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To investigate the physiological function of the VAMP3 vesicle SNARE (v-SNARE) isoform in the regulation of GLUT4 vesicle trafficking, we generated homozygotic VAMP3 null mice by targeted gene disruption. The VAMP3 null mice had typical growth rate and weight gain, with normal maintenance of fasting serum glucose and insulin levels. Analysis of glucose disposal and insulin sensitivity demonstrated normal insulin and glucose tolerance, with no evidence for insulin resistance. Insulin stimulation of glucose uptake in isolated primary adipocytes was essentially the same for the wild-type and VAMP3 null mice. Similarly, insulin-, hypoxia-, and exercise-stimulated glucose uptake in isolated skeletal muscle did not differ significantly. In addition, other general membrane trafficking events including phagocytosis, pinocytosis, and transferrin receptor recycling were also found to be unaffected in the VAMP3 null mice. Taken together, these data demonstrate that VAMP3 function is not necessary for either regulated GLUT4 translocation or general constitutive membrane recycling.Insulin increases glucose uptake in adipose and striated muscle tissues primarily by recruiting the GLUT4 glucose transporter protein to the cell surface (37). In the basal noninsulin-stimulated state, the majority of GLUT4 resides in one or more intracellular compartments (44,45). Upon addition of insulin, the signaling cascade triggered by the insulin receptor leads to rapid translocation of the GLUT4 transporter to the plasma membrane, thereby increasing the number of transporters at the cell surface and the rate of glucose uptake (12,27,38,41).The process of GLUT4 translocation shares important features with the exocytosis of synaptic vesicles during neurotransmitter release. For example, the plasma membrane docking and fusion of GLUT4 vesicles appears to require the t-SNARE protein isoforms syntaxin 4 and SNAP23 (9,37,48). GLUT4 vesicles contain the v-SNARE-interacting partners VAMP2 and VAMP3, both of which translocate to the plasma membrane in parallel with GLUT4 (33,47). Recent studies using various toxins and endosomal ablation techniques have indicated that VAMP2 is the predominant v-SNARE responsible for insulin-stimulated GLUT4 translocation in cultured 3T3-L1 adipocytes and in the L6 muscle cell line (9,32,33,40). In contrast, guanosine-5Ј-O-(3-thiotriphosphate) (GTP␥S)-stimulated GLUT4 translocation was found to be dependent on VAMP3, thereby suggesting the presence of two independently regulated pools of GLUT4 storage compartments (35). In this regard, skeletal muscle has also been shown to contain two pools of GLUT4 vesicles, one that responds to insulin and another that is responsive to exercise and contraction (1,11,39).In addition, the skeletal muscle exercise-contraction subpopulation utilizes a signaling pathway independent of the phosphatidylinositol (PI) 3-kinase (30, 31, 50). Similarly, GTP␥S stimulation in adipocytes is also independent of the PI 3-kinase, suggesting that the GTP␥S and exercise-contraction pathways may util...

Ca2؉ /calmodulin-dependent protein kinase II (CaMKII) activity is increased in heart failure (HF), a syndrome characterized by markedly increased risk of arrhythmia. Activation of CaMKII increases peak L-type Ca 2؉ current (I Ca ) and slows I Ca inactivation. Whether these events are linked mechanistically is unknown. I Ca was recorded in acutely dissociated subepicardial and subendocardial murine left ventricular (LV) myocytes using the whole cell patch clamp method. Pressure overload heart failure was induced by surgical constriction of the thoracic aorta. I Ca density was significantly larger in subepicardial myocytes than in subendocardial/myocytes. Similar patterns were observed in the cell surface expression of ␣1c, the channel pore-forming subunit. In failing LV, I Ca density was increased proportionately in both cell types, and the time course of I Ca inactivation was slowed. This typical pattern of changes suggested a role of CaMKII. Consistent with this, measurements of CaMKII activity revealed a 2-3-fold increase (p < 0.05) in failing LV. To test for a causal link, we measured frequency-dependentI Ca facilitation.InHFmyocytes,thisCaMKIIdependent process could not be induced, suggesting already maximal activation. Internal application of active CaMKII in failing myocytes did not elicit changes in I Ca . Finally, CaMKII inhibition by internal diffusion of a specific peptide inhibitor reduced I Ca density and inactivation time course to similar levels in control and HF myocytes. I Ca density manifests a significant transmural gradient, and this gradient is preserved in heart failure. Activation of CaMKII, a known pro-arrhythmic molecule, is a major contributor to I Ca remodeling in load-induced heart failure.Patients with heart failure are at increased risk of malignant arrhythmia, which accounts for a substantial component of the mortality associated with this disease (1). Mechanisms underlying these arrhythmias are multifactorial, but they stem, at least in part, from disordered electrical currents arising from prolongation of ventricular action potentials. The resulting delay in the recovery of excitability, a consistent feature of heart failure (2), predisposes to early and delayed after-depolarizations. Heart failure is also associated with myocardial fibrosis, altered electrotonic coupling between cells, slowed conduction, and dispersion of refractoriness, all of which predispose to reentrant mechanisms of arrhythmia. Together, these "electrical remodeling" responses underlie the propensity to arrhythmia, syncope, and sudden death.In recent years, electrical remodeling has emerged as an important pathophysiologic mechanism in many types of cardiac pathology. Whereas considerable progress has been made recently in elucidating the molecular pathogenesis of cardiac hypertrophy and failure (reviewed in Refs. 3 and 4), our understanding of mechanisms underlying disease-related action potential prolongation is limited. As a result, the means of treating heart failure-associated arrhythmias remain disappo...

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin-and exercisestimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.The stimulation of glucose uptake in adipose and muscle tissues primarily occurs through the translocation of the GLUT4 glucose transporter isoform from intracellular storage sites to the cell surface membranes (1-4). Insulin stimulation of this process requires the tyrosine phosphorylation of the insulin receptor substrate family of proteins and subsequent activation of the type 1 phosphatidylinositol (PI) 1 3-kinase (5-12). Although the precise signaling steps that occur downstream of the PI 3-kinase ultimately leading to GLUT4 translocation have remained elusive, recent studies have begun to resolve the specific trafficking, docking, and fusions events. In adipocytes, it is well established that GLUT4 storage compartments contain vesicle-localized proteins (v-SNAREs) that specifically interact with cognate cell surface target proteins (t-SNAREs) at the plasma membrane to promote vesicle docking and fusion (13,14). Insulin-stimulated GLUT4 translocation is dependent upon the interaction of the v-SNARE, VAMP2, with the plasma membrane t-SNAREs, syntaxin 4 and SNAP23 (15-21). Furthermore, the syntaxin 4-binding protein, Munc18c, has been shown to specifically modulate insulinsensitive GLUT4 translocation in 3T3L1 adipocytes (22-24). However, it is important to recognize that the majority of these studies have been performed in cultured 3T3L1 adipocytes and L6 myotubes due to the inherent technical limitations in the study of GLUT4 trafficking in adipocytes and skeletal muscle in vivo. Sk...

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.