposed to epinephrine after a 3-hour incubation period. The results indicated that epinephrine does not activate the lipolytic system in adipose tissue of reserpinized rats. Finally, some of the factors regulating the degree of inactivation of the epinephrine-sensitive lipase during incubation were investigated. Fat pads removed from rats which had been either anaesthetized or not anaesthetized prior to sacrifice were incubated for 3 hours. Data collected from a number of experiments indicated that there were virtually no differences in the extent of lipase inactivation between the two groups of rats.

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