The insulin-sensitive glucose transporter, GLUT4, is the most abundant facilitative glucose transporter in muscle and adipose tissue, the major sites for postprandial glucose disposal. To assess the role of GLUT4 in glucose homeostasis, we have disrupted the murine GLUT4 gene. Because GLUT4 has been shown to be dysregulated in pathological states such as diabetes and obesity, it was expected that genetic ablation of GLUT4 would result in abnormal glucose homeostasis. The mice deficient in GLUT4 (GLUT4-null) are growth-retarded and exhibit decreased longevity associated with cardiac hypertrophy and severely reduced adipose tissue deposits. Blood glucose levels in female GLUT4-null mice are not significantly elevated in either the fasting or fed state; in contrast, male GLUT4-null mice have moderately reduced glycaemias in the fasted state and increased glycaemias in the fed state. However, both female and male GLUT4-null mice exhibit postprandial hyperinsulinaemia, indicating possible insulin resistance. Increased expression of other glucose transporters is observed in the liver (GLUT2) and heart (GLUT1) but not skeletal muscle. Oral glucose tolerance tests show that both female and male GLUT4-null mice clear glucose as efficiently as controls, but insulin tolerance tests indicate that these mice are less sensitive to insulin action. The GLUT4-null mice demonstrate that functional GLUT4 protein is not required for maintaining nearly normal glycaemia but that GLUT4 is absolutely essential for sustained growth, normal cellular glucose and fat metabolism, and expected longevity.

GLUT4, the insulin-responsive glucose transporter, plays an important role in postprandial glucose disposal. Altered GLUT4 activity is suggested to be one of the factors responsible for decreased glucose uptake in muscle and adipose tissue in obesity and diabetes. To assess the effect of GLUT4 expression on whole-body glucose homeostasis, we disrupted the murine GLUT4 gene by homologous recombination. Male mice heterozygous for the mutation (GLUT4 +/-) exhibited a decrease in GLUT4 expression in adipose tissue and skeletal muscle. This decrease in GLUT4 expression did not result in obesity but led to increased serum glucose and insulin, reduced muscle glucose uptake, hypertension, and diabetic histopathologies in the heart and liver similar to those of humans with non-insulin-dependent diabetes mellitus (NIDDM). The male GLUT4 +/- mice represent a good model for studying the development of NIDDM without the complications associated with obesity.

Compartmentation and dynamics of cAMP and PKA signaling are important determinants of specificity among cAMP's myriad cellular roles. Both cardiac inotropy and the progression of heart disease are affected by spatiotemporal variations in cAMP/PKA signaling, yet the dynamic patterns of PKA-mediated phosphorylation that influence differential responses to agonists have not been characterized. We performed live-cell imaging and systems modeling of PKA-mediated phosphorylation in neonatal cardiac myocytes in response to G-protein coupled receptor stimuli and UV photolysis of ''caged'' cAMP. cAMP accumulation was rate-limiting in PKA-mediated phosphorylation downstream of the ␤-adrenergic receptor. Prostaglandin E 1 stimulated higher PKA activity in the cytosol than at the sarcolemma, whereas isoproterenol triggered faster sarcolemmal responses than cytosolic, likely due to restricted cAMP diffusion from submembrane compartments. Localized UV photolysis of caged cAMP triggered gradients of PKA-mediated phosphorylation, enhanced by phosphodiesterase activity and PKA-mediated buffering of cAMP. These findings indicate that combining live-cell FRET imaging and mechanistic computational models can provide quantitative understanding of spatiotemporal signaling.fluorescence imaging ͉ models ͉ signal transduction I ntracellular signaling through cAMP and its cAMP-dependent protein kinase (PKA) mediates hundreds of distinct cellular functions. Compartmentation and dynamics of cAMP͞PKA signaling are gaining increasing acceptance as general mechanisms used to maintain signaling specificity in a contextdependent manner. In the heart, compartmentation appears to contribute to functional differences between ␤ 1 -and ␤ 2 -adrenergic signaling and other stimuli that increase cAMP and thus has important consequences for understanding the role of ␤-adrenergic signaling in the development and treatment of heart failure (1, 2). Short-term ␤-adrenergic signaling increases heart contractility (3), whereas prolonged exposure to ␤ 1 -adrenergic agonists induces apoptosis (4). Recent live-cell imaging and electrophysiologic approaches are now providing direct measurements of compartmentation (5, 6) and cAMP signaling dynamics (7-10) in intact cells, and our increasing molecular understanding provides numerous candidate molecular mechanisms for compartmentation including caveolae (2, 11), ␤-arrestins (12, 13), and A-kinase anchoring proteins (AKAPs) (14, 15). A future challenge will be to understand quantitatively how these molecular signaling mechanisms orchestrate such precise context-dependent signaling in the cell.Here, we integrate fluorescent reporters of PKA-mediated phosphorylation (16) and mechanistic computational models to characterize rate-limiting biochemical reactions in ␤-adrenergic signaling and identify signaling mechanisms contributing to asynchronous and spatially heterogeneous PKA-mediated phosphorylation. This combination of techniques reveals restricted diffusion, phosphodiesterase (PDE)-mediated cAMP degradation, and c...

Background-␣-E-catenin is a cell adhesion protein, located within the adherens junction, thought to be essential in directly linking the cadherin-based adhesion complex to the actin cytoskeleton. Although ␣-E-catenin is expressed in the adherens junction of the cardiomyocyte intercalated disc, and perturbations in its expression are observed in models of dilated cardiomyopathy, its role in the myocardium remains unknown. Methods and Results-To determine the effects of ␣-E-catenin on cardiomyocyte ultrastructure and disease, we generated cardiac-specific ␣-E-catenin conditional knockout mice (␣-E-cat cKO). ␣-E-cat cKO mice displayed progressive dilated cardiomyopathy and unique defects in the right ventricle. The effects on cardiac morphology/function in ␣-E-cat cKO mice were preceded by ultrastructural defects in the intercalated disc and complete loss of vinculin at the intercalated disc. ␣-E-cat cKO mice also revealed a striking susceptibility of the ventricular free wall to rupture after myocardial infarction. Conclusions-These results demonstrate a clear functional role for ␣-E-catenin in the cadherin/catenin/vinculin complex in the myocardium in vivo. Ablation of ␣-E-catenin within this complex leads to defects in cardiomyocyte structural integrity that result in unique forms of cardiomyopathy and predisposed susceptibility to death after myocardial stress. These studies further highlight the importance of studying the role of ␣-E-catenin in human cardiac injury and cardiomyopathy in the future.

We have established recommendations on specific treatment of a pulmonary exacerbation and have outlined the areas where we need better information on appropriate therapies. Once we have a standardized definition of an exacerbation, we can proceed with clinical trials of therapies specific for its treatment.