No abstract

Abstract-Cardiovascular disease risk is higher in men than women, but the basis for this discrepancy remains controversial. Estrogenic stimulation of the myocardium or isolated cardiomyocytes has been purported to exert multiple beneficial effects associated with inhibition of maladaptive responses to pathogenic insults. This report describes a significant difference between the sexes in myocardial activation of Akt, a protein kinase that regulates a broad range of physiological responses including metabolism, gene transcription, and cell survival. We find that young women possess higher levels of nuclear-localized phospho-Akt 473 relative to comparably aged men or postmenopausal women. Both localization of phospho-Akt 473 in myocardial nuclei of sexually mature female mice versus males and Akt kinase activity in nuclear extracts of hearts from female mice versus males are elevated. Cytosolic localization of phospho-forkhead, a downstream nuclear target of Akt, is also increased in female relative to male mice, suggesting a potential mechanism for cardioprotective nuclear signaling resulting from Akt activation. Phospho-Akt 473 levels and localization at cardiac nuclei are similarly increased in transgenic mice with myocardium-specific expression of insulin-like growth factor I, a proven stimulus for Akt activation. Phospho-Akt 473 is also localized to the nucleus of cultured cardiomyocytes after exposure to 17␤-estradiol or genistein (a phytoestrogen in soy protein-based diets), and neonatal exposure of litters to genistein elevated nuclear phospho-Akt 473 localization. The activation of Akt in a gender-dependent manner may help explain differences observed in cardiovascular disease risk between the sexes and supports the potential beneficial effects of estrogenic stimulation.

Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51-56]. Here, we have identified this DGK activity as DGKθ, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKθ was present in nuclear, plasma membrane (and\or Golgi) and cytosolic fractions. Upon NA stimulation, DGKθ translocated towards the membrane and cytosol (155 and 153 % increases relative to the control, respectively) at 30 s, followed by a return to near-basal levels at 5 min ; AII was without effect. Translocation to the membrane was to both Triton-soluble and Abbreviations used : DGK, diacylglycerol kinase ; DG, 1,2-diacylglycerol ; PA, phosphatidic acid ; NA, noradrenaline ; AII, angiotensin II ; PKC, protein kinase C ; PH, pleckstrin homology ; OBG, octyl-β-glucoside ; PI 3-K, phosphoinositide 3-kinase ; PKB, protein kinase B. 1 To whom correspondence should be addressed (e-mail johanian!man.ac.uk).-insoluble fractions. NA, but not AII, transiently increased DGKθ activity in immunoprecipitates (126 % at 60 s). Membrane translocation and DGKθ activation were regulated differently : NA-induced DGKθ activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonistspecific activation of DGKθ suggests that this pathway may have a physiological role in vascular smooth-muscle responses.

This study shows that postnatal timing of altered signaling in cardiomyopathic transgenic mouse models is a pivotal part of determining outcome. In addition, intensifying hypertrophic stimulation exacerbates dilated cardiomyopathy, supporting the concept of shared molecular signaling between hypertrophy and dilation.