Quantification of fatty acid activation of the uncoupling protein in brown adipocytes and mitochondria from the guinea-pig

Abstract

Brown adipocytes from cold‐adapted guinea‐pigs (C‐cells) are more sensitive to uncoupling by exogenous palmitate than are cells from warm‐adapted animals (W‐cells) with much less uncoupling protein. Half‐maximal respiratory stimulation of C‐cells requires 80 nM free palmitate. Noradrenaline‐stimulated lipolysis is not rate‐limiting for the respiration of either C‐cells or W‐cells. Half‐maximal stimulation of fatty acid oxidation by mitochondria from warm‐adapted guinea‐pigs (W‐mitochondria) and cold‐adapted guinea‐pigs (C‐mitochondria) both require 12 nM free palmitate. Palmitate uncouples C‐mitochondria much more readily than M‐mitochondria, parallelling its action on the adipocytes. The uncoupling is partially saturable, about 100 nM free palmitate being required for half‐maximal response of C‐mitochondria. W‐ and C‐mitochondria show identical binding characteristics for palmitate. The respiratory increase of mitochondria is calculated as a function of bound palmitate. After correcting for the residual uncoupling protein present in W‐mitochondria, palmitate is estimated to be almost ineffective as an uncoupler of brown fat mitochondria in the absence of the protein. It is concluded that fatty acids display characteristics required of a necessary and sufficient physiological activator of the uncoupling protein.

The mitochondrial inner‐membrane anion channel possesses two mercurial‐reactive regulatory sites

“…Another pathway known to transport CI-in mitochondria is the uncoupling protein of brown-adipose-tissue mitochondria; however, in contrast with the inner membrane anion channel this pathway is regulated by fatty acids [20,21], GDP [22] (however, see [14]) and is not regulated by matrix protons or Mg". Recently, however, it has been shown that the activity of the uncoupling protein is modified by thiolreactive reagents.…”

Carnitine cycle in brown adipose tissue mitochondria as a tool for studying the regulatory role of fatty acids in the uncoupling protein function

“…Apart from other types of mitochondria they possess the uncoupling protein (UP), a unique proton translocator [2,3], converting A/~H + into heat. H + fluxes through the UP are inhibited by purine nucleotides [4][5][6][7][8] and activated by fatty acids [3,[8][9][10]. Both regulatory mechanisms were also demonstrated in a reconstituted system [3], but the activation by fatty acids was disputed in another laboratory [2].…”

“…Moreover, FA may play a role as endogenous natural protonophores in biological membranes [17] which is extremely important in considering dissipative H ÷ currents in mitochondria lacking UP. The stronger uncoupling effect of FA in BAT mitochondria clearly reflects activation of H ÷ transport through UP and correlates with the exclusive occurrence of UP in BAT [9,10]. In our work we used a controlled removal of endogenous fatty acid from BAT mitochondria by a carnitine cycle [8,9,[18][19][20] in order to estimate the interrelationship between effects of purine nucleotides and fatty acids on UP function.…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…Circulating fatty acids are bound to albumin, with typical molar ratios in the range 1-2 for the human (442) and up to 3 for the fasted rat (496). The free, unbound, fatty acid concentration under these conditions is only ϳ10 -20 nM (105,442). While the need for albumin binding is generally appreciated in the literature, molar ratios of 6:1 or even higher have been employed in vitro (144,285,332,472), producing unbound fatty acid concentrations close to 1 M, or 100 times higher than the normal physiological range (FIGURE 8A).…”