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Nitroalkene fatty acids are potent endogenous ligand activators of PPARγ-dependent transcription. Previous studies with the naturally occurring regioisomers of nitrolinoleic acid revealed that the isomers are not equivalent with respect to PPARγ activation. To gain further insight into the structure−activity relationships between nitroalkenes and PPARγ, we examined additional naturally occurring nitroalkenes derived from oleic acid, 9-nitrooleic acid (E-9-NO2-18:1 [1]) and 10-nitrooleic acid (E-10-NO2-18:1 [2]), and several synthetic nitrated enoic fatty acids of variable carbon chain length, double bonds, and nitration site. At submicromolar concentrations, E-12-NO2derivatives were considerably more potent than isomers nitrated at carbons 5, 6, 9, 10, and 13, and chain length (16 versus 18) or number of double bonds (1 versus 2) was of little consequence for PPARγ activation. Interestingly, at higher concentrations (>2 μM) the nitrated enoic fatty acids (E-9-NO2-18:1 [1], E-9-NO2-16:1 [3], E-10-NO2-18:1 [2], and E-12-NO2-18:1 [7]) deviated significantly from the saturable pattern of PPARγ activation observed for nitrated 1,4-dienoic fatty acids (E-9-NO2-18:2, E-10-NO2-18:2, E-12-NO2-18:2, and E-13-NO2-18:2). [ABSTRACT FROM AUTHOR]

A series of 3-acylindole-1-benzylcarboxylic acids were designed and synthesized while searching for a PPARγ modulator with additional moderate intrinsic PPARα agonistic activity. 2-[3-[[3-(4-Chlorobenzoyl)-2-methyl-6-(trifluoromethoxy)-1H-indol-1-yl]methyl]phenoxy]-(2R)-butanoic acid (12d) was identified as such an agent which demonstrated potent efficacy in lowering both glucose and lipids in multiple animal models with significantly attenuated side effects such as fluid retention and heart weight gain associated with PPARγ full agonists. The moderate PPARα activity of 12dnot only contributed to the agent’s ability to manage lipid profiles but also appears to have potentiated its PPARγ efficacy in lowering glucose levels in preclinical diabetic animal models. [ABSTRACT FROM AUTHOR]

GLUT4 (glucose transporter 4) plays important roles in glucose homoeostasis in vivo. GLUT4 expression and function are diminished in diabetic human and animal subjects. The goal of the present study is to develop a cell-based assay for identifying negative regulators of GLUT4 translocation as potential targets for the treatment of Type 2 diabetes. Traditional GLUT4 translocation assays performed in differentiated myocytes or adipocytes are difficult to perform, particularly in HTS (high-throughput screening) mode. In the present study, we stably co-expressed c-Myc and eGFP [enhanced GFP (green fluorescent protein)] dual-tagged recombinant GLUT4 with recombinant IRS1 (insulin receptor substrate 1) in HEK-293 cells (human embryonic kidney cells) (HEK-293.IRS1.GLUT4 cells). Insulin treatment stimulated both glucose uptake and GLUT4 translocation in these cells. GLUT4 translocation is quantified by a TRF (time-resolved fluorescence) assay in a 96-well HTS format. TRF assays confirmed insulin-stimulated GLUT4 translocation, which can be inhibited by PI3K (phosphoinositide 3-kinase) or Akt [also called PKB (protein kinase B)] inhibitors. Treatment with palmitate increased IRS1 serine phosphorylation and reduced insulin-stimulated Akt phosphorylation and GLUT4 translocation, indicating insulin resistance. Knockdown of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and PTP1B (protein tyrosine phosphatase 1B) gene expression by siRNA (small interfering RNA) treatment significantly increased GLUT4 translocation only in cells treated with palmitate but not in untreated cells. Similar results were obtained on treatment with siRNA of JNK1 (c-Jun N-terminal kinase 1), S6K1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) and PKCθ(protein kinase C θ). In summary, we have established and validated a novel GLUT4 translocation assay that is optimal for identifying negative regulators of GLUT4 translocation. In combination with more physiologically relevant secondary assays in myotubes and adipocytes, this assay system can be used to identify potential novel therapeutic targets for the treatment of Type 2 diabetes. [ABSTRACT FROM AUTHOR]