Impaired stimulation of glucose transport in cardiac myocytes exposed to very low-density lipoproteins.

We recently observed that free fatty acids impair the stimulation of glucose transport into cardiomyocytes in response to either insulin or metabolic stress. In vivo, fatty acids for the myocardium are mostly obtained from triglyceride-rich lipoproteins (chylomicrons and Very Low-Density Lipoproteins). We therefore determined whether exposure of cardiac myocytes to VLDL resulted in impaired basal and stimulated glucose transport. Primary adult rat cardiac myocytes were chronically exposed to VLDL before glucose uptake was measured in response to insulin or metabolic stress, provoked by the mitochondrial ATP synthase inhibitor oligomycin. Exposure of cardiac myocytes to VLDL reduced both insulin-and oligomycin-stimulated glucose uptake. The reduction of glucose uptake was associated with a moderately reduced tyrosine phosphorylation of the insulin receptor. No reduction of the phosphorylation of the downstream effectors of insulin signaling Akt and AS160 was however observed. Similarly only a modest reduction of the activating phosphorylation of the AMP-activated kinase (AMPK) was observed in response to oligomycin. Similar to our previous observations with free fatty acids, inhibition of fatty acid oxidation restored oligomycin-stimulated glucose uptake. In conclusions, VLDL-derived fatty acids impair stimulated glucose transport in cardiac myocytes by a mechanism that seems to be mediated by a fatty acid oxidation intermediate. Thus, in the clinical context of the metabolic syndrome high VLDL may contribute to enhancement of ischemic injury by reduction of metabolic stress-stimulated glucose uptake.

Impaired glucose metabolism in the heart of obese Zucker rats after treatment with phorbol ester.

OBJECTIVE: To investigate the influence of obesity on the regulation of myocardial glucose metabolism following protein kinase C (PKC) activation in obese (fa/fa) and lean (Fa/?) Zucker rats. DESIGN: Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) for different time periods prior to the evaluation of PKC and GLUT-4 translocation. For metabolic studies isolated hearts from 48 h starved Zucker rats were perfused with an erythrocytes-enriched buffer containing increased concentrations (10-100 nM) of PMA. MEASUREMENTS: Immunodetectable PKC isozymes and GLUT-4 were determined by Western blots. Glucose oxidation and glycolysis were evaluated by measuring the myocardial release of 14CO2 and 3H2O from [U-14C]glucose and [5-3H]glucose, respectively. RESULTS: PMA (200 nM) induced maximal translocation of ventricular PKCalpha from the cytosol to the membranes within 10 min. This translocation was 2-fold lower in the heart from obese rats when compared to lean rats. PMA also induced a significant translocation of ventricular GLUT-4 from the microsomal to the sarcolemmal fraction within 60 min in lean but not in obese rats. Rates of basal cardiac glucose oxidation and glycolysis in obese rats were approximately 2-fold lower than those of lean rats. Perfusion with increasing concentrations of PMA (10-100 nM) led to a significant decrease of cardiac glucose oxidation in lean but not in obese rats. CONCLUSION: Our results show that in the heart of the genetically obese Zucker rat, the impairment in PKCalpha activation is in line with a diminished activation of GLUT-4 as well as with the lack of PMA effect on glucose oxidation.

Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism.

OBJECTIVES: Increasing evidence suggests that left ventricular remodeling is associated with a shift from fatty acid to glucose metabolism for energy production. The aim of this study was to determine whether left ventricular remodeling with and without late-onset heart failure after myocardial infarction is associated with regional changes in the expression of regulatory proteins of glucose or fatty acid metabolism. METHODS: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary artery (LAD). In infarcted and sham-operated hearts the peri-infarction region (5-mm zone surrounding the region at risk), the interventricular septum and the right ventricular free wall were separated for analysis. RESULTS: At 8 and 20 weeks after LAD ligation, the peri-infarction region and the septum exhibited marked re-expression of atrial natriuretic factor [+252+/-37 and +1093+/-279%, respectively, in the septum (P<0.05)] and of alpha-smooth muscle actin [+34+/-10 and +43+/-14%, respectively, in the septum (P<0.05)]. At 8 weeks, when left ventricular hypertrophy was present without signs of heart failure, myocardial mRNA expression of glucose transporters (GLUT-1 and GLUT-4) was not altered, whereas mRNA expression of medium-chain acyl-CoA dehydrogenase (MCAD) was significantly reduced in the peri-infarction region (-25+/-7%; P<0.05). In hearts exhibiting heart failure 20 weeks after infarct-induction there was a change in all three ventricular regions of both mRNA and protein content of GLUT-1 [+72+/-28 and +121+/-15%, respectively, in the peri-infarction region (P<0.05)] and MCAD [-29+/-9 and -56+/-4%, respectively, in the peri-infarction region (P<0.05)]. CONCLUSION: In rats with large myocardial infarction, progression from compensated remodeling to overt heart failure is associated with upregulation of GLUT-1 and downregulation of MCAD in both the peri-infarction region and the septum.

Altered expression of proteins of metabolic regulation during remodeling of the left ventricle after myocardial infarction.

Non-infarcted myocardium after coronary occlusion undergoes progressive morphological and functional changes. The purpose of this study was to determine whether non-infarcted myocardium exhibits (1) alteration of the substrate pattern of myocardial metabolism and (2) concomitant changes in the expression of regulatory proteins of glucose and fatty acid metabolism. Myocardial infarction was induced in rats by ligation of the left coronary artery. One day and eight weeks after coronary occlusion, glucose and palmitate oxidation were measured. Expression of selected proteins of metabolism were determined one day to 12 weeks after infarction. One day after coronary occlusion no difference of glucose and palmitate oxidation was detectable, whereas after eight weeks, glucose oxidation was increased (+84%, P<0.05) and palmitate oxidation did not change significantly (-19%, P=0.07) in infarct-containing hearts, compared with hearts from sham-operated rats. One day after coronary occlusion, myocardial mRNA expression of the glucose transporter GLUT-1 was increased (+86%, P<0.05) and the expression of GLUT-4 was decreased (-28%, P<0.05) in surviving myocardium of infarct-containing hearts. Protein level of GLUT-1 was increased (+81%, P<0.05) and that of GLUT-4 slightly, but not significantly, decreased (-16%, P=NS). mRNA expressions of heart fatty acid binding protein (H-FABP), and of medium chain acyl-CoA dehydrogenase (MCAD), were decreased by 36% (P<0.05) and 35% (P=0. 07), respectively. Eight weeks after acute infarction, the left ventricle was hypertrophied and, at this time-point, there was no difference in the expression of GLUT-1 and GLUT-4 between infarcted and sham-operated hearts. However, myocardial mRNA and protein content of MCAD were decreased by 30% (P<0.01) and 27% (P<0.05), respectively. In summary, in surviving myocardium, glucose oxidation was increased eight weeks after coronary occlusion. Concomitantly, mRNA and protein expression of MCAD were decreased, compatible with a role of altered expression of regulatory proteins of metabolism in post-infarction modification of myocardial metabolism.

Postischemic recovery of heart metabolism and function: role of mitochondrial fatty acid transfer.

Postischemic recovery of contractile function is better in hearts from fasted rats than in hearts from fed rats. In this study, we examined whether feeding-induced inhibition of palmitate oxidation at the level of carnitine palmitoyl transferase I is involved in the mechanism underlying impaired recovery of contractile function. Hearts isolated from fasted or fed rats were submitted to no-flow ischemia followed by reperfusion with buffer containing 8 mM glucose and either 0.4 mM palmitate or 0.8 mM octanoate. During reperfusion, oxidation of palmitate was higher after fasting than after feeding, whereas oxidation of octanoate was not influenced by the nutritional state. In the presence of palmitate, recovery of left ventricular developed pressure was better in hearts from fasted rats. Substitution of octanoate for palmitate during reperfusion enhanced recovery of left ventricular developed pressure in hearts from fed rats. However, the chain length of the fatty acid did not influence diastolic contracture. The results suggest that nutritional variation of mitochondrial fatty acid transfer may influence postischemic recovery of contractile function.

Activation of Ras by phorbol esters in cardiac myocytes. Role of guanine nucleotide exchange factors.

The relationship between protein kinase C (PKC) activation and Ras function was investigated in cardiac cells. Ras function was required for ERK activation by phorbol esters in cardiac myocytes, but not in cardiac fibroblasts. Accordingly, treatment with phorbol esters resulted in GTP loading of Ras in cardiac myocytes, but not fibroblasts. Ras activation by phorbol esters was abolished by a PKC specific inhibitor, but was insensitive to tyrosine kinase inhibitors. Ras activation was mediated by stimulation of guanine nucleotide exchange. These results suggest the existence of a novel pathway for Ras activation, specific to cardiac myocytes, with implications for myocardial hypertrophy.

Effect of transient ischemia on the expression of glucose transporters GLUT-1 and GLUT-4 in rat myocardium.

A number of observations indicate that myocardial glucose utilization is increased late during post-ischemic reperfusion. The present study was designed to examine whether transient ischemia elicits altered expression of glucose transporters GLUT-1 and GLUT-4. In rats, the left anterior descending coronary artery was occluded for 20 min followed by reperfusion for 1, 3 or 7 days. Regional myocardial uptake and phosphorylation of glucose was determined based on myocardial accumulation of 2-deoxy-D-[2, 6-3H]glucose-6-phosphate. In hearts from fasted rats, after 3 days of reperfusion, myocardial uptake and phosphorylation of glucose was 48% higher in the reperfused region compared to a remote control region. No regional difference in myocardial glucose uptake and phosphorylation was detectable in hearts from fed rats. After 1 day of reperfusion, expression of myocardial glucose transporter GLUT-1 mRNA was increased to 195+/-24% (mean+/-SEM) of the value measured in the remote region and the expression of GLUT-4 mRNA was decreased to 58+/-7%. After 3 days of reperfusion both mRNA and protein of GLUT-1 were higher in the reperfused region, averaging 133+/-23% and 249+/-36%, respectively. The corresponding values for GLUT-4 mRNA and protein were 77+/-7% and 62+/-6%, respectively. The results indicate that a short period of ischemia alters the expression of glucose transporter isoforms GLUT-1 and GLUT-4. Observed changes may be involved in the mechanisms underlying late changes of substrate metabolism during reperfusion.

Transcriptional activation of the glucose transporter GLUT1 in ventricular cardiac myocytes by hypertrophic agonists.

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 12-O-tetradecanoylphorbol-13-acetate or phenylephrine increased expression of Glut1 mRNA relative to Glut4 mRNA. To study the transcriptional regulation of GLUT1 expression, myocytes were transfected with luciferase reporter constructs under the control of the Glut1 promoter. Stimulation of the cells with 12-O-tetradecanoylphorbol-13-acetate or phenylephrine induced transcription from the Glut1 promoter, which was inhibited by cotransfection with the mitogen-activated protein kinase phosphatases CL100 and MKP-3. 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.

Post-ischemic stimulation of 2-deoxyglucose uptake in rat myocardium: role of translocation of Glut-4.

Myocardial ischemia elicits translocation of the insulin-sensitive glucose transporter GLUT-4 from intracellular membrane stores to the sarcolemma. Because glucose metabolism is of crucial importance for post-ischemic recovery of the heart, myocardial uptake of [3H]-labeled 2-deoxyglucose and subcellular localization of GLUT-4 were determined during reperfusion in isolated rat hearts perfused with medium containing 0.4 mm palmitate and 8 mm glucose. Hearts were subjected to 20 min of no-flow ischemia, followed by reperfusion for up to 60 min. Subcellular localization of GLUT-4 was determined by cell fractionation followed by immunoblotting. After 15 and 60 min of reperfusion uptake of 2-deoxyglucose was significantly higher (91+/-9 and 96+/-8 nmol/min/g wet weight, respectively) as compared to control values (65+/-1 nmol/min/g wet weight). Ischemia elicited translocation of GLUT-4 to the sarcolemma, which persisted after 15 min of reperfusion. However, after 60 min of reperfusion the subcellular distribution of GLUT-4 was similar to control hearts. In conclusion, reversal of ischemia-induced translocation of GLUT-4 to the sarcolemma is rather slow, possibly facilitating glucose uptake early during reperfusion. However, myocardial uptake and phosphorylation of 2-deoxyglucose remains enhanced late during reperfusion, when pre-ischemic distribution of GLUT-4 is almost completely restored, indicating that additional mechanisms are likely to be involved in post-ischemic stimulation of glucose uptake.

[Cellular recovery after ischemia: physiopathologic aspects]. / La récupération cellulaire après une ischémie: aspects physiopathologiques.

Myocytes that have survived a period of transient ischemia may present prolonged alterations of cellular function, detectable during several days. The early period of postischemic reperfusion is characterized by restoration of ion homeostasis mediated by rapid resumption of function of ion pumps (Na+/K(+)-ATPase, Ca(2+)-ATPase) and transsarcolemmal ion exchange mechanisms (H+/Na+, Na+/Ca2+ exchangers). There is experimental evidence that cellular injury may be enhanced during the initial seconds or minutes of reperfusion, depending on the conditions of reperfusion. During the late phase of reperfusion mRNA expression of a number of key proteins of myocyte function is altered. The pattern of gene expression during reperfusion exhibits features of cellular adaptation and/or dedifferentiation in addition to cell repair.

[Hypertrophy due to left cardiac insufficiency: role of the neurohumoral system]. / De l'hypertrophie à l'insuffisance cardiaque gauche: rôle du système neuro-humoral.

Left ventricular hypertrophy in patients with hypertensive heart disease is associated with impaired relaxation and myocardial interstitial fibrosis leading to enhanced filling pressure, referred to as left ventricular diastolic dysfunction. Impairment of systolic function, characterized by reduced ejection fraction occurs at a later stage. Activation of the renin-angiotensin-aldosterone system contributes to progression to heart failure by at least two mechanisms: (1) increased left ventricular loading conditions due to vasoconstriction and retention of sodium; (2) direct effects on the myocardium resulting in myocyte hypertrophy and interstitial fibrosis.

Effect of nutritional state on substrate metabolism and contractile function in postischemic rat myocardium.

The pattern of substrate utilization may influence postischemic myocardial injury. To characterize the effect of nutritional state on substrate selection and contractile function during control conditions and postischemic reperfusion, hearts from fed and fasted rats were perfused retrogradely with 0.4 mM palmitate, 8 mM glucose, and 175 mU/l insulin. Under control conditions, hearts from fasted rats exhibited lower glucose oxidation (-59%) and higher palmitate oxidation (+191%) than hearts from fed rats. During reperfusion, postischemic hearts exhibited stimulation of glucose-oxidation, with no difference between hearts from fasted and fed rats. However, oxidation of palmitate remained higher after fasting (+68%). Hearts from fasted rats exhibited lower left ventricular diastolic pressure and higher left ventricular systolic pressure development during reperfusion. The results indicate that 1) substrate selection in myocardium is influenced by the nutritional state independently of substrate availability, 2) during postischemic reperfusion, inhibition of glucose oxidation is removed in hearts from fasted rats, whereas inhibition of fatty acid oxidation in hearts from fed rats is maintained, and 3) myocardial injury is lower after fasting.

Expression and regulation of Na-dependent P(i) transport in matrix vesicles produced by osteoblast-like cells.

Extracellular matrix vesicles (MV) are the loci of initial mineralization in several calcifying tissues. We recently reported that MV isolated from chicken epiphyseal cartilage are equipped with a Na-dependent P(i) transport (NaPiT) system. The activity of the NaPiT system appeared to be crucial for the development of MV-mediated calcification. In the present study we investigated the expression of NaPiT activity in MV produced by the osteoblast-like cells MC3T3-E1. The relationship between changes in NaPiT activity in the intact cells and in the released MV was also examined. NaPiT activity in MV harvested from cultured MC3T3-E1 cells was transiently expressed. It was markedly increased between Days 8 and 10 (5- to 6-fold), and then gradually decreased. NaPiT activity was enriched in MV as compared with the parent osteoblast-like cells, while the Na-dependent transport system for alanine (NaAlaT) was not. When NaPiT activity was enhanced in osteoblast-like cells by fetal calf serum (FCS) or P(i) depletion, P(i) transport stimulation was observed in the derived MV as well. Alkaline phosphatase (AP) was differentially expressed and regulated in MV from MC3T3-E1 cell cultures, as compared with NaPiT. In contrast to the transient expression of NaPiT, AP activity in MV increased continuously with time in culture. It was stimulated by FCS treatment of the parent cells, but decreased in MV obtained from P(i)-depleted cultures. These results suggest that the presence in osteogenic cells of selective regulatory mechanisms for the insertion and enrichment of P(i) transport activity in released MV.(ABSTRACT TRUNCATED AT 250 WORDS)

Pi transport regulation by chicken growth plate chondrocytes.

Inorganic phosphate (Pi) is a key element for the growth and mineralization of the epiphyseal cartilage. In this study, the characteristics of the transport of Pi in growth plate chondrocytes have been determined using primary cultures of chicken growth plate cartilage cells. The uptake of Pi was significantly increased in the presence of extracellular sodium. The kinetic parameters of the saturable sodium-dependent Pi transport (NaPiT) were determined. The Michaelis constant for Pi was 0.443 +/- 0.095 mM, and the concentration of sodium with which half-maximal Pi transport was observed was 48.0 +/- 8.7 mM. Stoichiometric analysis suggested that more than one sodium ion was cotransported with each Pi molecule. NaPiT was sensitive to inhibition by Pi analogues such as phosphonoformic acid and arsenate. These data strongly suggest that Pi uptake by chicken growth plate chondrocytes is a carrier-mediated process driven by the transmembrane electrochemical gradient of sodium. Two important regulators of biosynthetic activities of growth plate chondrocytes, insulin-like growth factor I (IGF-I) and parathyroid hormone (PTH), selectively regulated Pi transport. With IGF-I, maximal stimulation (117 +/- 7% above control) was observed at doses > 5 nM, with an half-maximal effective concentration of 0.46 +/- 0.18 nM. A significant effect was observed after 1 h of exposure and was maintained for up to 24 h. PTH increased Pi transport with a biphasic dose-response curve. The change in NaPiT was transient, being maximally observed after 8 h (58 +/- 8%) and unexpressed after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a Pi transport system in cartilage matrix vesicles. Potential role in the calcification process.

The mechanisms by which calcium (Ca2+) and inorganic phosphate (Pi) accumulate into matrix vesicles (MV) have not been elucidated. In the present study the characteristics of Pi uptake into MV isolated from mildly rachitic chicken growth plate cartilage have been investigated. The results indicate that Pi accumulates into MV mainly via a Na(+)-dependent Pi transport system. In the absence of NaCl in the extravesicular medium, Pi uptake was a nonsaturable process. In the presence of 150 mM NaCl, the initial rate of Pi uptake was 4.38 +/- 1.02-fold higher than with 150 mM choline chloride (mean +/- S.E., n = 8, p less than 0.005). Other cations showed partial activity to drive Pi into MV as compared to Na+:Li+ (64.4%) greater than K+ (39.8%) greater than choline (39.0%) greater than tetramethylammonium (30.0%) greater than N-methylglucamine (26.3%). Na(+)-dependent Pi transport activity displayed saturability towards increasing extra-vesicular concentrations of Na+ and Pi. The apparent Km for Pi was 0.68 +/- 0.16 mM. The Na+ concentration producing half-maximum Pi transport activity was 106.2 +/- 11.0 mM. Kinetic analysis suggests that Na+ interacts with the Pi carrier with a stoichiometry of more than one Na+ ion with one Pi molecule. In MV isolated from normal chicken growth plate cartilage, this Na(+)-dependent Pi transport system was barely expressed. In contrast to the effect on Pi uptake by MV, the activity of alkaline phosphatase was not changed when NaCl was substituted for choline chloride in the assay medium. In addition to this observation which suggests that this enzyme is not related to the Pi transport activity described in this study, levamisole, which inhibited alkaline phosphatase activity did not affect the Na(+)-dependent uptake of Pi. Both arsenate and phosphonoformic acid, two inhibitors of the epithelial Na(+)-dependent Pi transport systems, were active inhibitors of the Na(+)-dependent Pi uptake by MV with a higher potency for phosphonoformic acid. Associated with the expression of a facilitated Na(+)-coupled Pi transport in MV, in vitro calcification assessed by 45Ca2+ uptake also showed a marked dependence on extravesicular sodium. This relationship was markedly attenuated in MV isolated from normal chicken growth plate cartilage expressing a weak Na(+)-facilitated Pi transport activity. In conclusion, a saturable Na(+)-dependent Pi carrier has been characterized which facilitates Pi transport in MV. Its potential role for Ca-Pi accumulation into MV and subsequent development of vesicular calcification followed by mineralization of the osteogenic matrix is proposed and remains to be further investigated.

Stimulatory effect of insulin-like growth factor-1 on renal Pi transport and plasma 1,25-dihydroxyvitamin D3.

Administration of GH increases both the tubular reabsorption of inorganic phosphate (Pi) and the plasma level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. These two effects could be induced by a common mediator, possibly the GH-generated insulin-like growth factor 1 (IGF-1). In the present work, the influence of recombinant human IGF-1 on renal Pi transport and plasma 1,25-(OH)2D3 was examined in hypophysectomized (HPX) rats. IGF-1, infused by miniosmotic pump at the dose of 10 micrograms/h for 6 days, significantly increased the maximal tubular reabsorption of Pi per unit volume of glomerular filtrate (max TRPi/m1GFR): IGF-1 3.50 +/- 0.16; vehicle: 2.78 +/- 0.14 mumol/m1GFR, P less than 0.005. The response was associated with a marked stimulation of plasma 1,25-(OH)2D3 (IGF-1; 409 +/- 23; vehicle: 208 +/- 22 pmol/liter, P less than 0.001). As previously reported for GH, IGF-1 also increased GFR and reduced urinary sodium excretion. In brush border membrane vesicles isolated from renal cortex of HPX rats, the Na-dependent Pi transport was stimulated by IGF-1. Neither the Na-dependent glucose transport nor that of alanine was affected by the growth factor. The stimulatory effect of IGF-1 on maxTRPi/m1GFR was also expressed in thyroparathyroidectomized (TPTX) HPX rats (IGF-1: 5.20 +/- 0.29; vehicle: 3.88 +/- 0.37 mumol/m1GFR, P less than 0.025). In conclusion, administration of IGF-1 in HPX rats mimics the stimulatory effects of GH on maxTRPi/m1GFR and on plasma 1,25-(OH)2D3. As described for GH the change in maxTRPi/m1GFR is mediated by a PTH independent mechanism and is expressed at the level of the luminal membrane of proximal tubules. These results suggest that IGF-1 could be an important factor in the control of Pi metabolism, particularly during growth, and might play a significant role in mediating the effect of GH on the renal handling of Pi and production of 1,25-(OH)2D3.