Date of Award

1995

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Professor G.E. Gerber

Abstract

Long chain fatty acids (LCFA's) are important metabolic substrates for mammalian cells. They are utilized as energy sources, through β-oxidation, and as substrates for the synthesis of lipids, including phospholipids and triglycerides, as well as lipids involved in cellular signalling. LCFAs, themselves can also participate in a variety of crucial cell signalling cascades. Adipocytes, through the storage of LCFAs as triglycerides and the mobilization of stored LCFAs are important regulators of energy balance in mammals. A variety of pathogenic conditions involve impaired storage or utilization of LCFAs.

The involvement of LCFAs in these events involves their initial interaction with, uptake by and trafficking within cells. These processes were studied in murine 3T3-L1 cells, which differentiate from preadipocytes to adipocytes. Upon differentiation, they acquired increased levels of LCFA uptake, which was saturable, exhibiting high affinities both for oleate, a natural LCFA, and for 11-m-diazirinophenoxy-[11-³H]undecanoate, a photoreactive LCFA. This established them as a convenient cellular model in which to study LCFA uptake and trafficking. Furthermore, it established the photoreactive LCFA as an analogue of LCFAs which could prove useful for the identification and characterization of cellular LCFA binding proteins which might be involved in LCFA uptake and trafficking.

It has been proposed that cellular uptake and trafficking of LCFAs is a multi-step process involving the delivery of LCFAs to the cell surface, their movement across the plasma membrane and their delivery to intracellular sites. The involvement of each step in LCFA uptake and trafficking in 3T3-L1 adipocytes were studied. Serum albumin, the major carrier of LCFA's in plasma had a saturable, stimulatory effect on oleate uptake by 3T3-L1 adipocytes. Serum albumin which had been photo-labeled with the photoreactive LCFA, bound to 3T3-L1 adipocytes saturably. This implicated the involvement of cellular receptors for serum albumin in the delivery of LCFAs to the cell surface.

The photoreactive LCFA was also used to identify a novel 22 kDa protein as the only high affinity LCFA receptor in the plasma membrane of 3T3-L1 cells. It was localized to the caveolae fraction of the plasma membrane and was identified immunologically as caveolin, a resident component of caveolae. Caveolin was resolved by 2-dimensional polyacrylamide gel electrophoresis into at least two forms, differing in their reaction towards an antibody recognizing a stretch of amino acids found in the predicted amino-terminal sequence of caveolin. Both forms, however had similar affinities toward the photoreactive LCFA (Kd's of 373 and 388 nM). Caveolae in the plasma membrane are believed to act as sites of transport of a variety of compounds; the localization of the only high affinity, plasma membrane LCFA receptor within caveolae suggested that they may also be sites of LCFA transport or signalling events.

A 15 kDa, cytoplasmic protein was also labeled by the photoreactive LCFA. It was identified immunologically as the adipocyte lipid binding protein (ALBP), a member of the family of low molecular weight cytoplasmic fatty acid binding proteins. Its affinities for the photoreactive probe and natural LCFAs were determined by photoaffinity labeling and by ligand displacement, respectively. The labeling of ALBP by the photoreactive LCFA in intact 3T3-L1 adipocytes was dynamic. The time-dependence of its labeling revealed that the newly internalized photoreactive probe initially bound to and then became released by ALBP, consistent with a role for ALBP in the delivery of newly internalized LCFAs to sites of utilization within cells.

The labeling of ALBP by the photoreactive LCFA in intact cells was sensitive to inhibitors of different steps in LCFA uptake was also demonstrated. This confirmed that LCFA binding to ALBP occurred as an intermediate step in LCFA uptake. It also provided a means of discriminating between effects of various manipulations on early steps (upstream of LCFA binding to ALBP) and late steps (downstream of LCFA binding to ALBP) in LCFA uptake. Using this approach, the dependence of the movement of the photoreactive LCFA across the plasma membrane on ΔpH was demonstrated. This indicated that it traversed the membrane as the protonated species by diffusion across the lipid bilayer. Using this approach, α-iodopalmitate, an inhibitor of LCFA uptake originally thought to affect the movement of LCFAs across the plasma membrane, was shown to inhibit uptake by acting at a step downstream of LCFA binding to ALBP. This led to the identification of the specific target for delivery of LCFAs as the lipid droplet. This was the first in vivo demonstration of the delivery of LCFAs and of a specific target organelle for the delivery of newly internalized LCFAs by the ALBP in intact cells. It also led to the demonstration of the importance of long chain fatty acyl-CoA synthetase in LCFA uptake. This enzyme catalyzes the first step in LCFA metabolism, namely, the activation of LCFAs by conversion to their CoA thioester derivative. Its inhibition by α-iodopalmitate was shown to result in the inhibition of LCFA uptake, suggesting that the uptake process was driven by the conversion of LCFAs to their CoA thioester derivatives.

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