Uncoupling Protein 3 (UCP3) Stimulates Glucose Uptake in Muscle Cells through a Phosphoinositide 3-Kinase-dependent Mechanism

Abstract

UCP3 is a mitochondrial membrane protein expressed in humans selectively in skeletal muscle. To determine the mechanisms by which UCP3 plays a role in regulating glucose metabolism, we expressed human UCP3 in L6 myotubes by adenovirus-mediated gene transfer and in H 9 C 2 cardiomyoblasts by stable transfection with a tetracycline-repressible UCP3 construct. Expression of UCP3 in L6 myotubes increased 2-deoxyglucose uptake 2-fold and cell surface GLUT4 2.3-fold, thereby reaching maximally insulin-stimulated levels in control myotubes. Wortmannin, LY 294002, or the tyrosine kinase inhibitor genistein abolished the effect of UCP3 on glucose uptake, and wortmannin inhibited UCP3-induced GLUT4 cell surface recruitment. UCP3 overexpression increased phosphotyrosine-associated phosphoinositide 3-kinase (PI3K) activity 2.2-fold compared with control cells (p < 0.05). UCP3 overexpression increased lactate release 1.5-to 2-fold above control cells, indicating increased glucose metabolism. In H 9 C 2 cardiomyoblasts stably transfected with UCP3 under control of a tetracycline-repressible promotor, removal of doxycycline resulted in detectable levels of UCP3 at 12 h and 2.2-fold induction at 7 days compared with 12 h. In parallel, glucose transport increased 1.3-and 2-fold at 12 h and 7 days, respectively, and the stimulation was inhibited by wortmannin or genistein. p85 association with membranes was increased 5.5-fold and phosphotyrosine-associated PI3K activity 3.8-fold. In contrast, overexpression of UCP3 in 3T3-L1 adipocytes did not alter glucose uptake, suggesting tissue-specific effects of human UCP3. Thus, UCP3 stimulates glucose transport and GLUT4 translocation to the cell surface in cardiac and skeletal muscle cells by activating a PI3K dependent pathway.Uncoupling proteins (UCPs) 1 are mitochondrial inner membrane proteins proposed to be central to the regulation of energy expenditure. Energy expenditure is composed of the resting metabolic rate, physical activity, and thermogenesis and can be increased by energy dissipation due to futile metabolic processes. Uncoupling protein 1 (UCP1), the first uncoupling protein to be identified, is selectively expressed in brown adipose tissue (BAT), a major site of thermogenesis in rodents. UCP1 uncouples mitochondrial respiration from ATP synthesis by dissipating the transmembrane proton gradient, releasing energy as heat (1). The importance of UCP1 in energy expenditure in adult humans is less clear, because little BAT is present. Recently UCP2 and UCP3 were identified with 59 and 57% amino acid identity to UCP1, respectively (2-4). UCP3, unlike UCP1 and UCP2, exists as short and long form transcripts (5). The long form of UCP3 was shown to be an uncoupling protein, because it increases O 2 consumption and decreases the mitochondrial electrochemical potential when expressed in yeast or C 2 C 12 myoblasts (2, 4, 6, 7). Reconstitution of UCPs into liposomes showed that UCP2 and UCP3, like UCP1, mediate electrophoretic proton flux across lipid bilayers (8), providing furthe...

The Basal Proton Conductance of Skeletal Muscle Mitochondria from Transgenic Mice Overexpressing or Lacking Uncoupling Protein-3

“…However, the uncoupling of yeast mitochondria by mammalian UCP3 (6, 7, 11-13) is an expression artifact (13, 15-18) that provides no evidence for a physiological uncoupling function in mammalian cells. Uncoupling following UCP3 expression in transgenic mammalian systems (9,30,50) may also be an expression artifact that provides no evidence for physiological uncoupling by UCP3, as shown for the uncoupling of mouse skeletal muscle in UCP3-tg mice in the present paper. Experiments in which UCP3 concentrations have been changed by physiological manipulations (19 -27), and the lack of effect of UCP3 knockout on whole animal energy metabolism and basal metabolic rate (31,32) despite the importance of muscle proton leak to physiological energetics (49), support the conclusion that UCP3 is not a significant contributor to the basal proton conductance of muscle mitochondria.…”

“…Proton leak in muscle is a significant contributor to standard metabolic rate (49), and modulation of mitochondrial proton conductance in muscle constitutes a mechanism of regulation of energy dissipation and standard metabolic rate. Whether the uncoupling caused by transfection of UCP3 into myotubes and cardiac muscle cells (50) or by transgenic expression of UCP1 in mouse muscle (47) is a result of native function of the UCPs or an expression artifact remains to be determined by direct tests of the GDP and superoxide sensitivity of mitochondria isolated from the relevant cells.…”

Developmental programming of adult hyperinsulinemia, increased proinflammatory cytokine production, and altered skeletal muscle expression of SLC2A4 (GLUT4) and uncoupling protein 3

“…UCP3 is best known for its roles in promoting fatty acid oxidation (MacLellan et al 2005) and limiting mitochondrial damage by suppression of lipid peroxidation and the production of reactive oxygen species (Vidal-Puig et al 2000, Brand et al 2002. But, UCP3 can also stimulate glucose uptake and recruitment of SLC2A4 to the cell surface (Huppertz et al 2001) and so its decline in programmed offspring may contribute to the observed accumulation of cytoplasmic SLC2A4. Interestingly, reduced skeletal muscle Ucp3 expression has also been observed in diabetic human subjects (Krook et al 1998, Schrauwen et al 2001, and a recent report indicates that this is already the case in prediabetic subjects with impaired glucose tolerance (Schrauwen et al 2006).…”

“…The skeletal muscle expression of SLC2A4 mRNA and protein, including its subcellular localization, and peroxisome proliferator-activated receptor (PPAR)-d, PPAR-g coactivator-1a (PPARGC1A), and uncoupling protein 3 (UCP3) mRNAs, were also determined. These genes were targeted because PPARD is a key regulator of fuel metabolism (Brunmair et al 2006), the effects of which involve coactivation by PPARGC1A (Finck & Kelly 2006), whereas UCP3 promotes fatty acid oxidation (MacLellan et al 2005) and glucose uptake (Huppertz et al 2001), and limits mitochondrial damage via effects on lipid peroxidation (Brand et al 2002). The interactive effects of a postnatal, high u-3 fatty acid (high n-3, Hn3) diet were also determined, since our previous studies show that this dietary intervention markedly reduces other adverse phenotypic outcomes programmed by fetal glucocorticoid excess (Wyrwoll et al 2006(Wyrwoll et al , 2007.…”

Uncoupling Protein 3 Content Is Decreased in Skeletal Muscle of Patients With Type 2 Diabetes

“…Treatment of L6 myotubes with the strong chemical uncoupler dinitrophenol stimulated glucose uptake (3). Recently, Huppertz et al (15) showed that overexpression of UCP3 in L6 myotubes increases glucose uptake through increased recruitment of GLUT4 to the cell surface. Overexpression of UCP3 in transgenic mice resulted in increased glucose tolerance and reduced fasting plasma glucose levels (2).…”