Transforming growth factor-β1 (TGF-

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Although the peroxisome proliferator-activated receptor (PPAR␣) binds and is activated by a variety of synthetic xenobiotics, the identity of the high affinity endogenous ligand(s) is incompletely resolved. Likewise, it is not known how putative endogenous ligands alter PPAR␣ conformation in order to affect transcriptional regulation. Direct fluorescence binding and fluorescence displacement assays showed for the first time that PPAR␣ exhibits high affinity (1-14 nM K d values) for unsaturated long chain fatty acyl-CoAs as well as unsaturated long chain fatty acids commonly found in mammalian cells. Fluorescence resonance energy transfer between PPAR␣ aromatic amino acids and bound corresponding naturally occurring fluorescent ligands (i.e. cis-parinaroyl-CoA, trans-parinaric acid) yielded intermolecular distances of 25-29 Å, confirming close molecular interaction. Interestingly, although PPAR␣ also exhibited high affinity for saturated long chain fatty acylCoAs, regardless of chain length (1-13 nM K d values), saturated long chain fatty acids were not significantly bound. In contrast to the similar affinities of PPAR␣ for fatty acyl-CoAs and unsaturated fatty acids, CoA thioesters of peroxisome proliferator drugs were bound with 5-6-fold higher affinities than their free acid forms. Circular dichroism demonstrated that high affinity ligands (long chain fatty acyl-CoAs, unsaturated fatty acids), but not weak affinity ligands (saturated fatty acids), elicited conformational changes in PPAR␣ structure, a hallmark of ligand-activated nuclear receptors. Finally, these ligand specificities and induced conformational changes correlated functionally with co-activator binding. In summary, since nuclear concentrations of these ligands are in the nanomolar range, long chain fatty acyl-CoAs and unsaturated fatty acids may both represent endogenous PPAR␣ ligands. Furthermore, the finding that saturated fatty acylCoAs, rather than saturated fatty acids, are high affinity PPAR␣ ligands provides a mechanism accounting for saturated fatty acid transactivation in cell-based assays.

While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them for rapid removal in oxidative (mitochondria, peroxisomes) or storage (endoplasmic reticulum, lipid droplets) organelles. Mammals have a large (15 member) family of FABPs with multiple members occurring within a single cell type. The first described FABP, liver-FABP (L-FABP, or FABP1), is expressed in very high levels (2-5% of cytosolic protein) in liver as well as intestine and kidney. Since L-FABP facilitates uptake and metabolism of LCFAs in vitro and in cultured cells, it was expected that abnormal function or loss of L-FABP would reduce hepatic LCFA uptake/ oxidation and thereby increase LCFAs available for oxidation in muscle and/or storage in adipose. This prediction was confirmed in vitro with isolated liver slices and cultured primary hepatocytes from L-FABP gene-ablated mice. Despite unaltered food consumption when fed a control diet ad libitum, the L-FABP null mice exhibited age-and sex-dependent weight gain and increased fat tissue mass. The obese phenotype was exacerbated in L-FABP null mice pair-fed a high fat diet. Taken together with other findings, these data suggest that L-FABP could have an important role in preventing age-or diet-induced obesity.