Jacob Bar-Tana

Fatty acyl-CoA thioesters are ligands of hepatic nuclear factor-4α

Dietary fatty acids specifically modulate the onset and progression of various diseases, including cancer,, atherogenesis, hyperlipidaemia, insulin resistance and hypertension, as well as blood coagulability and fibrinolytic defects; their effects depend on their chain length and degree of saturation. Hepatocyte nuclear factor-4α (ref. 8) (HNF-4α) is an orphan transcription factor of the superfamily of nuclear receptors and controls the expression of genes (reviewed in ref. 9) that govern the pathogenesis and course of some of these diseases. Here we show that long-chain fatty acids directly modulate the transcriptional activity of HNF-4α by binding as their acyl-CoA thioesters to the ligand-binding domain of HNF-4α. This binding may shift the oligomeric–dimeric equilibrium of HNF-4α or may modulate the affinity of HNF-4α for its cognate promoter element, resulting in either activation or inhibition of HNF-4α transcriptional activity as a function of chain length and the degree of saturation of the fatty acyl-CoA ligands. In addition to their roles as substrates to yield energy, as an energy store, or as constituents of membrane phospholipids, dietary fatty acids may affect the course of a disease by modulating the expression of HNF-4α-controlled genes.

Peroxisome proliferator-activated receptor (PPAR) alpha activation and its consequences in humans.

Amphipathic carboxylates collectively defined as peroxisome proliferators (PP) induce in rodents a pleiotropic effect, mediated by the peroxisome proliferator-activated receptor alpha (PPAR alpha). Treatment with PP results in rodents in hypolipidemia, peroxisome proliferation and liver hypertrophy and hyperplasia leading to non-genotoxic hepatocarcinogenesis. In contrast to rodents, the hypolipidemic effect exerted by PP in humans is not accompanied by peroxisome proliferation nor by induction of peroxisomal beta-oxidation or other activities induced by PP in rodents. Non-responsiveness in humans may be ascribed to a missing liver component in the PPAR alpha transduction pathway specifically involved with transcriptional modulation of chromosomal PPAR alpha responsive genes. Hence, biological effects exerted by PP in the human liver are likely to be mediated by a transduction pathway independent of PPAR alpha.

Kinase-independent transcriptional co-activation of peroxisome proliferator-activated receptor α by AMP-activated protein kinase

AMPK (AMP-activated protein kinase) responds to intracellular ATP depletion, while PPARalpha (peroxisome proliferator-activated receptor alpha) induces the expression of genes coding for enzymes and proteins involved in increasing cellular ATP yields. PPARalpha-mediated transcription is shown here to be co-activated by the alpha subunit of AMPK, as well as by kinase-deficient (Thr172Ala) and kinase-less (Asp157Ala, Asp139Ala) mutants of AMPKalpha. The Ser452Ala mutant of mPPARalpha mutated in its putative consensus AMPKalpha phosphorylation site is similarly co-activated by AMPKalpha. AMPKalpha or its kinase-less mutants bind to PPARalpha; binding is increased by MgATP, to a lesser extent by MgADP, but not at all by AMP or ZMP [AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) monophosphate]. ATP-activated binding of AMPKalpha to PPARalpha is mediated primarily by the C-terminal regulatory domain of AMPKalpha. PPARalpha co-activation by AMPKalpha may, however, require its secondary interaction with the N-terminal catalytic domain of AMPKalpha, independently of its kinase activity. While AMPK catalytic activity is activated by AICAR, PPARalpha co-activation and PPARalpha-controlled transcription are robustly inhibited by AICAR, with concomitant translocation of nuclear AMPKalpha or its kinase-less mutants to the cytosol. In conclusion, AMPKalpha, independently of its kinase activity, co-activates PPARalpha both in primary rat hepatocytes and in PPARalpha-transfected cells. The kinase and transcriptional co-activation modes of AMPKalpha are both regulated by the cellular ATP/AMP ratio. Co-activation of PPARalpha by AMPKalpha may transcriptionally complement AMPK in maintaining cellular ATP status.

The effect of bezafibrate and long-chain fatty acids on peroxisomal activities in cultured rat hepatocytes

Peroxisomal activities have been evaluated in cultured rat hepatocytes in the presence of bezafibrate or long-chain fatty acids added to the culture medium. All activities decreased continuously over a time period of 100 h in culture but selected activities were relatively increased as a function of the added effectors. This relative increase in peroxisomal activities was dose-dependent, discernible within the first 24 h in culture and consisted of activities related specifically to peroxisomal fatty acyl beta-oxidation, e.g., cyanide-insensitive palmitoyl-CoA oxidation, H2O2-forming palmitoyl-CoA oxidase and heat-labile enoyl-CoA hydratase. Peroxisomal catalase or mitochondrial fatty acyl beta-oxidation (cyanide-sensitive) remained relatively unchanged. The relative increase in peroxisomal activities was accompanied by a respective increase in the number of peroxisomes as well as in thymidine incorporation rate.

Gating of the mitochondrial permeability transition pore by thyroid hormone

The calorigenic‐thermogenic activity of thyroid hormone (T3) has long been ascribed to uncoupling of mitochondrial oxidative phosphorylation. However, the mode of action of T3 in promoting mitochondrial proton leak is still unresolved. Mitochon‐drial uncoupling by T3 is reported here to be transduced in vivo in rats and in cultured Jurkat cells by gating of the mitochondrial permeability transition pore (PTP). T3‐induced PTP gating is shown here to be abrogated in inositol 1,4,5‐trisphosphate (IP3) receptor 1 (D?3R1)_/_ cells, indicating that the endoplasmic reticulum IP3R1 may serve as upstream target for the mitochondrial activity of T3. IP3R1 gating by T3 is due to its increased expression and truncation into channel‐only peptides, resulting in IP3‐independent Ca2+ efflux. Increased cytosolic Ca2+ results in activation of protein phosphatase 2B, dephosphorylation and depletion of mitochondrial Bcl2 (S70), and increase in mitochondrial free Bax leading to low‐conductance PTP gating. The T3 transduction pathway integrates genomic and nongenomic activities of T3 in regulating mitochondrial energetics and may offer novel targets for thyromimetics designed to modulate energy expenditure.—Yehuda‐Shnaidman, E., Kalderon, B., Azazmeh, N., Bar‐Tana, J. Gating of the mitochondrial permeability transition pore by thyroid hormone. FASEB J. 24, 93–104 (2010). www.fasebj.org

Sensitization to Insulin Induced by β,β′-Methyl-Substituted Hexadecanedioic Acid (MEDICA 16) in Obese Zucker Rats In Vivo

β,β′-methyl-substituted hexadecanedioic acid (MEDICA 16) consists of a nonmetabolizable long-chain fatty acid designed to probe the effect exerted by fatty acids on insulin sensitivity. The effect of MEDICA 16 was evaluated in insulin-resistant Zucker (fa/fa) rats in terms of liver, muscle, and adipose tissue response to clamped euglycemic hyperinsulinemia in vivo. Nontreated Zucker rats were insulin resistant, maintaining basal rates of total-body glucose disposal, glucose production in liver, free fatty acid (FFA) flux into plasma, and FFA reesterification in adipose tissue, irrespective of the insulin levels induced. MEDICA 16 treatment resulted in an insulin-induced decrease in hepatic glucose production, together with an insulin-induced increase in total-body glucose disposal. Intracellular reesterification of lipolysed FFA in adipose tissue was specifically activated by MEDICA 16, resulting in a pronounced decrease in FFA release, with a concomitant decrease in plasma FFA. In conclusion, MEDICA 16 treatment results in the sensitization of liver, muscle, and adipose tissue to insulin in an animal model for obesity-induced insulin resistance.

Within brown-fat cells, UCP1-mediated fatty acid-induced uncoupling is independent of fatty acid metabolism

In the present investigation, we have utilized the availability of UCP1(-/-) mice to examine a wide range of previously proposed lipid activators of Uncoupling Protein 1 (UCP1) in its native environment, i.e. in the brown-fat cells. A non-metabolizable fatty acid analogue, beta,beta cent-methyl-substituted hexadecane alpha,omega-dicarboxylic acid (Medica-16) is a potent UCP1 (re)activator in brown-fat cells, despite its bipolar structure. All-trans-retinoic acid activates UCP1 within cells, whereas beta-carotene only does so after metabolism. The UCP1-dependent effects of fatty acids are positively correlated with their chain length. Medium-chain fatty acids are potent UCP1 activators in cells, despite their lack of protonophoric properties in mitochondrial membranes. Thus, neither the ability to be metabolized nor an innate uncoupling/protonophoric ability is a necessary property of UCP1 activators within brown-fat cells.

AMPK activation by long chain fatty acyl analogs

The antidiabetic efficacy of first-line insulin sensitizers (e.g., metformin, glitazones) is accounted for by activation of AMP-activated protein kinase (AMPK). Long chain fatty acids (LCFA) activate AMPK, but their putative antidiabetic efficacy is masked by their beta-oxidized or esterified lipid products. Substituted alpha,omega-dicarboxylic acids of 14-18 carbon atoms in length (MEDICA analogs) are not metabolized beyond their acyl-CoA thioesters, and may therefore simulate AMPK activation by LCFA while avoiding LCFA turnover into beta-oxidized or esterified lipid products. MEDICA analogs are shown here to activate AMPK and some of its downstream targets in vivo, in cultured cells and in a cell-free system consisting of the (alpha(1)beta(1)gamma(1))AMPK recombinant and LKB1-MO25-STRAD (AMPK-kinase) recombinant proteins. AMPK activation by MEDICA is accompanied by normalizing the hyperglycemia-hyperinsulinemia of diabetic db/db mice in vivo with suppression of hepatic glucose production in cultured liver cells. Activation of AMPK by MEDICA or LCFA is accounted for by (a) decreased intracellular ATP/AMP ratio and energy charge by the free acid, (b) activation of LKB1 phosphorylation of AMPK(Thr172) by the acyl-CoA thioester. The two activation modes are complementary since LKB1/AMPK activation by the CoA-thioester is fully evident under conditions of excess AMP. MEDICA analogs may expand the arsenal of AMPK activators used for treating diabetes type 2.

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.