Recent studies have indicated that insulin activates endothelial nitric-oxide synthase (eNOS) by protein kinase B (PKB)-mediated phosphorylation at Ser1177 in endothelial cells. Because hyperglycemia contributes to endothelial dysfunction and decreased NO availability in types 1 and 2 diabetes mellitus, we have studied the effects of high glucose (25 mM, 48 h) on insulin signaling pathways that regulate NO production in human aortic endothelial cells. High glucose inhibited insulin-stimulated NO synthesis but was without effect on NO synthesis stimulated by increasing intracellular Ca 2؉ concentration. This was accompanied by reduced expression of IRS-2 and attenuated insulin-stimulated recruitment of PI3K to IRS-1 and IRS-2, yet insulin-stimulated PKB activity and phosphorylation of eNOS at Ser 1177 were unaffected. Inhibition of insulin-stimulated NO synthesis by high glucose was unaffected by an inhibitor of PKC. Furthermore, high glucose down-regulated the expression of CAP and Cbl, and insulin-stimulated Cbl phosphorylation, components of an insulin signaling cascade previously characterized in adipocytes. These data suggest that high glucose specifically inhibits insulin-stimulated NO synthesis and down-regulates some aspects of insulin signaling, including the CAP-Cbl signaling pathway, yet this is not a result of reduced PKBmediated eNOS phosphorylation at Ser 1177 . Therefore, we propose that phosphorylation of eNOS at Ser 1177 is not sufficient to stimulate NO production in cells cultured at 25 mM glucose.

BackgroundTranslocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.Methodology/Principal FindingsHere we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.Conclusion/SignificanceOur reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.

Interaction of SM (Sec1/Munc18) proteins with their cognate syntaxins represents an important regulatory mechanism of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-mediated membrane fusion. Understanding the conserved mechanisms by which SM proteins function in this process has proved challenging, largely due to an apparent lack of conservation of binding mechanisms between different SM-syntaxin pairs. In the present study, we have identified a hitherto uncharacterized mode of binding between syntaxin 4 and Munc18c that is independent of the binding mode shown previously to utilize the N-terminal peptide of syntaxin 4. Our data demonstrate that syntaxin 4 and Munc18c interact via two distinct modes of binding, analogous to those employed by syntaxin 1a-Munc18a and syntaxin 16-Vps45p (vacuolar protein sorting 45). These data support the notion that all syntaxin/SM proteins bind using conserved mechanisms, and pave the way for the formulation of unifying hypotheses of SM protein function.

Insulin stimulates the movement of the facilitative glucose transporter glucose transporter-4 (Glut4) from an intracellular compartment to the plasma membrane in adipocytes and muscle cells, resulting in an increased rate of glucose uptake. Insulin-stimulated Glut4 translocation and glucose transport are abolished by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Here, we demonstrate that neomycin, a drug that masks the cellular substrate of PI3K, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), prevents wortmannin inhibition of insulin-stimulated Glut4 translocation and glucose transport without activating protein kinase B, a downstream effector of PI3K. These results suggest that PIP 2 may have an important regulatory function in insulin-stimulated Glut4 translocation and glucose transport.

Background: Assessing the pre and post test probability of disease in the context of routine health care is challenging. We wished to study how test performance parameters relating to clinical utility vary by clinical indication in a ‘real world’ setting. Methods: The diagnostic accuracy of serum total B₁₂ and Active-B₁₂® (holotranscobalamin) was evaluated in a primary care population, using serum methylmalonic acid as the reference standard. We used electronic requesting to establish the clinical indication for each request. Routine requests from primary care for serum total B₁₂ were included if creatinine was also measured and estimated glomerular filtration rate was at least 60 ml/min/1.73m². Results Clinical indications included peripheral neuropathy (n=168), anaemia (n=168), cognitive decline (n=125), suspected dietary deficiency (n=76), other (n=362). For peripheral neuropathy, the area under the receiver operator curve ± 95% confidence interval (AUC±CI) was 0.63 (0.54–0.71)(p=0.002) for total B₁₂ and 0.68 (0.60–0.77) (p<0.0001) for Active-B₁₂®. For anaemia, AUC±CI was 0.56 (0.47–0.66)(p=0.10) for total B₁₂ and 0.69 (0.59–0.78) (p<0.0001) for Active-B₁₂®. For cognitive decline, AUC±CI was 0.54 (0.43–0.65)(p=0.26) for total B₁₂ and 0.69 (0.58–0.80) (p=0.0002) for Active-B₁₂®. The pre-post test change in probability of disease varied by clinical indication. Conclusion Combining diagnostic accuracy studies and electronic testing allows clinical utility to be assessed by clinical indication. Wider application of this would permit more personalised laboratory medicine. In this study, diagnostic performance of total B₁₂ and Active-B₁₂® varied across all indications. Active-B₁₂® provided better discrimination, but this may have reflected the cut-offs used.