Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT)--relationship to newly identified mitochondrial pyruvate carrier proteins.

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 (13)C-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.

Opposing effect by cytokines on Fas-mediated apoptosis in A549 lung epithelial cells.

Tissue repair is determined by many signals provided in the local environment. Central to this process is the commitment of the parenchymal cell to undergo apoptosis, survive, or proliferate following inflammation. We hypothesize that lung epithelial cell apoptosis is influenced by exposure to cytokines released into the alveolar microenvironment during the inflammatory process. In this investigation we demonstrate that interferon (IFN)-gamma and interleukin (IL)-1beta have opposing effects on Fas-mediated apoptosis in A549 cells, a human lung epithelial cell line. Exposure to IFN-gamma before Fas activation significantly increased caspase activity, caspase processing of CK-18, a key cytoskeletal protein in epithelial cells, and increased the appearance of apoptotic nuclei. Induction of Fas-mediated death by IFN-gamma was 3-fold higher than with Fas activation alone. In contrast, pretreatment with IL-1beta before Fas activation completely inhibited apoptosis. Furthermore, our results demonstrate that IFN-gamma and IL-1beta induce opposite effects at multiple checkpoints during Fas-mediated apoptosis. Most striking, IL-1beta prevented the activation of caspases involved in Fas-mediated death by inducing an anti-apoptotic effect proximal to or at the point of caspase-8 activation. Finally, our investigation demonstrates that the differential impact of IL-1beta and IFN-gamma on Fas-mediated apoptosis are in part dependent on modulation of the PI 3-K/Akt survival pathway.