Tbc1d1 mutation in lean mouse strain confers leanness and protects from diet-induced obesity

We previously identified Nob1 as a quantitative trait locus for high-fat diet-induced obesity and diabetes in genome-wide scans of outcross populations of obese and lean mouse strains. Additional crossbreeding experiments indicated that Nob1 represents an obesity suppressor from the lean Swiss Jim Lambert (SJL) strain. Here we identify a SJL-specific mutation in the Tbc1d1 gene that results in a truncated protein lacking the TBC Rab-GTPase-activating protein domain. TBC1D1, which has been recently linked to human obesity, is related to the insulin signaling protein AS160 and is predominantly expressed in skeletal muscle. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, whereas overexpression of TBC1D1 had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation and reduced glucose uptake in isolated skeletal muscle. Our data strongly suggest that mutation of Tbc1d1 suppresses high-fat diet-induced obesity by increasing lipid use in skeletal muscle.     

Insulin action in cultured human skeletal muscle cells during differentiation: assessment of cell surface GLUT4 and GLUT1 content

In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via the GLUT4 glucose transporter. However, in contrast to mature skeletal muscle, cultured muscle expresses significant levels of the GLUT1 glucose transporter. To assess the relative contribution of these two glucose transporters, we used a novel photolabelling techniques to assess the cell surface abundance of GLUT1 and GLUT4 specifically in primary cultures of human skeletal muscle. We demonstrate that insulin-stimulated glucose transport in cultured human skeletal muscle is mediated by GLUT4, as no effect on GLUT1 appearance at the plasma membrane was noted. Furthermore, GLUT4 mRNA and protein increased twofold (p < 0.05), after differentiation, whereas GLUT1 mRNA and protein decreased 55% (p < 0.005). Incubation of differentiated human skeletal muscle cells with a non-peptide insulin mimetic significantly (p < 0.05) increased glucose uptake and glycogen synthesis. Thus, cultured myotubes are a useful tool to facilitate biological and molecular validation of novel pharmacological agents aimed to improve glucose metabolism in skeletal muscle.     

Specific cleavage of insulin-like growth factor-binding protein-1 by a novel protease activity

Insulin-like growth factor-binding protein-1 (IGFBP-1) is secreted in a highly phosphorylated form that binds IGF-I with high affinity and is resistant to proteolysis. We have purified IGFBP-1-specific protease activity from the urine of an individual with multiple myeloma. This protease efficiently cleaves both phosphorylated and non-phosphorylated IGFBP-1 at Ile130-Ser131, generating fragments that together have higher association and dissociation rates for IGFs compared with intact IGFBP-1. The proteolytic fraction contained azurocidin, a protease homologue hitherto considered inactive. After cleavage of IGFBP-1, there was a lower affinity, but higher capacity for IGF-I binding, suggesting both N- and C-terminal fragments may interact with ligand independently. There was decreased inhibition of IGF-II-stimulated cell growth and glucose uptake. Alone, proteolysed IGFBP-1 stimulated glucose uptake in muscle. We conclude that specific cleavage of IGFBP-1 at target tissues is important in cellular growth and metabolism and opens novel strategies for targeting IGFBP-1 in treatment of disease.