Regulated secretion of EC (endothelial cell) vWF (von Willebrand factor) is part of the haemostatic response. It occurs in response to secretagogues that raise intracellular calcium or cAMP. Statins are cholesterol-lowering drugs used for the treatment of cardiovascular disease. We studied the effect of fluvastatin on regulated secretion of vWF from HUVEC (human umbilical-vein ECs). Secretion in response to thrombin, a protease-activated receptor-1 agonist peptide, histamine, forskolin and adrenaline (epinephrine) was inhibited. This inhibition was reversed by mevalonate or geranylgeranyl pyrophosphate, and mimicked by a geranylgeranyl transferase inhibitor, demonstrating that the inhibitory mechanism includes inhibition of protein geranylgeranylation. To investigate this mechanism further, calcium handling and NO (nitric oxide) regulation were studied in fluvastatin-treated HUVEC. Intracellular calcium mobilization did not correlate with vWF secretion. Fluvastatin increased eNOS [endothelial NOS (NO synthase)] expression, but NOS inhibitors failed to reverse the effect of fluvastatin on vWF secretion. Exogenous NO did not inhibit thrombin-induced vWF secretion. Many small GTPases are geranylgeranylated and some are activated by secretagogues. We overexpressed DN (dominant negative) Rho GTPases, RhoA, Rac1 and Cdc42 (cell division cycle 42), in HUVEC. DNCdc42 conferred inhibition of thrombin- and forskolin-induced vWF secretion. We conclude that, via inhibition of protein geranylgeranylation, fluvastatin is a broadspectrum inhibitor of regulated vWF secretion. Geranylgeranylated small GTPases with functional roles in regulated secretion, such as Cdc42, are potential targets for the inhibitory activity of fluvastatin.

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Stimulation of glucose transport is an important determinant of myocardial susceptibility to ischemia and reperfusion. Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FFA). Deactivation of the Focal Adhesion Kinase (FAK) by FFA contributes to glucose transport impairment, and could be corrected by chronic treatment with the phorbol ester TPA. However, TPA must have effects in addition to FAK reactivation to restore stimulated glucose transport. Chronic treatment with TPA improved basal and stimulated glucose transport in FFA-exposed, but not in control cardiomyocytes. Chronic FFA exposure induced the activation of PKCδ and PKCϵ. TPA markedly downregulated the expression of PKCα, PKCδ, and PKCϵ, suggesting that PKCδ or PKCϵ activation could contribute to inhibition of glucose transport by FFA. Rottlerin, a specific PKCδ inhibitor, improved glucose transport in FFA-exposed cardiomyocytes; and PKCδ was reduced in the particulate fraction of FFA + TPA-exposed cardiomyocytes. TPA also activated Protein Kinase D 1(PKD1) in FFA-exposed cardiomyocytes, as assessed by autophosphorylation of PKD1 on Y916. Pharmaceutical inhibition of PKD1 only partially prevented the improvement of glucose transport by TPA. Chronic TPA treatment also increased basal and stimulated glycolysis and favored accumulation of lipid droplets in FFA-exposed cardiomyocytes. In conclusion, basal and stimulated glucose transport in cardiomyocytes is reduced by chronic FFA exposure, but restored by concomitant treatment with a phorbol ester. The mechanism of action of phorbol esters may involve downregulation of PKCδ, activation of PKD1 and a general switch from fatty acid to glucose metabolism. J. Cell. Biochem. 9999: 4716-4727, 2017. © 2017 Wiley Periodicals, Inc.

Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FFA), despite relative preservation of canonical insulin- or metabolic stress signaling. We determined whether Focal Adhesion Kinase (FAK) activity is required for stimulation of glucose transport in cardiomyocytes, and whether FAK downregulation participates in FFA-induced impairment of glucose transport stimulation. Glucose transport, measured in isolated cultured cardiomyocytes, was acutely stimulated either by insulin treatment, or by metabolic inhibition with oligomycin resulting in AMP-activated kinase (AMPK) activation. FAK activity was inhibited pharmacologically by preincubation with PF-573,228 (PF). FAK activity was assessed from its autophosphorylation on residue Y397, and from the phosphorylation of its target paxillin on Y118. Y397 FAK phosphorylation was reduced in cultured cardiomyocytes chronically exposed to FFA. Preincubation with PF prior to determination of glucose transport resulted in a significant reduction of oligomycin-stimulated glucose transport, with a lesser reduction in insulin-stimulated glucose transport. Insulin and AMPK signaling was unaffected by PF preincubation. siRNA-mediated FAK knockdown also resulted in reduced oligomycin-stimulated glucose transport. Chronic treatment of FFA-exposed cardiomyocytes with phenylephrine or a phorbol ester restored FAK activity and improved glucose transport. In conclusion, stimulation of glucose transport in cardiomyocytes requires FAK activity prior to stimulation. The chronic reduction of FAK activity in cardiomyocytes exposed to FFA contributes to the loss of glucose transport responsiveness to insulin or metabolic inhibition. J. Cell. Biochem. 118: 670-677, 2017. © 2016 Wiley Periodicals, Inc.

Myocardial metabolism shifts during the perinatal period from predominant utilization of glucose towards oxidation of fatty acids. Expression of enzymes of the fatty acid oxidation (FAO) pathway is under the control of the nuclear receptor/transcription factor peroxisome proliferator-activated receptor α (PPARα). Insulinlike Growth Factor-I (IGF-I) plays an important role in the post-natal growth and differentiation of the heart. We determined the influence of IGF-I on the maturation of myocardial metabolism. In neonatal rat cardiac myocytes, expression of the FAO enzymes MCAD and M-CPT I was induced by treatment with the specific PPARα agonist WY-14643. Concomitant treatment with IGF