J. Patrick Kampf

Different Mechanisms of Free Fatty Acid Flip-Flop and Dissociation Revealed by Temperature and Molecular Species Dependence of Transport across Lipid Vesicles

The mechanism of free fatty acid (FFA) transport across membranes is a subject of intense investigation. We have demonstrated recently that flip-flop is the rate-limiting step for transport of oleic acid across phospholipid vesicles (Cupp, D., Kampf, J. P., and Kleinfeld, A. M. (2004) Biochemistry 43, 4473-4481). To better understand the nature of the flip-flop barrier, we measured the temperature dependence of a series of saturated and monounsaturated FFA. We determined the rate constants for flip-flop and dissociation for small (SUV), large (LUV), and giant (GUV) unilamellar vesicles composed of egg phosphatidylcholine. For all FFA and vesicle types, dissociation was faster than flip-flop, and for all FFA, flip-flop and dissociation were faster in SUV than in LUV or GUV. Rate constants for both flip-flop and dissociation decreased exponentially with increasing FFA size. However, only the flip-flop rate constants increased significantly with temperature; the barrier to flip-flop was virtually entirely due to an enthalpic activation free energy. The barrier to dissociation was primarily entropic. Analysis in terms of a simple free volume (Vf) model revealed Vf values for flip-flop that ranged between ∼12 and 15Å3, with larger values for SUV than for LUV or GUV. Vf values increased with temperature, and this temperature dependence generated the enthalpic barrier to flip-flop. The barrier for dissociation and its size dependence primarily reflect the aqueous solubility of FFA. These are the first results to distinguish the energetics of flipflop and dissociation. This should lead to a better understanding of the mechanisms governing FFA transport across biological membranes.

Fatty Acid Transport in Adipocytes Monitored by Imaging Intracellular Free Fatty Acid Levels

Transport of free fatty acids (FFA) across the adipocyte plasma membrane is critical for maintaining homeostasis. To determine the membranes role in regulating transport we describe here the first measurements of the intracellular (unbound) FFA concentration ([FFAi]) and their use in monitoring transport of FFA across 3T3F442A adipocytes. [FFAi] was measured by microinjecting cells with ADIFAB, a fluorescently labeled fatty acid-binding protein that is used to measure unbound FFA levels. We used ratio fluorescence microscopy of intracellular ADIFAB to image unbound FFA levels and determined the time course of [FFAi] in response to changing the extracellular unbound FFA concentration ([FFAo]). [FFAo] was clamped at defined levels using complexes of FFA and bovine serum albumin. We show that FFA influx is slow, requiring about 300 s to reach steady state (rate constant ∼ 0.02 s-1) and saturable (Ko ∼ 200 nm). Efflux is twice as fast as influx, for zero [FFAo], but decreases with increasing [FFAo]. Surprisingly, at steady state [FFAi] is 2–5-fold (average 2-fold) greater than [FFAo] and this [FFAi]/[FFAo] gradient is abolished by depleting cellular ATP. Our results indicate that FFA transport across adipocyte membranes is highly regulated, involving an ATP-driven pump and a mechanism for gating efflux that is sensitive to [FFAo]. These characteristics are well described by a membrane carrier model but are not consistent with FFA transport across the membranes lipid phase. We suggest that these characteristics are important in regulating circulating FFA levels by the adipocyte.