Essential role of protein kinase C zeta in the impairment of insulin-induced glucose transport in IRS-2-deficient brown adipocytes

Insulin receptor substrate-2-deficient (IRS-2(-/-)) mice develop type 2 diabetes. We have investigated the molecular mechanisms by which IRS-2(-/-) immortalized brown adipocytes showed an impaired response to insulin in inducing GLUT4 translocation and glucose uptake. IRS-2-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity was blunted in IRS-2(-/-) cells, total PI 3-kinase activity being reduced by 30%. Downstream, activation of protein kinase C (PKC) zeta was abolished in IRS-2(-/-) cells. Reconstitution with retroviral IRS-2 restores IRS-2/PI 3-kinase/PKC zeta signalling, as well as glucose uptake. Wild-type cells expressing a kinase-inactive mutant of PKC zeta lack GLUT4 translocation and glucose uptake. Our results support the essential role played by PKC zeta in the insulin resistance and impaired glucose uptake observed in IRS-2-deficient brown adipocytes.     

tmRDB (tmRNA database)

Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.html with mirror sites located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Bioinformatics Research Center, Aarhus, Denmark (http://www.bioinf.au.dk/tmRDB/). The tmRDB collects and distributes information relevant to the study of tmRNA. In trans-translation, this molecule combines properties of tRNA and mRNA and binds several proteins to form the tmRNP. Related RNPs are likely to be functional in all bacteria. In this release of tmRDB, 186 new entries from 10 bacterial groups for a total of 274 tmRNA sequences have been added. Lists of the tmRNAs and the corresponding tmRNA-encoded tag-peptides are presented in alphabetical and phylogenetic order. The tmRNA sequences are aligned manually, assisted by computational tools, to determine base pairs supported by comparative sequence analysis. The tmRNA alignment, available in a variety of formats, provides the basis for the secondary and tertiary structure of each tmRNA molecule. Three-dimensional models of the tmRNAs and their associated proteins in PDB format give evidence for the recent progress that has been made in the understanding of tmRNP structure and function.     

Ras-GRF1 signaling is required for normal beta-cell development and glucose homeostasis

Development of diabetes generally reflects an inadequate mass of insulin-producing beta-cells. beta-cell proliferation and differentiation are regulated by a variety of growth factors and hormones, including insulin-like growth factor I (IGF-I). GRF1 is a Ras-guanine nucleotide exchange factor known previously for its restricted expression in brain and its role in learning and memory. Here we demonstrate that GRF1 is also expressed in pancreatic islets. Interestingly, our GRF1-deficient mice exhibit reduced body weight, hypoinsulinemia and glucose intolerance owing to a reduction of beta-cells. Whereas insulin resistance is not detected in peripheral tissues, GRF1 knockout mice are leaner due to increased lipid catabolism. The reduction in circulating insulin does not reflect defective glucose sensing or insulin production but results from impaired beta-cell proliferation and reduced neogenesis. IGF-I treatment of isolated islets from GRF1 knockouts fails to activate critical downstream signals such as Akt and Erk. The observed phenotype is similar to manifestations of preclinical type 2 diabetes. Thus, our observations demonstrate a novel and specific role for Ras-GRF1 pathways in the development and maintenance of normal beta-cell number and function.     

Type A and type B botulism in the North: first reported cases due to toxin other than type E in Alaskan Inuit.

Botulism outbreaks shown to be due to type A and type B toxin occurred in Alaska, a region previously known for only type E botulism. The outbreak due to type A toxin involved three people, two of whom died. The outbreak due to type B toxin involved nine people, none of whom died. Both outbreaks were in Inuit villages, and native foods were incriminated. The occurrence of these outbreaks strongly suggests that Clostridium botulinum, types A and B are indigenous to Alaska. The outbreaks underscore the need for initial treatment of patients with antitoxin that is trivalent (ABE), even in Arctic regions.     

A single mRNA encodes multiple copies of the egg peptide speract

A complementary DNA clone (2.3 kb) that encodes the egg peptide speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) has been isolated from an ovary cDNA library of the sea urchin. Stronglyocentrotus purpuratus. The DNA sequence predicts an open reading frame of 296 amino acids. The likely site of initiation, however, is a downstream in-frame translation initiation codon that would result in a polypeptide of 260 amino acids containing 10 decapeptides, each separated by a single lysine residue. Four of the peptides are speract, and six have the predicted structures of Gly-Phe-Ala-Leu-Gly-Gly-Gly-Gly-Val-Gly (occurs twice), Gly-Phe-Asn-Leu-Asn-Gly-Gly-Gly-Val-Gly, Gly-Phe-Ser-Leu-Thr-Gly-Gly-Gly-Val-Gly, Gly-Thr-Met-Pro-Thr-Gly-Ala-Gly-Val-Asp, and Ile-Asp-His-Asp-Thr-Leu-Ala-Ser-Val-Ser. The isolated cDNA insert hybridized to two species of ovarian mRNA (1.2 and 2.3 kb) obtained from species known to produce speract or speract-like peptides, but failed to hybridize to RNA from other species. Subsequently, a second ovarian cDNA clone (1.2 kb) was isolated and sequenced; this clone contained two additional potential decapeptides: Ser-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly and Ser-Thr-Met-Pro-Thr-Gly-Ala-Gly-Val-Asp. The various speract and speract-like peptides found in egg-conditioned media, therefore, reflect, in part, variable structures within a single copy of mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gab-1-mediated IGF-1 signaling in IRS-1-deficient 3T3 fibroblasts

The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70-80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration of egr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation.     

Comparative 3-D Modeling of tmRNA

Background

Trans-translation releases stalled ribosomes from truncated mRNAs and tags defective proteins for proteolytic degradation using transfer-messenger RNA (tmRNA). This small stable RNA represents a hybrid of tRNA- and mRNA-like domains connected by a variable number of pseudoknots. Comparative sequence analysis of tmRNAs found in bacteria, plastids, and mitochondria provides considerable insights into their secondary structures. Progress toward understanding the molecular mechanism of template switching, which constitutes an essential step in trans-translation, is hampered by our limited knowledge about the three-dimensional folding of tmRNA.

Results

To facilitate experimental testing of the molecular intricacies of trans-translation, which often require appropriately modified tmRNA derivatives, we developed a procedure for building three-dimensional models of tmRNA. Using comparative sequence analysis, phylogenetically-supported 2-D structures were obtained to serve as input for the program ERNA-3D. Motifs containing loops and turns were extracted from the known structures of other RNAs and used to improve the tmRNA models. Biologically feasible 3-D models for the entire tmRNA molecule could be obtained. The models were characterized by a functionally significant close proximity between the tRNA-like domain and the resume codon. Potential conformational changes which might lead to a more open structure of tmRNA upon binding to the ribosome are discussed. The method, described in detail for the tmRNAs of Escherichia coli, Bacillus anthracis, and Caulobacter crescentus, is applicable to every tmRNA.

Conclusion

Improved molecular models of biological significance were obtained. These models will guide in the design of experiments and provide a better understanding of trans-translation. The comparative procedure described here for tmRNA is easily adopted for the modeling the members of other RNA families.     

Stromal Cells Derived from Visceral and Obese Adipose Tissue Promote Growth of Ovarian Cancers

Obesity, and in particular visceral obesity, has been associated with an increased risk of developing cancers as well as higher rates of mortality following diagnosis. The impact of obesity on adipose-derived stromal cells (ASC), which contribute to the formation of tumor stroma, is unknown. Here we hypothesized that visceral source and diet-induced obesity (DIO) changes the ASC phenotype, contributing to the tumor promoting effects of obesity. We found that ASC isolated from subcutaneous (SC-ASC) and visceral (V-ASC) white adipose tissue(WAT) of lean(Le) and obese(Ob) mice exhibited similar mesenchymal cell surface markers expression, and had comparable effects on ovarian cancer cell proliferation and migration. Obese and visceral derived ASC proliferated slower and exhibited impaired differentiation into adipocytes and osteocytes in vitro as compared to ASC derived from subcutaneous WAT of lean mice. Intraperitoneal co-injection of ovarian cancer cells with obese or visceral derived ASC, but not lean SC-ASC, increased growth of intraperitoneal ID8 tumors as compared to controls. Obese and V-ASC increased stromal infiltration of inflammatory cells, including CD3+ T cells and F4/80+ macrophages. Obese and visceral derived ASC, but not lean SC-ASC, increased expression of chemotactic factors IL-6, MIP-2, and MCP-1 when cultured with tumor cells. Overall, these results demonstrate that obese and V-ASC have a unique phenotype, with more limited proliferation and differentiation capacity but enhanced expression of chemotactic factors in response to malignant cells which support infiltration of inflammatory cells and support tumor growth and dissemination.