Background: Thiazolidinediones may have insulin-sensitizing effects independent of the nuclear receptor PPAR␥. Results: A novel PPAR␥-sparing thiazolidinedione ameliorated insulin resistance and inflammation in obese mice. Conclusion:The insulin-sensitizing effects of thiazolidinediones are separable from the ability to bind PPAR␥. Significance: Identification of other molecular targets of thiazolidinediones may generate new therapeutics for treatment of insulin resistance and diabetes.
The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor g and CCAAT/enhancer binding protein a. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an ?25% reduction in basal 3 H-labeled fatty acid uptake and a complete loss of insulin-stimulated 3 H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.
Lipin 1 is a bifunctional intracellular protein that regulates fatty acid metabolism in the nucleus via interactions with DNAbound transcription factors and at the endoplasmic reticulum as a phosphatidic acid phosphohydrolase enzyme (PAP-1) to catalyze the penultimate step in triglyceride synthesis. However, livers of 8-day-old mice lacking lipin 1 (fld mice) exhibited normal PAP-1 activity and a 20-fold increase in triglyceride levels. We sought to further analyze the hepatic lipid profile of these mice by electrospray ionization mass spectrometry. Surprisingly, hepatic content of phosphatidate, the substrate of PAP-1 enzymes, was markedly diminished in fld mice. Similarly, other phospholipids derived from phosphatidate, phosphatidylglycerol and cardiolipin, were also depleted. Another member of the lipin family (lipin 2) is enriched in liver, and hepatic lipin 2 protein content was markedly increased by lipin 1 deficiency, food deprivation, and obesity, often independent of changes in steady-state mRNA levels. Importantly, RNAi against lipin 2 markedly reduced PAP-1 activity in hepatocytes from both wild type and fld mice and suppressed triglyceride synthesis under conditions of high fatty acid availability. Collectively, these data suggest that lipin 2 plays an important role as a hepatic PAP-1 enzyme.Spontaneously arising mutations in the gene encoding lipin 1 (Lpin1) cause severe metabolic abnormalities in fatty liver dystrophic (fld) mice (1), which exhibit neonatal growth retardation, fatty liver, and dyslipidemia (2). These phenotypic manifestations abruptly resolve at ϳ14 days of age. However, adult fld mice exhibit lipodystrophy, insulin resistance, and susceptibility to atherosclerotic lesion formation (3, 4). Recent evidence regarding the molecular functions of lipin 1 has begun to explain the severe metabolic phenotype of these mice.Lipin 1 catalyzes the Mg 2ϩ -dependent dephosphorylation of phosphatidic acid (phosphatidic acid (PA) 2 phosphohydrolase (PAP-1) (5)) to form diacylglycerol (DG), the penultimate step in the Kennedy pathway of triglyceride (TG) synthesis (Fig. 1A). As proof of the importance of lipin 1-mediated PAP-1 activity, fld mice almost completely lack this enzymatic activity in adipose tissue, skeletal muscle, and heart (6), and the inability to synthesize TG in adipocytes may explain the defect in adipogenesis in fld mice.Interestingly, lipin 1 is a soluble protein, and significant lipin 1 localization to the nucleus has also been demonstrated (1). Moreover, recent evidence suggests that lipin 1 functions as a regulator of gene transcription via protein-protein interactions with DNA-bound transcription factors (7, 8), including the peroxisome proliferator-activated receptor ␣ (PPAR␣) and its coactivator protein PPAR␥-coactivator 1␣ (PGC-1␣) (7). PAP-1 catalytic activity is not required for peroxisome proliferator-activated receptor ␣ coactivation, and these two molecular functions seem to be completely separable. Thus, lipin 1 is poised to play important roles in controlling the ...
Fatty acid transport protein 1 (FATP1) is an ϳ63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16 -24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the K m values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.
Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using ␣-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V max /K m (C16:0)/ V max /K m (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.
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