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Myocardial hypertrophy is associated with increased basal glucose metabolism. Basal glucose transport into cardiac myocytes is mediated by the GLUT1 isoform of glucose transporters, whereas the GLUT4 isoform is responsible for regulatable glucose transport.
Treatment of neonatal cardiac myocytes with the hypertrophic agonist O -tetradecanoylphorbolacetate or phenylephrine increased expression of Glut1 mRNA relative to Glut4 mRNA. To study the transcriptional regulation of GLUT1 expression, myocytes were transfected 90012 luciferase reporter constructs under the control of the Glut1 promoter.
Stimulation of the cells with O -tetradecanoylphorbolacetate or phenylephrine induced transcription from the Glut1 promoter, which was inhibited by cotransfection with the mitogen-activated protein kinase phosphatases CL and MKP Cotransfection of the myocytes with constitutively active versions of Ras and MEK1 or an estrogen-inducible version of Raf1 also stimulated transcription from the Glut1 promoter. Hypertrophic induction of the Glut1 promoter was also partially sensitive to inhibition of the phosphatidylinositol 3-kinase pathway and was strongly inhibited by cotransfection with dominant-negative Ras.
Thus, Ras activation and pathways downstream of Ras mediate induction of the Glut1 promoter during myocardial hypertrophy. Myocardial cells can use a wide variety of substrates for energy production, including free fatty acids, glucose, lactate, and ketone bodies. Substrate selection by cardiac myocytes is developmentally regulated. During the perinatal period, substrate metabolism shifts from predominant non-oxidative glucose utilization to predominant fatty acid oxidation 1.
This shift is associated with a shift in the expression of several regulatory proteins involved in glucose and fatty acid metabolismincluding GLUT glu cose t ransport proteins. Such alterations of the metabolic behavior could be explained by a resumption of the fetal expression pattern of proteins involved in glucose and fatty acid metabolism.
Two preliminary reports have described increased expression of the Glut1 isoform mRNA in myocardial hypertrophy induced in adult rats by pressure overload 11 or a large infarct of the left ventricle In response to treatment with a variety of different agonists, primary cultures of ventricular myocytes isolated from neonatal rat hearts display many of the features associated with hypertrophy in vivo and provide a useful model to study this nonproliferative growth response We have used this model to investigate the regulation of GLUT1 expression in cardiac myocytes.
Cardiac myocytes were transfected with a reporter construct encoding luciferase under the control of the Glut1 promoter. The expression of this reporter in response to the hypertrophic agonists O -tetradecanoylphorbolacetate TPA 1 and phenylephrine was assessed, and the signaling pathways responsible for increased expression of GLUT1 were investigated.
For transfection experiments, cells were plated at a density of fcz. Cardiac fibroblasts cultures were prepared by two passages of the cells adherent to the culture dish during the pre-plating procedure. Cells for morphological analysis were plated on glass coverslips coated with gelatin and laminin. Cells were treated for 48 h and then fixed and stained with fca isothiocyanate-conjugated phalloidin to show filamentous actin.
Cells were pretreated with or without PD for 30 min and then treated with either TPA or phenylephrine for 10 min. The plasmid containing 1. ER expresses the kinase domain of Raf1 fused to the steroid-binding domain of the human estrogen receptor Transient transfections of myocytes were performed using the calcium phosphate precipitation method as described previously 141920using the amounts of plasmid DNA indicated in the figure legends.
To evaluate the relative expression of the endogenous glucose transporter genes Glut1 and Glut4 by RT-PCR, we took advantage of regions of structural similarity and differences between the two isoforms Primers capable of amplifying both Glut1 and Fc cDNAs such that their respective products could be resolved on the basis of a base pair size difference were used Fca products were labeled by adding 0.
The gels were dried and exposed to storage phosphor screens. Detection of Ras-GTP in cells extract was performed as described Blots were probed using a rabbit anti-Ha-Ras polyclonal antibody Santa Cruz sc As described previously 13, cells treated with either agonist for 48 h showed a dramatic increase in size, together with increased organization of myofibrils, two hallmarks of hypertrophy of ventricular myocytes.
TPA and phenylephrine induce hypertrophy of rat neonatal ventricular myocytes. Cells were plated onto glass coverslips coated with gelatin and laminin and left untreated or treated with TPA or PE for 48 h. Cells were then stained fcx fluorescein isothiocyanate-labeled phalloidin to show filamentous actin.
This increase was mainly achieved by overexpression 0912 Glut1 and to a minor extent by a decrease in Glut4 expression. TPA and phenylephrine stimulate expression of the Glut1 gene. RT-PCR was gcs with 1. As shown in Fig.
TPA also induced transcription in cardiac fibroblasts, albeit to a 902 extent, whereas PE did not affect transcription in these cells. Induction by TPA was already detectable after 6 h of treatment and reached a plateau by 12 h, whereas induction by phenylephrine was slower, being detectable after 12 h and reaching a 99012 by 48 h only. Therefore, an incubation time of 48 h was selected for subsequent experiments.
TPA and phenylephrine increase transcription from the Glut1 promoter. Stimulation of the MAP kinase pathways plays an important role in the development of hypertrophy of myocardial cells.
We therefore assessed the involvement of MAP kinase pathways in the overexpression of Glut1.
Treatment with PD did not affect base-line expression of either the Glut1 or Glut4 endogenous gene. However, treatment with PD markedly reduced expression of the Glut1 gene induced by either TPA or phenylephrine, without affecting expression of the Glut4 gene Fig.
PD also inhibited the response to both hypertrophic agonists, confirming participation of the ERK pathway. In contrast, ccs p38 inhibitor SB did not affect induction of the Glut1 promoter by TPA or phenylephrine data not shown.
MAP kinase inhibitors block induction of the Glut1 promoter.
ERK activation is required for induction of the Glut1 gene. The results shown in Fig. Furthermore, expression induced by all of these agonists was inhibited by cotransfection with the broad specificity MAP kinase phosphatase CL Constitutively active mutants of the ERK pathway induce the Glut1 promoter. ER-transfected cells were then treated with either 0. Ras activation is required for phenylephrine-induced hypertrophy and is sufficient to induce both morphological and genetic markers of hypertrophy 1929 This result suggests that Ras activation is required for transduction of the signal elicited by both TPA and phenylephrine in cardiac myocytes.
These results suggest that Ras does not participate in signal transduction activated by TPA in cardiac fibroblasts, although it does participate in induction by TPA in cardiac myocytes. Ras activity is required for induction of the Glut1 promoter in myocytes.
Therefore, TPA can induce Ras in muscle cells, but not in fibroblasts, thus explaining the Ras requirement only in muscle cells. We therefore investigated whether activation of the PI3K pathway was required for induction of the Glut1 promoter by hypertrophic agonists.
LY did not inhibit induction of the Glut1 promoter by V12Ras. These results suggest that PI3K activation contributes to the induction of the Glut1 promoter in response to TPA and phenylephrine and that it acts upstream of Ras. Inhibition of the phosphatidylinositol 3-kinase pathway reduces hypertrophic Glut1 induction. In vivohypertrophic hearts have a pattern of substrate metabolism resembling that observed in fetal hearts, with increased reliance on glycolysis for energy production and reduced oxidation of fatty acids In this study, we observed that hypertrophy of rat neonatal ventricular myocytes is associated with increased expression of the glucose transporter Glut1 isoform mRNA.
GLUT1 is the principal isoform expressed in the fetal heart, and its expression is down-regulated following birth in normal myocardium 236concomitantly with the shift from glycolytic to oxidative metabolism 1. Our results suggest that regulation of Glut1 expression during hypertrophy is primarily achieved at the transcriptional level. Transient transfection experiments with a luciferase reporter under the control of the mouse Glut1 promoter indicated that treatment of myocytes with hypertrophic agonists resulted in increased transcription from the Glut1 promoter occurring between 6 and 48 h following addition of the agonist.
The two agonists we used TPA and phenylephrine activated the promoter with different kinetics, with TPA acting more rapidly and efficiently than phenylephrine.
The mode of action of these agonists is different. Ras activation, in turn, can trigger a variety of downstream signaling pathways, including Raf and phosphatidylinositol 3-kinase for a review, see Ref.
TPA belongs to the phorbol ester family, a class of compounds able to directly stimulate the classical and novel protein kinase C PKC isoforms. Thus, the modes of action of both agonists may converge onto Raf. Our data indicate that primary ventricular myocytes fall into this class see below.
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GLUT1 is a ubiquitous isoform of the glucose transporter, expressed at a significant level in virtually every tissue of the body. Therefore, it was a potential concern that the effect of TPA, a non-tissue-specific protein kinase C agonist, could be due to increased expression of the Glut1 promoter in contaminating non-myocyte cardiac cells, mainly cardiac fibroblasts.
We are confident, however, that this is not the case, for the following reasons. This observation is corroborated by the finding that TPA treatment resulted in activation of Ras in myocytes, but not in fibroblasts see below. Activation of the MAP kinase pathways is involved in the process of myocardial hypertrophy.
Previous results have shown that Ras or Raf-1 activity is required for expression of the c- fosatrial natriuretic factor, and myosin light chain-2 promoters in phenylephrine-induced hypertrophy 14 In addition, active MAP kinase is required for induction of the c- fos and fcz natriuretic factor promoters by phenylephrine 4142and expression of constitutively active MEK1 results in overexpression of hypertrophic genes In this study, we found that activation of the ERK mitogen-activated protein kinase pathway is required for activation of the Glut1 9021 during myocardial hypertrophy.
Because of the low transfection efficiency in primary myocytes, it was not possible to assess the effect of transfection with these molecules on expression of endogenous Glut1 mRNA. Conversely, cotransfection of the cells with a dominant-negative version of Ras A15Ras strongly inhibits induction of the Glut1 promoter by either TPA or phenylephrine, suggesting that activation of Ras is not only sufficient, fsc also necessary for induction of the Glut1 promoter.
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However, Marais et al. These results therefore suggest that Ras-mediated Raf activation, rather than PKC-mediated Raf phosphorylation, is the main pathway leading to stimulation of ERK activity in myocytes treated with phorbol esters. The reason why TPA activates Ras in myocytes but not in fibroblasts remains unknown, but could be related to expression of different PKC isoforms. We have also shown that PI3K activation is involved, at least in part, in the transduction of the signal fcd hypertrophic agonists to the Glut1 promoter.
The exact position of PI3K in these signal transduction pathways remains somewhat controversial. Although an initial report described PI3K as being a direct target of Ras 32other studies have suggested that PI3K could act upstream of Ras 46 The present study does not allow us to draw firm conclusions on this issue.
Experiments using the specific p38 inhibitor SB 5253 or cotransfection with the JNK inhibitory protein JIP data not shown have shown that the p38 pathway was not significantly involved and the JNK pathway contributed very little to induction of Glut1 transcription during myocardial hypertrophy.
Although this study used pharmacological agonists to study the in vitro hypertrophic response of glucose transporters, it has implications for the understanding of glucose metabolism in pathophysiological situations. Interestingly the signaling pathways that are used in different cell types in the heart to activate the ERK molecules are different.
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