Identification of a Mitochondrial Target of Thiazolidinedione Insulin Sensitizers (mTOT)—Relationship to Newly Identified Mitochondrial Pyruvate Carrier Proteins (original) (raw)
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 [11] 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.