Depletion of mtDNA in myocytes causes insulin resistance and alters nuclear gene expression that may be involved in rescuing processes against cellular stress. Here we show that the expression of C1q tumor necrosis factor ␣-related protein isoform 5 (C1QTNF5) is drastically increased following depletion of mtDNA in myocytes. C1QTNF5 is homologous to adiponectin in respect to domain structure, and its expression and secretion from myocytes correlated negatively with the cellular mtDNA content. Similar to adiponectin, C1QTNF5 induced the phosphorylation of AMP-activated protein kinase (AMPK), leading to increased cell surface recruitment of GLUT4 and increased glucose uptake. Treatment of cells with purified recombinant C1QTNF5 increased the phosphorylation of acetyl-CoA carboxylase and stimulated fatty acid oxidation. C1QTNF5-mediated phosphorylation of AMPK or acetyl-CoA carboxylase was unaffected by depletion of adiponectin receptors such as AdipoR1 or AdipoR2, which indicated that adiponectin receptors do not participate in C1QTNF5-induced activation of AMPK. Serum C1QTNF5 levels were significantly higher in obese/diabetic animals (OLETF rats, ob/ob mice, and db/db mice). These results highlight C1QTNF5 as a putative biomarker for mitochondrial dysfunction and a potent activator of AMPK.

Abbreviations: GLUT, glucose transporter; HBP, hexosamine biosynthetic pathway; IRS, insulin receptor substrate; PDK-1, phosphatidylinositol dependent protein kinase-1; PKC-λ/ζ, protein kinase C-λ/ζ; PI3K, phospatidylinositol 3-kinase; PM, plasma membrane IntroductionThe pathophysiology of non-insulin-dependent diabetes mellitus (NIDDM) results from impaired peripheral tissue sensitivity to insulin and reduction of insulin secretion. Glucose uptake in mammalian cells is mediated by a family of intrinsic membrane proteins known as facilitative glucose transporters, GLUTs (Mueckler, 1994). GLUT4, the insulin-responsive glucose transporter, is selectively expressed in adipocytes and muscle (Bryant et al., 2002). GLUT4 in these cells constantly recycles between the plasma membrane (PM) and intracellular storage pools (Cushm an and W ardzala, 1980). About 90 percent of GLUT4 is sequestered intracellularly in the absence of insulin or other stimuli such as exercise (Cushman and Wardzala, 1980). Insulin stimulates glucose uptake in these cells primarily by inducing net translocation of GLUT4 from the intracellular storage sites to the PM (Lee et al., 1999, Lee et al., 2000b. The translocation of GLUT4 by insulin results from a cascade of signal transduction, which is composed of a series of molecules (Pessin et al., 1999). Insulin-stimulated GLUT4 translocation and glucose uptake are mediated largely through the activation/phosphorylation of IRS-1 and its downstream effectors, such as PI3K, PDK-1, PKC-λ/ζ, and Akt2 (Saltiel and Pessin, 2002;Kanzaki and Pessin, 2003). An impaired GLUT4 translocation and/or insulin signaling pathway would result in insulin resistance and hyperglycemia, the primary characteristics and hallmarks of NIDDM (Watson and Pessin, 2001). O-GlcNAc m odification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat prim ary adipocytes O-GlcNAc modification and insulin resistance 221It has been known that O-GlcNAc modification of protein plays an important role in transcription, translation, nuclear transport and cytoskeletal assembly (Comer and Hart, 2000;Wells et al., 2001). Recently, O-GlcNAc modification as well as phosphorylation/ dephosphorylation has been focused on as a main regulation mechanism of cellular signal transduction pathways. Post-translational O-GlcNAc modification on proteins is defined as the O-linked attachment of single GlcNAc moiety to specific hydroxyl groups of Ser or Thr residue (Buse et al., 2002). Since Oglycosylation of cellular proteins via Ser/Thr takes place dynamically in various ways and the modification site is usually located near the O-phosphorylation site or sometimes at the same site, O-GlcNAc modification plays as a dynamic negative regulator against protein phosphorylation (Snow and Hart, 1998, Zachara and. Therefore, O-glycosylation and O-phosphorylation are known to participate in the modification of protein interaction and regulation of signal transduction by changing the protein reaction site through the mutua...

The prompt clearance of cells undergoing apoptosis is critical during embryonic development and normal tissue turnover, as well as during inflammation and autoimmune responses. We recently demonstrated that stabilin-2 is a phosphatidylserine receptor that mediates the clearance of apoptotic cells, thereby releasing the anti-inflammatory cytokine, transforming growth factor-␤. However, the downstream signaling components of stabilin-2-mediated phagocytosis are not known. Here, we provide evidence that the adaptor protein, GULP, physically and functionally interacts with the stabilin-2 cytoplasmic tail. Using fluorescent resonance energy transfer analysis and biochemical approaches, we show that GULP directly binds to the cytoplasmic tail of stabilin-2. Knockdown of endogenous GULP expression significantly decreased stabilin-2-mediated phagocytosis. Conversely, overexpression of GULP caused an increase in aged cell engulfment. The phosphotyrosine binding (PTB) domain of GULP was sufficient for the interaction with stabilin-2; therefore, transduction of TAT fusion PTB domain acts as a dominant negative, resulting in impaired engulfment of aged red blood cells in stabilin-2 expressing cells. In addition, the PTB domain of GULP was able to specifically interact with the NPXY motif of the stabilin-2 cytoplasmic tail. Taken together, these results indicate that GULP is a likely downstream molecule in the stabilin-2-mediated signaling pathway and plays an important role in stabilin-2-mediated phagocytosis.

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