Mitofusin-2 (Mfn2) is a mitochondrial membrane protein that participates in mitochondrial fusion in mammalian cells and mutations in the Mfn2 gene cause Charcot-Marie-Tooth neuropathy type 2A. Here, we show that Mfn2 loss-of-function inhibits pyruvate, glucose and fatty acid oxidation and reduces mitochondrial membrane potential, whereas Mfn2 gain-of-function increases glucose oxidation and mitochondrial membrane potential. As to the mechanisms involved, we have found that Mfn2 loss-of-function represses nuclear-encoded subunits of OXPHOS complexes I, II, III and V, whereas Mfn2 overexpression induced the subunits of complexes I, IV and V. Obesity-induced Mfn2 deficiency in rat skeletal muscle was also associated with a decrease in the subunits of complexes I, II, III and V. In addition, the effect of Mfn2 overexpression on mitochondrial metabolism was mimicked by a truncated Mfn2 mutant that is inactive as a mitochondrial fusion protein. Our results indicate that Mfn2 triggers mitochondrial energization, at least in part, by regulating OXPHOS expression through signals that are independent of its role as a mitochondrial fusion protein.

Cystinuria is a classic heritable aminoaciduria that involves the defective transepithelial transport of cystine and dibasic amino acids in the kidney and intestine. Six missense mutations in the human rBAT gene, which is involved in high-affinity transport of cystine and dibasic amino acids in kidney and intestine, segregate with cystinuria. These mutations account for 30% of the cystinuria chromosomes studied. Homozygosity for the most common mutation (M467T) was detected in three cystinuric siblings. Mutation M467T nearly abolished the amino acid transport activity induced by rBAT in Xenopus oocytes. These results establish rBAT as a cystinuria gene.

We have isolated a cDNA clone by screening a rabbit kidney cortex cDNA library for expression of sodiumindependent transport of L-arginine and L-alanine in Xenopus laevis oocytes. Expressed uptake relates to a single component of sodium-independent transport for dibasic and neutral amino acids. This transport activity resembles the functionally defined system b0'+ and carries cystine and dibasic amino acids with high affmity. The rBAT (bO'+ amino acid transporterrelated) mRNA is found mainly in kidney and intestinal mucosa. It encodes a predicted 77.8-kDa protein with only one putative transmembrane domain and seven potential N-glycosylation sites. This protein could either be a constitutive element or a specific activator of system b°'+.

The previous characterization of an abundant population of non-adrenergic imidazoline-I 2 binding sites in adipocytes and the recent demonstration of the interplay between these binding sites and amine oxidases led us to analyze the amine oxidase activity in membranes from isolated rat adipocytes. Adipocyte membranes had substantial levels of semicarbazide-sensitive amine oxidase (SSAO). SSAO activity and immunoreactive SSAO protein were maximal in plasma membranes, and they were also detectable in intracellular membranes. Vesicle immunoisolation analysis indicated that GLUT4-containing vesicles from rat adipocytes contain substantial levels of SSAO activity and immunoreactive SSAO protein. Immunotitration of intracellular GLUT4 vesicles indicated that GLUT4 and SSAO colocalize in an endosomal compartment in rat adipocytes. SSAO activity was also found in GLUT4 vesicles from 3T3-L1 adipocytes and rat skeletal muscle.Benzylamine, a substrate of SSAO activity, caused a marked stimulation of glucose transport in isolated rat adipocytes in the presence of very low vanadate concentrations that by themselves were ineffective in exerting insulin-like effects. This synergistic effect of benzylamine and vanadate on glucose transport was totally abolished in the presence of semicarbazide, a specific inhibitor of SSAO. Subcellular membrane fractionation revealed that the combination of benzylamine and vanadate caused a recruitment of GLUT4 to the plasma membrane of adipose cells. The stimulatory effects of benzylamine and vanadate on glucose transport were blocked by catalase, suggesting that hydrogen peroxide production coupled to SSAO activity plays a crucial regulatory role. Based on these results we propose that SSAO activity might contribute through hydrogen peroxide production to the in vivo regulation of GLUT4 trafficking in adipose cells.

Skeletal muscle differentiation involves myoblast alignment, elongation, and fusion into multinucleate myotubes, together with the induction of regulatory and structural muscle-specific genes. Here we show that two phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, blocked an essential step in the differentiation of two skeletal muscle cell models. Both inhibitors abolished the capacity of L6E9 myoblasts to form myotubes, without affecting myoblast proliferation, elongation, or alignment. Myogenic events like the induction of myogenin and of glucose carrier GLUT4 were also blocked and myoblasts could not exit the cell cycle, as measured by the lack of mRNA induction of p21 cyclindependent kinase inhibitor. Overexpresssion of MyoD in 10T1/2 cells was not sufficient to bypass the myogenic differentiation blockade by LY294002. Upon serum withdrawal, 10T1/2-MyoD cells formed myotubes and showed increased levels of myogenin and p21. In contrast, LY294002-treated cells exhibited none of these myogenic characteristics and maintained high levels of Id, a negative regulator of myogenesis. These data indicate that whereas phosphatidylinositol 3-kinase is not indispensable for cell proliferation or in the initial events of myoblast differentiation, i.e. elongation and alignment, it appears to be essential for terminal differentiation of muscle cells.