Chronic AICAR treatment prevents metabolic changes in cardiomyocytes exposed to free fatty acids.

The stimulation of glucose transport by metabolic stress is an important determinant of myocardial susceptibility to ischemia and reperfusion injury. Stimulation of glucose transport is markedly impaired in cardiomyocytes chronically exposed to excess free fatty acids (FFA), as occurs in vivo in type 2 diabetes. To determine whether chronic low-grade activation of AMP-activated kinase (AMPK) improves substrate metabolism in cardiomyocytes exposed to FFA, isolated cultured cardiomyocytes were exposed for 7 days to FFA ± the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Glucose transport and glycolysis were then measured during acute metabolic stress provoked by oligomycin. Chronic treatment with AICAR improved basal and oligomycin-stimulated glucose transport in FFA-exposed but not in control cardiomyocytes. Similarly, basal and oligomycin-stimulated glycolysis was reduced in FFA-exposed cardiomyocytes but restored by chronic AICAR treatment. Conversely, fatty acid oxidation was increased in FFA-exposed cardiomyocytes and reduced by chronic AICAR treatment. Chronic AICAR treatment induced in FFA-exposed cardiomyocytes the biogenesis of numerous lipid droplets. Curiously, whereas acute treatment of cardiomyocytes with AICAR increased phosphorylation of the AMPKα subunit on T172, a classical marker of AMPK activation, chronic AICAR treatment almost completely obliterated T172 phosphorylation. However, phosphorylation of the AMPK target protein raptor on S792 was reduced in FFA-exposed cardiomyocytes but restored by AICAR treatment. In conclusion, chronic AICAR treatment induces a metabolic shift in FFA-exposed cardiomyocytes, characterized by improved glucose transport and glycolysis and redirection of fatty acids towards neutral storage. Such metabolic changes in vivo could protect the hearts of patients with type 2 diabetes against ischemia-reperfusion injury.

Pleiotropic Effects of Chronic Phorbol Ester Treatment to Improve Glucose Transport in Insulin-Resistant Cardiomyocytes.

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.

A Role for Focal Adhesion Kinase in the Stimulation of Glucose Transport in Cardiomyocytes.

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.

Fluvastatin inhibits regulated secretion of endothelial cell von Willebrand factor in response to diverse secretagogues.

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.

Effects of insulin-like growth factor-I on the maturation of metabolism in neonatal rat cardiomyocytes.

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 alpha (PPARalpha). Insulin-like 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 PPARalpha agonist WY-14643. Concomitant treatment with IGF-I enhanced the expression of both FAO enzymes. By comparison, treatment with FGF-2, which is required for myocyte differentiation of cardiac precursors, did not increase WY-14643-induced expression of FAO enzymes. Despite stimulation of FAO enzyme expression, IGF-I did not further enhance WY-14643-stimulated palmitate oxidation. In contrast, IGF-I relieved WY-14643-mediated inhibition of glucose uptake and promoted storage of fatty acids into cellular neutral lipids. In conclusion, IGF-I promotes a more mature pattern of FAO gene expression but, because of insulin-like metabolic effects, does not concomitantly enhance oxidation of fatty acids.