Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site VSports app下载. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2013 May 15;8(5):e61551.
doi: 10.1371/journal.pone.0061551. Print 2013.

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

Jerry R Colca  1 , William G McDonald, Gregory S Cavey, Serena L Cole, Danielle D Holewa, Angela S Brightwell-Conrad, Cindy L Wolfe, Jean S Wheeler, Kristin R Coulter, Peter M Kilkuskie, Elena Gracheva, Yulia Korshunova, Michelle Trusgnich, Robert Karr, Sandra E Wiley, Ajit S Divakaruni, Anne N Murphy, Patrick A Vigueira, Brian N Finck, Rolf F Kletzien
Affiliations

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

Jerry R Colca et al. PLoS One. .

Abstract

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 VSports手机版. 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. .

PubMed Disclaimer

"VSports在线直播" Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: Elena Gracheva, Yulia Korshunova, Michelle Trusgnich, and Robert Karr are employed by Medros. Medros performed the experiments in Drosophila under contract to Metabolic Solutions Development Company. This fact did not influence their interpretation of the results and they have no other conflicts in this regard. The authors confirm that this does not alter their adherence to all PLOS ONE policies on sharing data and materials V体育安卓版.

"V体育安卓版" Figures

Figure 1
Figure 1. Selective crosslinking with photo affinity probe.
(A) 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) 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). (C) Liver mitochondrial fractions from wild type or mitoNEET null mice 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. (D) 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.
Figure 2
Figure 2. Crosslinked protein is BRP44.
(A) Blue Native electrophoresis of crosslinked proteins showing the stain pattern (left) and the corresponding autoradiogram image for the specifically crosslinked protein (without, “−”on the left and with MSDC-0160 competition, “+”, on the right). The <150 kDa band is marked by the arrow. (B) Second dimension SDS separation of Blue Native gel without (left) and with (right) MSDC-0160 competition. Upper figures are silver stained images of the gels and lower figures are corresponding autoradiograms. Areas of interest are marked by the yellow arrows. (C) Expanded view of silver-stained images showing the three areas submitted for MS/MS analysis followed by the respective trypsin peptide sequences. These peptides (shown also in red, bottom frame D) identify BRP44 (Q9D023).
Figure 3
Figure 3. BRP44 family.
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.
Figure 4
Figure 4. Expressed BRP44 goes to mitochondrion and is crosslinked selectively by the TZD probe.
(A) 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) 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. (C) 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.
Figure 5
Figure 5. Crosslinking of the Drosophila ortholog in wild type and knockdown lines.
(A) Wild-type larvae and adult flies were homogenized and mitochondrial pellets were crosslinked with and without the addition of MSDC-0160 as in figure 1. 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) 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). (C) 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).
Figure 6
Figure 6. BPR44 and BRP44L are involved in pyruvate transport.
(A) 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) 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. (C) A separate experiment similar to that shown in B with other TZDs. (D) 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 Figure 1B.
Figure 7
Figure 7. Insulin sensitizer effects in wild type and knockdown flies.
(A) Acute insulin action was measured in inverted larvae in the absence (−) or presence (+) of 2 µM recombinant insulin. The larvae were either grown under control conditions (left) or in the presence of high sucrose (right). Representative Western blots are shown below and the calculated areas are shown above for pAKT (left side) or total AKT (right side) normalized to actin in the absence (open bars) or presence of insulin (upper bars). An unpaired, two-tailed t-test was used to derive p-values. Error bars are ± SE (N = 3). (B) Effect of incubation with DMSO, MSDC-1473 or MSDC-0160 on the amount of insulin-stimulated phosphorylation of AKT under the high sucrose conditions (detected and analyzed as in A; mean and SE, N = 3). (C) Wild-type female flies or flies with knockdown of CG14290 (as in Figure 5) were raised on a 0.75M sucrose matrix and aged for 7 days with or without MSDC-0160 added to the matrix. RNA from flies grown under these conditions was extracted and full genome expression analysis was performed using the Agilent system. Conditions in the presence of the high sugar included control flies in addition to knock down of CG14290 (line 15858). MSDC-0160 was provided to either control or line 15859 knockdown flies. The data shown reflect changes that were more than 1.5 fold different and t-test value <9E-03 relative to the high sucrose DMSO control in terms of increase (green) or decrease (red). The list of genes, along with the mammalian ortholog names, are shown on the left in the case of where the expression was decreased in the presence of high sucrose and thus increased with the treatment of MSDC-0160 and on the right for the transcripts that were increased in the presence of high sucrose and thus reduced with the treatment with MSDC-0160. All MSDC-0160-induced changes were lost in the knockdown strain.
See this image and copyright information in PMC

"V体育安卓版" References

    1. Wild S, Roglic G, Green A, Sicree R, King H (2004) Global prevalence of diabetes. Estimates for the year 2000 and projections for 2030. Diabetes Care 27: 1047–1053. - "VSports app下载" PubMed
    1. DeFronzo RA, Abdul-Ghani M (2011) Type 2 Diabetes can be prevented with early pharmacological intervention. Diabetes Care 34: S202–S209. - PMC - PubMed
    1. Ryder REJ (2011) Pioglitazone: an agent which reduces stroke, myocardial infarction and death and is also a key component of the modern paradigm for the optimum management of type 2 diabetes. The British Journal of Diabetes & Vascular Disease 11: 113–120.
    1. Colca JR, Kletzien RF (2006) What has prevented the expansion of insulin sensitisers? Expert Opin. Investig. Drugs 15: 205–210. - PubMed
    1. Kahn BB, McGraw TE (2010) Rosiglitazone, PPARγ, and type 2 diabetes. N Engl J Med 363: 2667–2669. - PMC - PubMed

Publication types

"V体育平台登录" MeSH terms