Ruth Brandes

Induction of fatty acid binding protein by peroxisome proliferators in primary hepatocyte cultures and its relationship to the induction of peroxisomal β-oxidation

The induction of liver fatty acid binding protein (L-FABP) by the peroxisome proliferators bezafibrate and clofibrate was compared with the induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation in cultured rat hepatocytes maintained on a substratum of laminin-rich (EHS) gel. This substratum was chosen because marked induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was effected by bezafibrate in hepatocytes supported on EHS gel, whereas only peroxisomal palmitoyl-CoA oxidation was induced in hepatocytes maintained on collagen-coated plates. In control cells on EHS, activity of peroxisomal palmitoyl-CoA oxidation remained stable, while L-FABP abundance declined with time, and L-FABP mRNA was undetectable after 5 days. In cultures exposed to bezafibrate or clofibrate, peroxisomal palmitoyl-CoA oxidation activity was induced earlier and more rapidly than L-FABP. When fibrates were withdrawn, peroxisomal palmitoyl-CoA oxidation declined rapidly, whereas L-FABP continued to increase. L-FABP induction was accompanied by a striking increase in mRNA specifying this protein. Tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I, effectively doubled peroxisomal palmitoyl-CoA oxidation activity. However, tetradecylglycidic acid markedly inhibited fibrate induction of L-FABP and peroxisomal palmitoyl-CoA oxidation but, unexpectedly, did not prevent the fibrate-induced proliferation of peroxisomes. Maximal induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was produced at a bezafibrate concentration in the culture medium (0.05 mM) much lower than that of clofibrate (0.3 mM). Also, bezafibrate, but not clofibrate, inhibited [1-14C]oleic acid binding to L-FABP with a Ki = 9.5 microM. We conclude that hepatocytes maintained on EHS gel provide an important tool for investigating the regulation of L-FABP. These studies show that the induction of peroxisomal beta-oxidation and L-FABP by peroxisome proliferators are temporally consecutive but closely related processes which may be dependent on a mechanism distinct from that which leads to peroxisome proliferation. Furthermore, the mechanism of action of the more potent peroxisome proliferator, bezafibrate, may be mediated, in part, by interaction of this agent with L-FABP.

Liver cytosolic fatty acid-binding proteins: Effect of diabetes and starvation

The binding of [1-(14)C]oleate to rat liver cytosol was studied, using gel filtration on Sephadex G-75 and G-50. In liver cytosols from control rats, most of the high-affinity oleate binding was in the region of 12 000-dalton proteins. In liver cytosols from diabetic and starved rats, a second peak of radioactivity appeared in the void volume. This peak was shown to be associated with a component having the molecular weight of 400 000. Evidence suggesting that a change in the composition of cytosolic binding proteins is involved is presented.

Inducers of adipose conversion activate transcription promoted by a peroxisome proliferators response element in 3T3-L1 cells

Nonmetabolizable fatty acids are shown here to induce adipose conversion of 3T3-L1 preadipocytes as well as to activate transcription of a reporter plasmid promoted by a peroxisome proliferators response element. This dual activity was also observed using the peroxisome proliferator bezafibrate or the differentiation inducer isobutyl methylxanthine. The data suggest a role for a peroxisome proliferators activated receptor (PPAR) in adipose conversion induced by fatty acids, isobutyl methylxanthine, or xenobiotic amphipathic carboxylates.

Adipose conversion of cultured rat primary preadipocytes by hypolipidemic drugs

Cultured rat epididymal preadipocytes exposed for 24-72 h to either bezafibrate or clofibrate added to the culture medium were extensively converted to fat-loaded adipocytes. Adipocyte conversion increased during the first 5-7 days following plating, reaching a level of 100% and 60% conversion with bezafibrate and clofibrate, respectively, as compared to 10% conversion in their absence. Adipocyte conversion in culture was a saturable function of the hypolipidemic effectors and was associated with an increase in the incorporation rate of exogenous palmitate into triacylglycerols, in glycerol-3-phosphate dehydrogenase and hormone-sensitive lipase activities but not in lipoprotein lipase activity. Adipocyte conversion by hypolipidemic drugs was much more prominent than that exerted by dibutyryl cAMP, and the relative conversion efficiency of the two fibrate drugs did not correlate with their respective cAMP content of the culture. Hence, hypolipidemic drugs and dibutyryl cAMP appear to act independently in initiating adipose conversion in primary epididymal preadipocytes.

Adipocyte conversion of cultured 3T3-Ll preadipocytes by bezafibrate

Transient exposure of cultured 3T3-L1 preadipocytes to hypolipidemic fibrate drugs results in extensive adipocyte conversion. Adipocyte conversion in culture was characterized by an increase in neutral lipids content and in adipocyte marker enzymes like hormone-sensitive lipase and glycerol-3-phosphate dehydrogenase. Adipocyte conversion in culture was also accompanied by induction of cyanide-insensitive peroxisomal palmitoyl-CoA oxidation. The conversion pattern exerted by fibrate drugs in 3T3-L1 cells was similar to that reported previously for primary cultured epididymal preadipocytes (R. Brandes, R. Arad and J. Bar-Tana, Biochim. Biophys. Acta, 877, 314-321 (1986)), and seems to refute clonal selection in the conversion sequel initiated by fibrate drugs in primary cultured preadipocytes.

The induction of adipose conversion by bezafibrate in 3T3-L1 cells. Synergism with dibutyryl-cAMP

The induction of adipose conversion in 3T3-L1 cells by bezafibrate (Brandes, R., Hertz, R. Arad R., Naishtat S., Weil, S. and Bar-Tana, J. (1987) Life Sci., 40, 935-941) was enhanced by dibutyryl-cAMP as well as forskolin, theophylline or isobutylmethylxanthine added to the incubation medium together with the bezafibrate inducer. The synergistic effect of bezafibrate and dibutyryl-cAMP resulted in enhancing the expression of late markers of adipose conversion, e.g., lipid accumulation or glycerol-3-phosphate dehydrogenase activity and its mRNA. This enhanced expression of late markers was reflected in shortening the time period required for their first appearance as well as increasing their yield during the course of adipose conversion. By following the accumulation of glutamine synthetase mRNA serving as an early marker for adipose conversion, the synergistic effect of bezafibrate and dibutyryl-cAMP was already evident as early as 5 h following their addition to confluent 3T3-L1 cells. Hence, the induction of adipose conversion by bezafibrate in 3T3-L1 cells appears to involve an early event which is rate-limited by the availability of intracellular cAMP.

Translocation of long chain fatty acids into lecithin liposomes containing the long chain fatty acyl-CoA synthetase

Transport of long chain fatty acids through biological membranes was reported to be mediated either by adsorption to the cell membrane followed by simple diffusion [I ,2], carrier-facilitated transport [3,4] or translocation catalyzed by long chain fatty acylCoA synthetase [5-71. The simple diffusion of long chain fatty acids requires flip-flop of the transported acid within the bilayer membrane. However, the flipflop diffusion rate for free fatty acids was found to be relatively low [8,9] to account for the high metabolic turnover of long chain fatty acid used as substrate in vivo [lo], in organ perfusion [ 1 l] or in cultured cells [ 121. The possible role of a specific protein carrier in facilitated diffusion of long chain fatty acids is still unresolved due to methodological difficulties presented by the nonspecific absorption of long chain fatty acids to biological membranes, the limited concentration range of free fatty acid which may be used for binding studies in the absence of added detergent, as well as the micellar character of the fatty acid substrate in the presence of added detergent. Vectorial translocation of long chain fatty acids catalyzed by the ATP-dependent long chain fatty acyl-CoA synthetase (EC 6.2.1.3) was first suggested [S ] in &cherichiu coli K12. This claim was based on the inability of a mutant lacking the acyl-CoA synthetase to take up fatty acids, the absence of an efflux reaction for the entrapped fatty acid moiety and the substrate specificity for uptake as compared to the substrate specificity for the synthetase reaction. However, it is worth noting that the isolated permeation step was not analyzed specifically but was followed in terms of total uptake as reflected in esterification or oxidation. Whatever the mode of

Induction of adipose conversion in 3T3-L1 cells is associated with an early phosphorylation of a protein partly homologous with mouse vimentin

Induction of adipose conversion in 3T3‐L1 by bezafibrate has been previously shown to be enhanced by dibutyryl cAMP and to be associated with an early phosphorylation of a 60 kDa acidic protein [Biochim. Biophys. Acta 1054 (1990) 219‐224; FEBS Lett. 285 (1991) 63‐65]. We describe here the isolation and sequencing of two peptides of the protein concerned. Both appear to be homologous with two respective amino acid sequences of the mouse intermediate filament protein vimentin.

The induction of adipose conversion in 3T3-L1 cells is associated with early phosphorylation of a 60-kDa nuclear protein

The induction of adipose conversion in 3T3‐L1 cells by bezafibrate has been shown to be enhanced by dibutyryl‐cAMP. We here report that the induction of adipose conversion in 3T3‐L1 cells, by either bezafibrate and dibutyryl cAMP, or isobutylmethyxanthine alone, is associated with a very early phosphorylation of a 60‐kDa acidic protein found in the nuclear fraction.

The specificity of triacylglycerol synthesis for medium-chain fatty acids in rat and human adipose preparations

The availability of medium-chain fatty acids as substrates for triacylglycerol synthesis was studied in cultured and suspended rat adipocytes and in pieces of human adipose tissue. Octanoate was virtually excluded from glycerol 3-phosphate esterification while serving as a substrate for diacylglycerol esterification. This specificity was similar to that of cultured rat hepatocytes.