Human TBC1D4 blocking peptide

Whether the well-known metabolic switch AMP-activated protein kinase (AMPK) is involved in the insulin-sensitizing effect of calorie restriction (CR) is unclear. In this study, we investigated the role of AMPK in the insulin-sensitizing effect of CR in skeletal muscle. Wild-type (WT) and AMPK-alpha 2(-/-) mice received ad libitum (AL) or CR (8 weeks at 60% of AL) feeding. CR increased the protein level of AMPK-alpha 2 and phosphorylation of AMPK-a2. In WT and AMPK-alpha 2(-/-) mice, CR induced comparable changes of body weight, fat pad weight, serum triglycerides, serum nonesterified fatty acids, and serum leptin levels. However, decreasing levels of fasting/fed insulin and fed glucose were observed in WT mice but not in AMPK-alpha 2(-/-) mice. Moreover, CR-induced improvements of whole-body insulin sensitivity (evidenced by glucose tolerance test/insulin tolerance test assays) and glucose uptake in skeletal muscle tissues were abolished in AMPK-alpha 2(-/-) mice. Furthermore, CR-induced activation of Akt-TBC1D1/TBC1D4 signaling, inhibition of mammalian target of rapamycin - S6K1 - insulin receptor substrate-1 pathway, and induction of nicotinamide phosphoribosyltransferase - NAD(+)-sirtuin-1 cascade were remarkably impaired in AMPK-alpha 2(-/-) mice. CR serum increased stability of AMPK-alpha 2 protein via inhibiting the X chromosome-linked ubiquitin-specific protease 9-mediated ubiquitylation of AMPK-alpha 2. Our results suggest that AMPK may be modulated by CR in a ubiquitylation-dependent manner and acts as a chief dictator for the insulin-sensitizing effects of CR in skeletal muscle. Diabetes 61:1051-1061, 2012

Glucose uptake by mammalian cells is a key mechanism to maintain cell and tissue homeostasis and relies mostly on plasma membrane-localized glucose transporter proteins (GLUTs). Two main cellular mechanisms regulate GLUT proteins in the cell: first, expression of GLUT genes is under dynamic transcriptional control and is used by cancer cells to increase glucose availability. Second, GLUT proteins are regulated by membrane traffic from storage vesicles to the plasma membrane (PM). This latter process is triggered by signaling mechanisms and well-studied in the case of insulin-responsive cells, which activate protein kinase AKT to phosphorylate TBC1D4, a RAB-GTPase activating protein involved in membrane traffic regulation. Previously, we identified protein kinase WNK1 as another kinase able to phosphorylate TBC1D4 and regulate the surface expression of the constitutive glucose transporter GLUT1. Here we describe that downregulation of WNK1 through RNA interference in HEK293 cells led to a 2-fold decrease in PM GLUT1 expression, concomitant with a 60% decrease in glucose uptake. By mass spectrometry, we identified serine (S) 704 in TBC1D4 as a WNK1-regulated phosphorylation site, and also S565 in the paralogue TBC1D1. Transfection of the respective phosphomimetic or unphosphorylatable TBC1D mutants into cells revealed that both affected the cell surface abundance of GLUT1. The results reinforce a regulatory role for WNK1 in cell metabolism and have potential impact for the understanding of cancer cell metabolism and therapeutic options in type 2 diabetes.