Free fatty acid and ATP levels in adipocytes during lipolysis

Abstract

Lipolytic hormones such as catecholamines and ACTH as well as lipolytic agents—e.g., 3′–5′ dibutyryl-cyclic AMP and methyl xanthines—are known to reduce ATP levels in adipose cells incubated in vitro. Considerable indirect evidence exists implicating intracellular FFA accumulation as a causative factor. To obtain more direct evidence for a causal relationship a simple and accurate method was developed for measuring intracellular FFA levels in isolated adipocytes using sucrose-U- 14C as an extracellular marker. Following incubation, medium and cells were separated by centrifugation and the infranatant medium was removed by aspiration. The volume of medium trapped between cells was determined by measuring the amount of sucrose- 14C retained in the packed adipose cell float. In this way the FFA content of the adipose cell float could be corrected for contamination by FFA bound to extracellular albumin. The basal FFA content of isolated adipocytes obtained from normal fed rats was 0.6 μEq./Gm. cell lipid. Addition of norepinephrine resulted in a rapid increase in intracellular FFA which plateaued at-22.5 μEq./Gm. cell lipid within 15 mins. Under optimal conditions of linear release of fatty acids—i.e., when medium albumin is not saturated by FFA—adipocyte ATP levels were not significantly depressed. Reduction of adipocyte ATP levels by lipolytic hormones correlated inversely with intracellular accumulation of FFA. Under these circumstances medium albumin approached saturation and intracellular FFA levels reached 6–9 μEq./Gm. cell lipid. Glucose, which is known to have a protective effect against ATP reduction, also prevented intracellular accumulation of FFA to cytotoxic levels. These results strongly support the view that the fall in adipocyte ATP after treatment with lipolytic hormone is the result of intracellular FFA accumulation, which depresses ATP synthesis by uncoupling oxidative phosphorylation. It is suggested that experiments undertaken to examine the relationship between effects of lipolytic hormones and other energy requiring processes must employ optimal in-vitro conditions—e.g., linear FFA release and nonsaturation of medium albumin—so as to prevent artifactual phenomena arising secondary to excessive intracellular FFA accumulation or ATP deficiency.