Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes.

Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4. We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4. Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4. In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation. These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation.     

Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin

Muscle and fat cells develop insulin resistance when cultured under hyperinsulinemic conditions for sustained periods. Recent data indicate that early insulin signaling defects do not fully account for the loss of insulin action. Given that cortical filamentous actin (F-actin) represents an essential aspect of insulin regulated glucose transport, we tested to see whether cortical F-actin structure was compromised during chronic insulin treatment. The acute effect of insulin on GLUT4 translocation and glucose uptake was diminished in 3T3-L1 adipocytes exposed to a physiological level of insulin (5 nm) for 12 h. This insulin-induced loss of insulin responsiveness was apparent under both low (5.5 mm) and high (25 mm) glucose concentrations. Microscopic and biochemical analyses revealed that the hyperinsulinemic state caused a marked loss of cortical F-actin. Since recent data link phosphatidylinositol 4,5-bisphosphate (PIP(2)) to actin cytoskeletal mechanics, we tested to see whether the insulin-resistant condition affected PIP(2) and found a noticeable loss of this lipid from the plasma membrane. Using a PIP(2) delivery system, we replenished plasma membrane PIP(2) in cells following the sustained insulin treatment and observed a restoration in cortical F-actin and insulin responsiveness. These data reveal a novel molecular aspect of insulin-induced insulin resistance involving defects in PIP(2)/actin regulation.     

Disruption of cortical actin in skeletal muscle demonstrates an essential role of the cytoskeleton in glucose transporter 4 translocation in insulin-sensitive tissues

Cell culture work suggests that signaling to polymerize cortical filamentous actin (F-actin) represents a required pathway for the optimal redistribution of the insulin-responsive glucose transporter, GLUT4, to the plasma membrane. Recent in vitro study further suggests that the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) mediates the effect of insulin on the actin filament network. Here we tested whether similar cytoskeletal mechanics are essential for insulin-regulated glucose transport in isolated rat epitrochlearis skeletal muscle. Microscopic analysis revealed that cortical F-actin is markedly diminished in muscle exposed to latrunculin B. Depolymerization of cortical F-actin with latrunculin B caused a time- and concentration-dependent decline in 2-deoxyglucose transport. The loss of cortical F-actin and glucose transport was paralleled by a decline in insulin-stimulated GLUT4 translocation, as assessed by photolabeling of cell surface GLUT4 with Bio-LC-ATB-BMPA. Although latrunculin B impaired insulin-stimulated GLUT4 translocation and glucose transport, activation of phosphatidylinositol 3-kinase and Akt by insulin was not rendered ineffective. In contrast, the ability of insulin to elicit the cortical F-actin localization of N-WASP was abrogated. These data provide the first evidence that actin cytoskeletal mechanics are an essential feature of the glucose transport process in intact skeletal muscle. Furthermore, these findings support a distal actin-based role for N-WASP in insulin action in vivo.     

Calmodulin antagonists inhibit insulin-stimulated GLUT4 (glucose transporter 4) translocation by preventing the formation of phosphatidylinositol 3,4,5-trisphosphate in 3T3L1 adipocytes

It has been previously reported that calmodulin plays a regulatory role in the insulin stimulation of glucose transport. To examine the basis for this observation, we examined the effect of a panel of calmodulin antagonists that demonstrated a specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4) but not insulin- or platelet-derived growth factor (PDGF)-stimulated GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or phosphotyrosine antibody immunoprecipitation of phosphatidylinositol (PI) 3-kinase activity was not affected. Despite the marked insulin and PDGF stimulation of PI 3-kinase activity, there was a near complete inhibition of protein kinase B activation. Using a fusion protein of the Grp1 pleckstrin homology (PH) domain with the enhanced green fluorescent protein, we found that the calmodulin antagonists prevented the insulin stimulation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo. Similarly, although PDGF stimulation increased PI 3-kinase activity in in vitro immunoprecipitation assays, there was also no significant formation of PI(3,4,5)P3 in vivo. These data demonstrate that calmodulin antagonists prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo production of PI(3,4,5)P3 without directly affecting IRS1- or phosphotyrosine-associated PI 3-kinase activity. This phenomenon is similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.     

The palatability of flavoured novel floating pellets made with brewer's spent grain to captive carp

Abstract

The palatability to common carp, Cyprinus carpio L. of three newly developed differently flavoured floating pellets made from a high proportion (40%) of brewer's spent grain (BSG) was tested using a multiple-offer feeding experiment. The addition of ‘bold’ flavours, such as vanilla or strawberry essence, may help mask the unpleasant taste of some piscicides; however, their inclusion must not compromise uptake by carp. There were no significant differences between the consumption rates of the three varieties, and all flavours were readily consumed. Therefore, it is suggested that highly flavoured pellets made with BSG have a strong potential to mask the flavour of an unpalatable toxin, and further research is now needed to test this hypothesis.     

The addition of CO2 to traditional taste solutions alters taste quality

Previous studies of the effect of carbonation on taste perception have suggested that it may be negligible, manifesting primarily in increases in the perceived intensity of weak salt and sour stimuli. Assuming CO2 solutions in the mouth stimulate only trigeminal nerve endings, this result is not altogether surprising; however, there are neurophysiological data indicating that CO2 stimulates gustatory as well as trigeminal fibers. In that case, carbonation might alter the quality profile of a stimulus without producing substantial changes in overall taste intensity--much as occurs when qualitatively different taste stimuli are mixed. To address this possibility, subjects were asked to rate the total taste intensity of moderate concentrations of stimuli representing each of the basic tastes and their binary combinations, with an without added carbonation. They then subdivided total taste intensity into the proportions of sweetness, saltiness, sourness, bitterness and 'other taste qualities' they perceived. The addition of carbonation produced only small increases in ratings of total taste intensity. However, rather dramatic alterations in the quality profiles of stimuli were observed, particularly for sweet and salty tastes. The nature of the interaction is consistent with a direct effect of carbonation/CO2 on the gustatory system, although the possibility that at least some of the observed effects reflect trigeminal-gustatory interactions cannot be ruled out.