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

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

Selective crosslinking with photo affinity probe.

<p>(<b>A</b>) Mitochondrial membranes (20 µg) from rat liver were incubated with the iodinated (¹²⁵I) photo-probe (MSDC-1101) in the absence of any competing compound (lane 1); in the presence of 25 µM MSDC-0160 (lane 2); in the presence of 25 µM MSDC-0602 (lane 3); or in the presence of 25 µM MSDC-1473 (lane 4). Following exposure to UV light, samples were separated on one dimensional SDS-PAGE and the dried gel was exposed to X-ray film. (<b>B</b>) Active TZDs, but not MSDC-1473, produce a dose-dependent increase in UCP1 as detected on Western blots from mouse BAT progenitor cells. The abscissa shows drug concentration (µM). The inset on the top of this figure shows a representative Western blot of the increase in UCP1 protein in cells treated with an active TZD (pioglitazone). The data below the blot show the dose dependent increases observed in a representative experiment from a scan of the Western blots (arbitrary units, Mean and SE; N = 3). (<b>C</b>) Liver mitochondrial fractions from wild type or mitoNEET null mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061551#pone.0061551-Bricker1" target="_blank">[11]</a> were crosslinked as in A without (−) or with (+) 25 µM MSDC-0160. The top figure is the resulting autoradiogram and the bottom is the Western blot for mitoNEET. (<b>D</b>) Mouse liver mitochondria were fractionated to generate fractions for outer membrane (OM), inter membrane space (IMS), inner membrane (IMM), and matrix. Total protein (10 µg) from each fraction was crosslinked as in A. The autoradiogram is shown at the top and a stain for total protein for the same 4 mitochondrial subfractions is shown below.</p

Expressed BRP44 goes to mitochondrion and is crosslinked selectively by the TZD probe.

<p>(<b>A</b>) Upper frame: C-terminally-tagged GFP BRP44 (green) in Cos7 cells is co-localized with endogenous BRP44 (red). Lower frame: C-terminally tagged 6×His BRP44 (green) is co-localized with Mitotracker Red confirming the mitochondrial localization. (<b>B</b>) A sequence encoding BRP44 C-terminally tagged with six histidine residues was transiently expressed in HEK293 cells. Subcellular fractions were prepared (P1, crude nuclear fraction; P2, crude mitochondrial fraction; S2, post mitochondrial fraction) and incubated with the radiolabeled photo-probe in the presence or absence of 25 µM MSDC-0160 as indicated. Samples were subjected to one dimensional SDS-PAGE and the dried gel was exposed to X-ray film. The red arrows point to the image of the selectively crosslinked native and C-terminal extension proteins. (<b>C</b>) Crosslinked BRP44 6×His is selectively immunoprecipitated by antibodies to 6×His. Membranes from HEK293 cells stably transfected with BRP44 6×His (right lanes, 6 His) or non transfected cells (left lanes, control) were crosslinked with or without competition with MSDC-0160 as in B. The membranes, all of which contained the specifically crosslinked band as shown in B, were then solubilized and immunoprecipitation was carried out with anti-His agarose beads. The resulting pellets were solubilized and submitted to SDS-PAGE. The corresponding autoradiogram is shown demonstrating that the radioactive band is only precipitated by anti-His tag in the tissue from the cells expressing His-6 BRP44.</p

Crosslinking of the <i>Drosophila</i> ortholog in wild type and knockdown lines.

<p>(<b>A</b>) Wild-type larvae and adult flies were homogenized and mitochondrial pellets were crosslinked with and without the addition of MSDC-0160 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061551#pone-0061551-g001" target="_blank">figure 1</a>. The autoradiograms of the resulting image of the specifically crosslinked proteins are shown for the flies on the left side compared to mouse liver in the lane on the right. (<b>B</b>) Fly stocks carrying RNAi constructs were crossed to Act-GAL4 driver and progeny tissues were processed and crosslinked as described in the text. Lanes marked “1” were from knockdowns of CG9399, lanes marked “4” were from knockdowns of CG14290, the other lanes were either unrelated genes or control strains. Two separate knockdown lines are represented. The complete list of the strains is shown in the text. Red arrows indicate reduced crosslinking of the specific CG9399 protein (BRP44 ortholog). (<b>C</b>) Membrane samples from CG9399 and CG14290 KD as in B were subjected to Western blot analysis for either the CG9399 (left) or CG14290 protein (right).</p

BRP44 family.

<p>Upper panel (A) is the multiple species sequence alignment of BRP44 and lower panel (B) shows the multiple species alignments for the family member protein BRP44L. Shaded amino acids show identical amino acids as compared to the human sequences.</p

BPR44 and BRP44L are involved in pyruvate transport.

<p>(<b>A</b>) UK5099 structure and effect of adding either 25 µM MSDC-0160 (lane 2) or UK5099 (lane 3) on crosslinking of BRP44 (Mpc2). Lane 1 is the DMSO control. (<b>B</b>) Incubation of mouse BAT cells with UK5099 effectively limits carbon flow from U-¹³C glucose into acetyl CoA (red line) while MSDC-0602 has a biphasic dose response. The non TZD insulin sensitizer MRL-24 also modulated flow of carbon into acetyl CoA under these conditions. Data are the ratio of Acetyl CoA (¹³C/¹²C) derived from U-¹³C glucose following a 2 hour pre-incubation and a 15 minute flux at 37C (n = 3; mean and SE; representative of 3 experiments) as described in the Methods. (<b>C</b>) A separate experiment similar to that shown in B with other TZDs. (<b>D</b>) BAT progenitor cells were incubated with the indicated concentrations of UK5099 with (+) or without (−) 3 µM MSDC-0160 for 5 days. Cells were lysed and Western blots were conducted for UCP1 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061551#pone-0061551-g001" target="_blank">Figure 1B</a>.</p