Methazolamide is a new hepatic insulin sensitizer that lowers blood glucose in vivo.

We previously used Gene Expression Signature technology to identify methazolamide (MTZ) and related compounds with insulin sensitizing activity in vitro. The effects of these compounds were investigated in diabetic db/db mice, insulin-resistant diet-induced obese (DIO) mice, and rats with streptozotocin (STZ)-induced diabetes. MTZ reduced fasting blood glucose and HbA(1c) levels in db/db mice, improved glucose tolerance in DIO mice, and enhanced the glucose-lowering effects of exogenous insulin administration in rats with STZ-induced diabetes. Hyperinsulinemic-euglycemic clamps in DIO mice revealed that MTZ increased glucose infusion rate and suppressed endogenous glucose production. Whole-body or cellular oxygen consumption rate was not altered, suggesting MTZ may inhibit glucose production by different mechanism(s) to metformin. In support of this, MTZ enhanced the glucose-lowering effects of metformin in db/db mice. MTZ is known to be a carbonic anhydrase inhibitor (CAI); however, CAIs acetazolamide, ethoxyzolamide, dichlorphenamide, chlorthalidone, and furosemide were not effective in vivo. Our results demonstrate that MTZ acts as an insulin sensitizer that suppresses hepatic glucose production in vivo. The antidiabetic effect of MTZ does not appear to be a function of its known activity as a CAI. The additive glucose-lowering effect of MTZ together with metformin highlights the potential utility for the management of type 2 diabetes.

Inhibition of inosine monophosphate dehydrogenase reduces adipogenesis and diet-induced obesity.

We previously described a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in lipid accumulation. Here we present data which demonstrate that IMPDH activity is required for differentiation of preadipocytes into mature, lipid-laden adipocytes and maintenance of adipose tissue mass. In 3T3-L1 preadipocytes inhibition of IMPDH with mycophenolic acid (MPA) reduced intracellular GTP levels by 60% (p<0.05) and blocked adipogenesis (p<0.05). Co-treatment with guanosine, a substrate in the salvage pathway of nucleotide biosynthesis, restored GTP levels and adipogenesis demonstrating the specificity of these effects. Treatment of diet-induced obese mice with mycophenolate mofetil (MMF), the prodrug of MPA, for 28 days did not affect food intake or lean body mass but reduced body fat content (by 36%, p=0.002) and adipocyte size (p=0.03) and number. These data suggest that inhibition of IMPDH may represent a novel strategy to reduce adipose tissue mass.

Neogenin and RGMa control neural tube closure and neuroepithelial morphology by regulating cell polarity.

In humans, neural tube closure defects occur in 1:1000 pregnancies. The design of new strategies for the prevention of such common defects would benefit from an improved understanding of the molecular events underlying neurulation. Neural fold elevation is a key morphological process that acts during neurulation to drive neural tube closure. However, to date, the molecular pathways underpinning neural fold elevation have not been elucidated. Here, we use morpholino knock-down technology to demonstrate that Repulsive Guidance Molecule (RGMa)-Neogenin interactions are essential for effective neural fold elevation during Xenopus neurulation and that loss of these molecules results in disrupted neural tube closure. We demonstrate that Neogenin and RGMa are required for establishing the morphology of deep layer cells in the neural plate throughout neurulation. We also show that loss of Neogenin severely disrupts the microtubule network within the deep layer cells suggesting that Neogenin-dependent microtubule organization within the deep cells is essential for radial intercalation with the overlying superficial cell layer, thereby driving neural fold elevation. In addition, we show that sustained Neogenin activity is also necessary for the establishment of the apicobasally polarized pseudostratified neuroepithelium of the neural tube. Therefore, our study identifies a novel signaling pathway essential for radial intercalation and epithelialization during neural fold elevation and neural tube morphogenesis.

Emerging roles for neogenin and its ligands in CNS development.

It is now well established that the netrin guidance cues and their receptors comprise a major molecular guidance system driving axon pathfinding during nervous system development. One netrin receptor, neogenin, is now emerging as a key regulator of many developmental processes throughout the embryo. Unexpectedly, a new family of neogenin ligands, the repulsive guidance molecule (RGM) family, has recently been identified. The functional outcome of neogenin activation is dictated by both the nature of the ligand as well as the developmental context. Netrin-1-neogenin interactions mediate chemoattractive axon guidance, while RGMa-neogenin interactions repel axons. Neogenin is required for the establishment of the pseudostratified epithelium of the neural tube, probably by promoting cell adhesion. In addition, a role for RGMa and neogenin in neuronal differentiation has been demonstrated. While neogenin signaling cascades are poorly understood, the opposing responses of neogenin to RGMa and netrin-1 in the context of axon guidance indicates that neogenin signaling is complex and subject to tight spatiotemporal regulation. In summary, neogenin is a multifunctional receptor regulating diverse developmental processes. Thus, its contribution to neural development is proving to be considerably more extensive than originally predicted.

Aplysia temptin - the 'glue' in the water-borne attractin pheromone complex.

Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aplysia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation. In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using a cold shock promoter and purified by RP-HPLC. CD spectra confirmed a predominantly beta-sheet structure. Two disulfide bonds were determined via limited proteolysis and MS. One internal disulfide (Cys57-Cys77) was predicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface. A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca(2+)-binding, EGF-like domain of the extracellular matrix protein fibrillin. Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a proposed metal-binding loop. The water-borne pheromone attractin in egg cordon eluates is complexed with other proteins. Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors. Gel shifts confirmed that temptin complexes with wild-type attractin. These results indicate that temptin, analogous to the role of fibrillin in controlling transforming growth factor-beta concentration, modulates pheromone signaling by direct binding to attractin.

Gene identification and evidence for expression of G protein alpha subunits, phospholipase C, and an inositol 1,4,5-trisphosphate receptor in Aplysia californica rhinophore.

In the marine mollusk Aplysia californica, waterborne protein pheromones that are released during egg laying act in concert to stimulate mate attraction. However, molecular information concerning the cellular receptors and signaling mechanisms that may be involved in waterborne peptide and protein pheromonal communication is lacking. As a first step toward examining whether members of the G protein family and phosphoinositide signaling pathway are present in the primary peripheral chemosensory organs (i.e., rhinophores), we isolated five full-length cDNA clones from an A. californica central nervous system cDNA library. These clones encoded (1) the G protein alpha subunits of the Gq, Gi, and Go families, (2) a protein with homology to phospholipase C (PLC) isoforms, and (3) an inositol 1,4,5-trisphosphate receptor (IP3R). The expression of these genes was examined using laser capture microdissection/reverse transcription-polymerase chain reaction and in situ hybridization. All of them are expressed in the rhinophore sensory epithelium, suggesting that Galphaq, Galphai, Galphao, PLC-like protein, and IP3R may be involved in waterborne protein pheromone detection in Aplysia-possibly via a phosphoinositide signaling mechanism.

Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold.

Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg(50) residue to generate a smaller 49-residue mature peptide. The Arg(50) cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly alpha-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.