OBJECTIVETo elucidate the molecular basis for mitochondrial dysfunction, which has been implicated in the pathogenesis of diabetes complications.RESEARCH DESIGN AND METHODSMitochondrial matrix and membrane fractions were generated from liver, brain, heart, and kidney of wild-type and type 1 diabetic Akita mice. Comparative proteomics was performed using label-free proteome expression analysis. Mitochondrial state 3 respirations and ATP synthesis were measured, and mitochondrial morphology was evaluated by electron microscopy. Expression of genes that regulate mitochondrial biogenesis, substrate utilization, and oxidative phosphorylation (OXPHOS) were determined.RESULTSIn diabetic mice, fatty acid oxidation (FAO) proteins were less abundant in liver mitochondria, whereas FAO protein content was induced in mitochondria from all other tissues. Kidney mitochondria showed coordinate induction of tricarboxylic acid (TCA) cycle enzymes, whereas TCA cycle proteins were repressed in cardiac mitochondria. Levels of OXPHOS subunits were coordinately increased in liver mitochondria, whereas mitochondria of other tissues were unaffected. Mitochondrial respiration, ATP synthesis, and morphology were unaffected in liver and kidney mitochondria. In contrast, state 3 respirations, ATP synthesis, and mitochondrial cristae density were decreased in cardiac mitochondria and were accompanied by coordinate repression of OXPHOS and peroxisome proliferator–activated receptor (PPAR)-γ coactivator (PGC)-1α transcripts.CONCLUSIONSType 1 diabetes causes tissue-specific remodeling of the mitochondrial proteome. Preservation of mitochondrial function in kidney, brain, and liver, versus mitochondrial dysfunction in the heart, supports a central role for mitochondrial dysfunction in diabetic cardiomyopathy.

Similar to insulin, osmotic shock of 3T3L1 adipocytes stimulated an increase in glucose transport activity and translocation of GLUT4 protein from intracellularly localized vesicles to the plasma membrane. The docking/ fusion of GLUT4 vesicles with the plasma membrane appeared to utilize a similar mechanism, since expression of a dominant interfering mutant of syntaxin-4 prevented both insulin-and osmotic shock-induced GLUT4 translocation. However, although the insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, activation by osmotic shock was wortmannin-insensitive. Furthermore, insulin stimulated the phosphorylation and activation of the Akt kinase, whereas osmotic shock was completely without effect. Surprisingly, treatment of cells with the tyrosine kinase inhibitor, genistein, or microinjection of phosphotyrosine antibody prevented both the insulin-and osmotic shock-stimulated translocation of GLUT4. In addition, osmotic shock induced the tyrosine phosphorylation of several discrete proteins including Cbl, p130 cas , and the recently identified soluble tyrosine kinase, calcium-dependent tyrosine kinase (CADTK). In contrast, insulin had no effect on CADTK but stimulated the tyrosine phosphorylation of Cbl and the tyrosine dephosphorylation of pp125 FAK and p130 cas . These data demonstrate that the osmotic shock stimulation of GLUT4 translocation in adipocytes occurs through a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.The facilitative glucose transporters are a family of related integral membrane proteins that are responsible for the regulation of whole body and cellular glucose homeostasis. Unlike other members of this family, the insulin-responsive glucose transporter isoform (GLUT4) 1 is predominantly expressed in adipose tissue and in skeletal and cardiac muscle (1, 2). In these tissues, insulin increases glucose uptake by regulating the intracellular trafficking of the GLUT4 protein. In the basal state, GLUT4 cycles continuously between the plasma membrane and one or more intracellular compartments, with the vast majority of the transporter residing within the cell interior (3, 4). Activation of the insulin receptor triggers a large increase in the rate of GLUT4 vesicle exocytosis in addition to a smaller decrease in the rate of internalization by endocytosis. This insulin-dependent shift in the cellular dynamics of GLUT4 vesicle trafficking results in a net increase of GLUT4 protein level on the cell surface, thereby increasing the rate of glucose uptake (for recent reviews, see Refs. 5-8).Activation of the insulin receptor by ligand binding initiates a cascade of signaling events, which include activation of the intrinsic receptor tyrosine kinase, autophosphorylation of the receptor, and phosphorylation of cellular substrates such as insulin receptor substrate (IRS)-1/2 and Shc (for recent reviews, see Refs. 9 and 10). Phosphorylation of these substrates provides docking s...

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Guanosine 5-O-(3-thiotriphosphate) (GTP␥S) treatment of permeabilized adipocytes results in GLUT4 translocation similar to that elicited by insulin treatment. However, although the selective phosphatidylinositol 3-kinase inhibitor, wortmannin, completely prevented insulin-stimulated GLUT4 translocation, it was without effect on GTP␥S-stimulated GLUT4 translocation. In addition, insulin was an effective stimulant, whereas GTP␥S was a very weak activator of the downstream Akt serine/threonine kinase. Consistent with an Akt-independent mechanism, guanosine 5-O-2-(thio) diphosphate inhibited insulin-stimulated GLUT4 translocation without any effect on the Akt kinase. Surprisingly, two functionally distinct tyrosine kinase inhibitors, genistein and herbimycin A, as well as microinjection of a monoclonal phosphotyrosine specific antibody, inhibited both GTP␥S-and insulin-stimulated GLUT4 translocation. Phosphotyrosine immunoblotting and specific immunoprecipitation demonstrated that GTP␥S did not elicit tyrosine phosphorylation of insulin receptor or insulin receptor substrate-1. In contrast to insulin, proteins in the 120 -130-kDa and 55-75-kDa range were tyrosine-phosphorylated following GTP␥S stimulation. Several of these proteins were identified and include protein-tyrosine kinase 2 (also known as CAK␤, RAFTK, and CADTK), pp125 focal adhesion tyrosine kinase, pp130 Crk-associated substrate, paxillin, and Cbl. These data demonstrate that the GTP␥S-stimulated GLUT4 translocation utilizes a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.

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It has been shown that Fructus Ligustri Lucidi (FLL), a promising traditional Chinese medicine, can inhibit the growth of tumors. However, the effective component and molecular mechanism of FLL act to inhibit tumor proliferation are unclear. In this study, we demonstrated that oleanolic acid (OA), a principal chemical component of FLL, inhibited the proliferation of human leukemia HL60 cells in culture. MTT assay showed that treatment of HL60 cells with FLL crude extracts or OA dramatically blocked the growth of target tumor cell in a time- and dose-dependent manner. Morphological changes of the nuclei and DNA fragmentation showed that apoptotic cell death occurred in the HL60 cells after treating with FLL extracts (20 mg/ml) or OA (3.65 x 10(-2) mg/ml). Furthermore, flow cytometry assay showed that treatment of HL60 cells with FLL or OA caused an increased accumulation of G(1) and sub-G(1) subpopulations. Western blot analysis showed that caspase-9 and caspase-3 were activated, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) in the target cells during FLL- or OA-induced apoptosis. These results suggest that OA acts as the effective component of FLL by exerting its cytotoxicity towards target tumor cells through activation of caspases and cleavage of PARP.

Taken together, our data provide strong evidence that epithelial-mesenchymal transition occurs in renal cell carcinoma. Thus, interference with epithelial-mesenchymal transition in renal cell carcinoma might represent a future therapeutic option.