Thiazolidinedione (TZD) insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44) and BRP44 Like (BRP44L), which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT) cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of 13C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT) and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

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Activation of the nuclear transcription factor-B is an early event in endothelial activation. NF-B activation is regulated by the inducible phosphorylation and subsequent degradation of the inhibitory subunit IB-␣. We identified two discrete kinases of approximately 36 and 41 kDa in the cytoplasm of human umbilical vein endothelial cells that specifically bind to and phosphorylate the IB-␣ subunit. IB-␣ kinase activity is transiently elevated following treatment with either tumor necrosis factor ␣, interleukin-1␤, or bacterial lipopolysaccharides and precedes activation of either mitogen-activated kinase or Jun kinase. Furthermore, activation of the IB-␣ kinases precedes both the appearance of hyperphosphorylated IB-␣ and its subsequent degradation, as well as the translocation of NF-B to the nucleus. Deletion mutagenesis of the IB-␣ polypeptide revealed that these kinases bind in or around the ankyrin repeat domains and phosphorylate residues within the C terminus. These kinases, however, were not identical to casein kinase II and displayed a pharmacologic profile distinct from other known kinases. These kinases may represent components of a signal transduction pathway regulating IB-␣ levels in vascular endothelium.

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