Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNF␣-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR-Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR ␤-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR-Cav1 complex by the interactions of IR with GM3 in microdomains.adipocyte ͉ caveolae microdomain ͉ lipid rafts ͉ live cell imaging ͉ type 2 diabetes

Leptin stimulates fatty acid oxidation in skeletal muscle through the activation of AMP-activated protein kinase (AMPK) and the induction of gene expression, such as that for peroxisome proliferator-activated receptor ␣ (PPAR␣). We now show that leptin stimulates fatty acid oxidation and PPAR␣ gene expression in the C2C12 muscle cell line through the activation of AMPK containing the ␣2 subunit (␣2AMPK) and through changes in the subcellular localization of this enzyme. Activated ␣2AMPK containing the ␤1 subunit was shown to be retained in the cytoplasm, where it phosphorylated acetyl coenzyme A carboxylase and thereby stimulated fatty acid oxidation. In contrast, ␣2AMPK containing the ␤2 subunit transiently increased fatty acid oxidation but underwent rapid translocation to the nucleus, where it induced PPAR␣ gene transcription. A nuclear localization signal and Thr 172 phosphorylation of ␣2 were found to be essential for nuclear translocation of ␣2AMPK, whereas the myristoylation of ␤1 anchors ␣2AMPK in the cytoplasm. The prevention of ␣2AMPK activation and the change in its subcellular localization inhibited the metabolic effects of leptin. Our data thus suggest that the activation of and changes in the subcellular localization of ␣2AMPK are required for leptin-induced stimulation of fatty acid oxidation and PPAR␣ gene expression in muscle cells.AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is thought to function in mammalian cells to yeast as a cellular "fuel gauge," signaling when energy stores are full or depleted (9, 16). The activation of AMPK results in the phosphorylation of several target molecules and consequent stimulation of fatty acid oxidation, glucose transport in muscle, and cardiac glycolysis as well as the inhibition of anabolic processes and ion channel activities (9, 16). AMPK is a heterotrimer consisting of a catalytic ␣ subunit (␣1 or ␣2) and regulatory ␤ (␤1 or ␤2) and ␥ (␥1, ␥2, or ␥3) subunits (9, 16). Phosphorylation of the ␣ subunit (on Thr 172 ) by the upstream kinase AMPKK or allosteric modulation by AMP binding is essential for the activation of AMPK (8, 38). Four kinases, LKB1 (1), Ca 2ϩ /calmodulin-dependent protein kinase kinase (10, 13, 39), ATM (32), and TAK1 (24), have been proposed to function as AMPKKs.The activity of AMPK is regulated by hormones (21, 22, 41) and growth factors (32, 33) as well as by changes in cellular energy status. Leptin is a hormone produced by adipocytes that regulates food intake and fuel metabolism (3). Leptin exerts metabolic effects in peripheral tissues both directly and through the central nervous system (22,25). We have previously shown that leptin stimulates fatty acid oxidation in skeletal muscle by activating the ␣2 subunit-containing AMPK (␣2AMPK) both directly at the muscle level and indirectly through the hypothalamus and the sympathetic nervous system (22). Activated ␣2AMPK phosphorylates acetyl coenzyme A (CoA) carboxylase (ACC), resulting in the inhibition of its activity and reduced formation of malonyl-CoA. ...

The Arabidopsis thaliana AtHKT1 protein, a Na ؉ ͞K ؉ transporter, is capable of mediating inward Na ؉ currents in Xenopus laevis oocytes and K ؉ uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K ؉ transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K ؉ channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na ؉ currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135-142 and 377-384, face the cytosol, whereas the region of residues 55-62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.