GLUT4--at the cross roads between membrane trafficking and signal transduction

GLUT4 is a mammalian facilitative glucose transporter that is highly expressed in adipose tissue and striated muscle. In response to insulin, GLUT4 moves from intracellular storage areas to the plasma membrane, thus increasing cellular glucose uptake. While the verification of this ‘translocation hypothesis’ (Cushman SW, Wardzala LJ. J Biol Chem 1980;255: 4758–4762 and Suzuki K, Kono T. Proc Natl Acad Sci 1980;77: 2542–2545) has increased our understanding of insulin-regulated glucose transport, a number of fundamental questions remain unanswered. Where is GLUT4 stored within the basal cell? How does GLUT4 move to the cell surface and what mechanism does insulin employ to accelerate this process? Ultimately we require a convergence of trafficking studies with research in signal transduction. However, despite more than 30 years of intensive research we have still not reached this point. The problem is complex, involving at least two separate signal transduction pathways which feed into what appears to be a very dynamic sorting process. Below we discuss some of these complexities and highlight new data that are bringing us closer to the resolution of these questions.     

Differing effects of endocrine-disrupting chemicals on basal and FSH-stimulated progesterone production in rat granulosa-luteal cells

Previous studies have shown that the phytoestrogen, genistein, inhibits basal and forskolin-stimulated progesterone synthesis in rat granulosa-luteal cells. Genistein, however, not only binds and activates the estrogen receptor (ER), but is also a potent inhibitor of tyrosine kinase. In these studies we have compared the effects of estradiol, two other phytoestrogens, apigenin and coumarin, the pesticide, [2-(chlorphenyl)-2-(4-chlorphenyl)-1,1,1-trichlorethan] (2,4'DDT), and the industrial chemical, 4-octyl-phenol, on basal and follicle stimulating hormone (FSH)-stimulated progesterone production in the same experimental system. Only a supraphysiological dose of estradiol (10(-5) M) significantly inhibited basal and forskolin-stimulated progesterone production in granulosa-luteal cells, but had no effect on FSH-stimulated production. In contrast, apigenin, DDT, and octyl-phenol stimulated basal progesterone production at doses around 10(-8) to 10(-7) M, but this effect was reversed at higher doses. Coumarin was without effect. Like basal production, the two phytoestrogens had opposing effects on FSH-stimulated progesterone production. Genistein at 10(-5) M was inhibitory, while apigenin significantly potentiated the response at 19(-7) M. In contrast, DDT had no effect on the FSH-induced response, though 10(-7) M octyl-phenol nearly doubled the response. While all these chemicals are known to interact with the estrogen receptor to a greater or lesser extent, these studies suggest that like genistein, these different endocrine-disrupting chemicals may have other actions apart from those on the estrogen receptor.     

Determinants of the interaction of the spinal muscular atrophy disease protein SMN with the dimethylarginine-modified box H/ACA small nucleolar ribonucleoprotein GAR1

Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.     

HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line

The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.     

Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.     

Double-blind comparison of absorbable colloidal bismuth subcitrate and nonabsorbable bismuth subnitrate in the eradication of Helicobacter pylori and the relief of nonulcer dyspepsia

Background. Bismuth is widely used for the eradication of H. pylori , especially in developing countries, although there are concerns over its neurotoxicity. Whether bismuth has to be absorbed in humans to act against H. pylori is not known. In this study, we compared "absorbable" (colloidal bismuth subcitrate) and "nonabsorbable" (bismuth subnitrate) bismuth as part of triple therapy in the eradication of H. pylori.
Materials and Methods. A double-blind, randomized, placebo-controlled trial was carried out with 120 H. pylori –positive patients with nonulcer dyspepsia. Group CBS + Ab (n = 35) received colloidal bismuth subcitrate (one tablet qds), amoxicillin (500 mg qds), and metronidazole (400 mg tds). Group BSN + Ab (n = 35) received bismuth subnitrate (two tablets tds) and the same antibiotics. Group Ab (n = 35) received placebo bismuth (two tablets tds) and the antibiotics. Group BSN (n = 15) received bismuth subnitrate (two tablets tds) and placebo antibiotics. Bismuth was taken for 4 weeks and the antibiotics for the first 2 weeks. H. pylori eradication, side effects, compliance, pre- and post-treatment symptom scores, and bismuth absorption were assessed.
Results. H. pylori eradication was 69%, 83%, 31%, and 0% in CBS + Ab, BSN + Ab, Ab, and BSN, respectively. Side effects, compliance, and symptom relief were similar in all groups, but blood bismuth levels were significantly greater in CBS + Ab than the other three groups.
Conclusion. The efficacy of bismuth-based therapies as part of triple therapy in the eradication of H. pylori is unrelated to absorption. Hence, the use of effective but poorly absorbed bismuth preparations should be encouraged for bismuth-based eradication therapies.     

Development of molecular methods for identification of Streptococcus bovis from human and ruminal origins

Streptococcus bovis has been identified as a causative agent in humans for a variety of diseases, including endocarditis, meningitis, and septicemia. Identification of S. bovis strains of human origin in clinical settings has been problematic due to variations in biochemical tests as compared to ruminal strains of S. bovis, and other streptococcal species. DNA-DNA hybridization with chromosomal DNA from various S. bovis strains indicates that strains of human origin are different from those of ruminal origin. Specific probes have been designed from S. bovis 16S rDNA gene sequences that differentiate strains of human and ruminal origin by direct hybridization and PCR analyses. These techniques now allow for rapid identification of S. bovis strains for clinical and other scientific investigations.     

Non-geographically based population structure of South Pacific sperm whales: dialects, fluke-markings and genetics

1. This study addresses the issue of structure in sperm whale ( Physeter macrocephalus Linnaeus) populations and whether it is geographically based.
2. During a survey around the South Pacific Ocean, we collected sloughed skin for genetic analyses, recorded coda vocalizations, and photographed fluke markings.
3. Groups of female and immature sperm whales had characteristic mitochondrial haplotypes, coda repertoires, and fluke-mark patterns, but there was no clear geographical structure in any of these attributes.
4. However, similarities of coda repertoire and mitochondrial haplotype distribution were significantly correlated among pairs of groups in a manner that was not geographically based. There was also a significant canonical correlation coefficient between coda repertoire and fluke-mark patterns.
5. These results suggest that attributes (such as vocal repertoire and techniques of predator defence) which are acquired matrilineally, and probably culturally, are conserved during the fission and dispersal of groups.