Deletion of C-terminal 12 amino acids of GLUT1 protein does not abolish the transport activity.

We engineered the GLUT1 cDNA to delete C-terminal 12 amino acids of encoded GLUT1 protein. This mutated GLUT1 protein expressed in CHO cells by transfection of its cDNA was demonstrated to reside on the plasma membrane by cell surface labeling technique, and retain the transport activity, similar to that of the wild-type GLUT1. In addition, metabolic labeling of the intact cells with 35S indicated that the half-life of the mutated GLUT1 was not significantly different from that of the wild-type GLUT1. These results suggest that C-terminal 12 amino acids of GLUT1 are not important for the transport activity and the stability of the protein. Taken together with our previous results on the mutant without C-terminal 37 amino acids, the amino acids between the 37th and the 13th from the C-terminus appear to be essential for the transport activity.     

Both cyclooxygenase and lipoxygenase inhibitor partially restore the anorexia by interleukin-1 beta.

Since the peripheral prostaglandin synthetizing system may at least partly involved in the anorexia that follows central interleukin-1 beta (IL-1) administration, this study was undertaken to investigate the effect of ibuprofen (ip), selective cyclooxygenase blocker and AA 861, selective lipoxygenase inhibitor, on changes of food and water intake by a single injection of IL-1 (2 micrograms/rat, ip). We demonstrated that food and water intake were suppressed by peripheral administration of IL-1. Throughout the entire observation periods, suppressed food intake was partially restored to control levels by ibuprofen, while water intake completely restored. In addition, no significant differences about water/food intake were observed in the IL-1 + ibuprofen-treated groups, respectively. In the next experiment, IL-1 induced anorexia was also partially restored to the control level following pretreatment with AA 861. These results may suggest that other mechanism including lipoxygenase blocker besides prostaglandin production may be involved in IL-1 induced anorexia.     

3-phosphoinositide-dependent protein kinase 1, an Akt1 kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.     

Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose

Lacto-N-tetraose (Galbeta1 -3GlcNAcbeta1-3Galbeta1-4Glc, LNT) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, LNnT) were enzymatically synthesized by consecutive additions of GlcNAc and Gal residues to lactose. Lacto-N-triose II (GlcNAcbeta1-3Galbeta1-4Glc) was prepared first by the transfer of GlcNAc from UDP-GlcNAc to lactose by beta-1,3-N-acetylglucosaminyltransferase from bovine serum. The resulting lacto-N-triose II was converted into LNT and LNnT utilizing two kinds of beta-D-galactosidase-mediated transglycosylations. Thus, beta-D-galactosidase from Bacillus circulans ATCC31382 induced regioselective galactosyl transfer from o-nitrophenyl beta-D-galactoside to the OH-3" position of lacto-N-triose II, and commercially available beta-D-galactosidase from B. circulans to the OH-4" position of lacto-N-triose II. These convenient processes are suitable for large-scale preparations of LNT and LNnT. As another method, LNT was directly synthesized from lactose as an initial substance, utilizing lacto-N-biosidase (Aureobacterium sp. L-101)-mediated transglycosylation with Galbeta1-3GlcNAcbeta-pNP donor.     

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473-484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.     

p110beta is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity

Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85alpha) and one of two widely distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110beta was up-regulated approximately 10-fold, whereas expression of p110alpha was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110beta and p110alpha in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110beta was lower than that of p110alpha. When p110alpha and p110beta were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85alpha and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110beta, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110alpha were observed. Interestingly, overexpression of p110beta increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110alpha increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110beta neutralizing antibody into 3T3-L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110alpha neutralizing antibody did only slightly. Together, these findings suggest that p110beta plays a crucial role in cellular activities evoked acutely by insulin.     

Dissection of a major QTL for photoperiod sensitivity in rice: its association with a gene expressed in an age-dependent manner

 A proposed major quantitative trait locus (QTL) for photoperiod sensitivity on chromosome 6 in rice was examined by introducing a chromosomal segment from a sensitive line into an insensitive one. The crossing experiments showed that a range of variation in heading date occurred in the later generations and that the region might contain at least a major gene and two additional recessive genes controlling photoperiod sensitivity. Gene mapping experiments showed that the major gene was Se-1 and that a recessive gene (tentatively named se-pat) was loosely linked to it. The responses to photoperiods were examined among the different genotypes under natural and controlled conditions. The two genes acted additively on the degree of photoperiod sensitivity. However, se-pat plants showed a response to photoperiods that differed from that of the other sensitive lines; a short-day treatment at the seedling stage delayed heading in the former plants, suggesting that the manner of its expression was age-dependent. A recessive gene similar to se-pat seemed to be widely distributed in wild and cultivated rice, suggesting that the gene complex in the region plays a significant role in response to photoperiod. Received: 8 October 1997 / Accepted: 1 April 1998     

LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells

LKB1 is a 50 kDa serine/threonine kinase that phosphorylates and activates the catalytic subunit of AMPK at its T-loop residue Thr 172. We prepared adenoviruses expressing the constitutive active (wild-type) form (CA) or dominant negative (kinase inactive, D194A mutant) form (DN) of LKB1 and overexpressed these proteins in cultured myotubes (C2C12 cells) and rat hepatoma cells (FAO cells). When analyzed by immunoblotting with the antibody against Thr172-phosphorylated AMPK, the phosphorylation of AMPK was increased (2.5-fold) and decreased (0.4-fold) in cells expressing CA and DN LKB1, respectively, as compared with Lac-Z expressing control cells. Immunoprecipitation experiments, using isoform-specific antibody, revealed these alterations of AMPK phosphorylation to be attributable to altered phosphorylation of AMPK alpha2, but not alpha1 catalytic subunits, strongly suggesting the alpha2 catalytic subunit to be the major substrate for LKB1 in mammalian cells. In addition, adiponectin or AICAR-stimulated AMPK phosphorylation was inhibited by overexpression of DN LKB1, while phenformin-stimulated phosphorylation was unaffected. These results may explain the difference in AMPK activation mechanisms between AMP and phenformin, and also indicate that AMPK phosphorylation by LKB1 is involved in AMP-stimulated AMPK activation. As a downstream target for AMPK, AICAR-induced glucose uptake and ACCbeta phosphorylation were found to be significantly reduced in DN LKB1 expressing C2C12 cells. The expression of key enzymes for gluconeogenesis, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was also dependent on LKB1 activities in FAO cells. These results demonstrate that LKB1 is a crucial regulator of AMPK activation in muscle and liver cells and, therefore, that LKB1 activity is potentially of importance to our understanding of glucose and lipid metabolism.