TY - JOUR
AU1 - Liang,, Qiangrong
AU2 - Donthi, Rajakumar, V
AU3 - Kralik, Patricia, M
AU4 - Epstein, Paul, N
AB - Abstract Objective: Cardiac glucose metabolism is critical to normal and pathological function. The significance of the first committed metabolic step, glucose phosphorylation, has not been established. In this study a new transgenic model was developed in order to investigate the importance of this enzymatic step in cardiac glycolysis. Methods: Transgenic mice were produced that overexpress yeast hexokinase B under the control of a cardiac specific promoter. Yeast hexokinase B is a high affinity enzyme that is not inhibited by glucose-6-phosphate. Hexokinase enzyme activity was measured by a modified radiometric procedure. Cardiac glucose metabolism and contractility were measured in the Langendorff mode. Cardiac glycogen content and glucose-6-phosphate independent glycogen synthase activity were also determined. Results: In transgenic hearts hexokinase activity was significantly elevated and increased glucose metabolism, particularly in the presence of insulin and during cardiac reperfusion. However during ischemic perfusion the effect of the transgene on glycolysis was minimal. Under all conditions tested there was no effect of hexokinase on contractility. Glycogen content of transgenic hearts was elevated 2-fold and glucose-6-phosphate independent glycogen synthase was also increased. Conclusion: These results demonstrate that glucose phosphorylation is a key step in determining cardiac glucose metabolism under oxidative conditions. Energy metabolism, Enzyme (kinetics), Glycolysis Time for primary review 21 days. 1 Introduction Myocardial fuel metabolism is tightly regulated and closely parallels the rate of cardiac work. Under most circumstances cardiac energy is derived primarily from fatty acids, while a small fraction is derived from glucose. However, under pathological conditions the significance of glucose metabolism increases. Hypertrophy is known to increase the reliance of the heart on glycolytic metabolism [1]. It has also been found that natural [2] or experimental [3] genetic mutations that impair glycolysis produce cardiac pathology. Similarly, diabetes produces a major decline in glycolysis [4] that is associated with diabetic cardiomyopathy. In ischemic [5] hearts, dependence on glucose and glycolysis increases. In fact, stimulation of glycolysis with glucose and insulin has been used successfully to protect patient hearts from ischemic damage [6,7]. Unfortunately, our current understanding of the relationship between glycolysis and cardiac pathology is limited. In part this is due to the fact that experimental manipulations employed to modify glycolysis, such as fasting and insulin treatment, affect many other systems. More specific manipulations of cardiac carbohydrate metabolism are needed to define its role in heart physiology and pathology. Glucose transport has been proposed to be rate limiting in skeletal and cardiac muscle glucose utilization. This conclusion was based largely on the finding that intracellular glucose concentrations are very low [8,9]. However, recent studies demonstrated that glucose concentrations in cardiac myocytes can reach several millimolar and rise still further when glucose transport is stimulated by insulin [10,11], indicating that some step beyond glucose transport becomes rate-limiting under conditions of hyperinsulinemia or hyperglycemia. Since intracellular glucose must first be phosphorylated for further metabolism, hexokinase is implicated as a critical step for control of glucose utilization. To confirm this role for glucose phosphorylation, transgenic animals have been bred that overexpress hexokinase II in skeletal and cardiac muscle [12,13]. Those studies observed relatively small effects of hexokinase overexpression on glucose homeostasis and cardiac glucose utilization [12,13]. Heikkinen et al. [14] produced heterozygous hexokinase II knockout animals with a 50% reduction in cardiac enzyme activity. These animals also exhibited only a minor impact on metabolism. Hexokinase II is strongly inhibited by its product, glucose-6-phosphate (g6p). This self-regulation may limit the impact of hexokinase II over- or under-expression on overall glycolysis. The significance of this allosteric regulation was highlighted by the studies of Jimenez-Chillaron et al. [15] who transiently overexpressed glucokinase, a g6p unregulated enzyme in hind-limb muscle. Glucokinase, despite a much lower affinity for glucose than hexokinase II, had a much greater impact on glucose metabolism in skeletal muscle. Those results suggested that there might be a significant effect on cardiac glucose metabolism if a hexokinase free of allosteric inhibition was overexpressed. To study the effects of glucose phosphorylation on cardiac glucose metabolism and to develop a model suitable for analyzing the impact of increased glucose phosphorylation on cardiac ischemia and health, we created a new transgenic mouse model that expresses yeast hexokinase B specifically in the heart. This hexokinase isozyme is free of significant g6p inhibition. Transgenic animals exhibited no cardiac abnormalities. However, the elevated hexokinase activity resulted in enhanced glycolysis and increased glycogen storage in the heart, indicating that glucose phosphorylation is an important step determining cardiac glucose utilization. 2 Methods 2.1 Development of transgenic mice The MyHEX transgene was produced by replacement of the catalase coding sequences in the transgene MyCat [16] with a 2700-bp fragment that contained all the coding sequences of the yeast hexokinase B gene [17], the SV40 early splice region and SV40 early polyadenylation site [18]. Prior to microinjection all plasmid sequences were removed by digestion with NotI. FVB mice were used to produce transgenic lines containing the MyHEX transgene. Standard embryo microinjection procedures were used for producing transgenic animals. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All animal procedures were approved by the USDA certified institutional animal care committee. 2.2 Analysis of transgene expression The activity and tissue specificity of the transgene were determined by measuring RNA on Northern blots and enzyme activity in tissue homogenates. RNA for Northern blots was prepared with RNazol as we have previously described [19,20] from heart, liver, skeletal muscle, brain, kidney and lung of transgenic and normal mice. A 1.3-kb NcoI/PstI fragment of the yeast hexokinase gene was labeled with 32P by random oligonucleotide priming and used as probe. This sequence had little detectable cross-reactivity with any other mouse messenger RNA. Hexokinase enzyme activity was measured by a modification of the radiometric procedure of Bedoya et al. [21]. Hearts were homogenized in 50 vol. of buffer containing 30 mM HEPES (pH 7.6), 4 mM MgCl2, 0.2% BSA, 130 mM KCl, 14 mM β-mercaptoethanol and 1 mM EDTA. Then 2 μl of extract were incubated for 60 min in 30 μl of buffer containing 50 mM HEPES (pH 7.6), 120 mM KCl, 5 mM ATP, 14 mM β-mercaptoethanol, 0.1% BSA, 0.5 μCi [2-3H]glucose, 1 mM glucose and various concentrations of glucose-6-phosphate (g6p). The incubation was then heated to 96°C and 2 U of phosphoglucoisomerase were added for an additional 60 min. After the addition of 20 μl of 0.5 M HCl the formation of tritiated water was assessed by diffusion overnight at 37°C into 1 ml of water and scintillation counting. Counts were adjusted for the efficiency of diffusion and background samples that contained no extract. 2.3 Cardiac perfusion Langendorff perfusions were carried out by a modification of a procedure previously reported by us [22]. Mice were anesthetized by i.p. injection of sodium pentobarbital 150 mg/kg administered with 100 IU of heparin. The left side of the chest was opened. Through the incision the left lung was moved to the right side of the chest and the descending aorta isolated on the back wall. A suture was then placed around the aorta in preparation for cannulation. A small incision was made in the descending aorta to allow insertion of a cannula. The innominate artery, common carotid artery and subclavian artery were then sutured and cut. The heart was removed from the chest and transferred to the perfusion apparatus. The heart was retrogradely perfused at 2 ml/min with Krebs-Henseleit buffer (KH) consisting of 120 mM NaCl, 20 mM NaHCO3, 4.6 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgCl2, 1.25 mM CaCl2 5 mM glucose and 0.25 μCi/ml of [5-3H]glucose. Throughout the perfusion KH buffer was continuously equilibrated with 95% O2/5% CO2 which maintained a pH of 7.4 and temperature was maintained at 37°C. The heart was paced throughout the procedure at 6 Hz (6 V, 3 ms). For studies on the effect of ischemia, baseline glycolysis was determined during the first 60 min. Next, the heart was subject to a 60-min period of low-flow ischemia. This was achieved by switching a three-way valve to another perfusion line running from a buffer reservoir bubbled with 95% N2 and 5% CO2 to ensure hypoxia and a buffer pH of 7.4. The ischemic flow rate was 0.15 ml/min. For the last hour the heart was again perfused with 95% O2 and 5% CO2 equilibrated KH buffer at 2.0 ml/min. For studies on the effect of insulin, baseline glycolysis was determined for the first 30 min followed by 50 min in the presence of 200 μU/ml insulin. Perfusion pressure was monitored on a Gould/Statham p23Db physiological pressure transducer. Contractile force was measured by means of a Grass FT03 force transducer hooked to the apex of the heart. Transducers were connected to a ETH 400 bridge amplifier which fed into a Powerlab/400 amplifier. Data was analyzed using AD Instruments Chart for Windows version 3.3.5. 2.4 Measurements of glycolysis and lactate production Tritiated water produced from [5-3H]glucose during the perfusion was determined by diffusion. A 400-μl sample of cardiac effluent and 25 μl of 0.6 N HCl were added to 1.5-ml tubes that were placed inside 20-ml scintillation vials containing 2 ml water. Vials were incubated for 72 h at 37°C. The inside tube was removed and scintillant added for counting. Effluent from each time point was assayed in triplicate. For each experiment, background counts were determined by performing the same equilibration on perfusion media that had not passed through the heart. Diffusion efficiency was also measured in each experiment using tritiated water. Lactate concentration was measured in 6-fold dilution of oxygenated or 24-fold dilution of ischemic effluent using Sigma kit 826-B. 2.5 Glycogen assay The heart was quickly removed, blotted dry, and frozen with liquid nitrogen. The frozen tissue was weighed, homogenized in 9 vol. of 0.03 N HCl at 0°C and then heated in a boiling water bath for 5 min. Then 10 μl of homogenate glycogen was hydrolyzed to glucose by the addition of 50 ng amyloglucosidase (Boehringer-Mannheim) in 100 μl of 0.1 M acetate buffer, pH 4.7. Glucose was then measured by fluorometric assay using hexokinase and glucose-6-phosphate dehydrogenase [23]. For each sample assays were performed with and without amyloglucosidase to distinguish glucose derived from glycogen from preexisting cardiac glucose. 2.6 Assay of glycogen synthase The heart was blotted dry and homogenized on ice in 9 vol. of 50 mM HEPES buffer, pH 7.4, containing 50 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM EDTA, and 5 μl/ml of protease inhibitor cocktail (Sigma, P 8340). The homogenate was centrifuged at 10,000×g, 0°C for 15 min. A 30-μl aliquot of supernatant was added to 60 μl of an assay buffer containing 50 mM Tris–HCl, pH 7.0, 20 mM EDTA, 25 mM potassium fluoride, bovine liver glycogen (5 mg/ml) and 8 mM uridine diphospho-d-[U-14C]glucose (0.05 μCi). Separate assays were performed at pH 8.5 with 2.0 mM glucose-6-phosphate as activator of the b form. The mixture was incubated at 30°C for 20 min, and then boiled in a water bath for 10 min to terminate the enzyme reaction. Then 75 μl of the reaction mixture was spotted onto a 2×2-cm2 filter paper, which was washed twice in 66% ethanol and once with acetone prior to liquid scintillation counting. 2.7 Assay of cardiac glucose-6-phosphate (g6p) Hearts were quickly blotted and frozen in liquid nitrogen. The frozen tissue was weighed and homogenized in 9 vol. of 1.0 M perchloric acid. The extract was centrifuged at 12,000×g for 5 min and the supernatant neutralized to pH 7.4. The concentration of g6p was measured against g6p standards by a fluorometric procedure [23] employing NADP and glucose 6-phosphate dehydrogenase. 2.8 Statistical analysis Statistical analysis of hexokinase activity was carried out by one-way ANOVA followed by Bonferroni post-hoc test. For comparisons of contractility, glycolysis and lactate production, values were subject to two-way ANOVA for repeated measures and post-hoc analysis by Student-Newman-Keuls test. Pair wise comparisons for values obtained under the same experimental conditions for glycogen content, glycogen synthase activity and perfused heart function, were made using Student's t-test. The accepted level of significance was 0.05. 3 Results 3.1 Generation of transgenic mice expressing yeast hexokinase B in the heart A transgene designated MyHEX was constructed with the yeast hexokinase B gene. To achieve cardiac expression, the transgene was regulated by a 5000-bp portion of the cardiac specific alpha myosin heavy chain (MHC) gene [24], consisting of the promoter and the first two introns. Four transgenic lines of mice were produced with the MyHEX gene. Line 188 demonstrated the clearest increase in cardiac enzyme activity, which was enhanced 2-fold when animals were bred to homozygosity (data not shown). In all subsequent assays homozygous line 188 mice were used. These mice were healthy with no indications of increased mortality or reduced fertility over a period of more than 1 year. No morphological abnormalities were noted and heart to body weight ratios did not suggest cardiac hypertrophy (ratios of heart to body weight in mg/g of 4.5±0.1 and 4.3±0.1 for control and MyHEX mice, respectively, n=11 per group, P>0.1). 3.2 Enzymatic and Northern analysis of transgene expression To estimate the increase in hexokinase activity in vivo we performed enzyme assays at three different g6p concentrations. As shown in Fig. 1, the hexokinase activity from transgenic hearts was increased 12, 67 and 680% over the non-transgenic controls when measured at 0, 0.46 and 3 mM g6p, respectively. Since 0.46 mM g6p approximates intracellular cardiac g6p level [25], these data suggest that in vivo activity was increased by ∼67% in transgenic hearts. The fact that the hexokinase activity in non-transgenic hearts was suppressed to a much greater extent by g6p than activity in transgenic hearts was consistent with the differential sensitivities of yeast hexokinase B and mammalian hexokinase to feed back inhibition by g6p. It is noteworthy that the increase in hexokinase activity in transgenic hearts increased gradually and reproducibly from 8 to 12.5 to 26 pmol glucose/μg tissue per h at 0, 0.46 and 3 mM g6p, respectively. Expression of yeast hexokinase mRNA was used to assess the tissue specificity of the transgene. The Northern blot shown in Fig. 2 demonstrated that the transgene was active primarily in the heart. Fig. 2 Open in new tabDownload slide Yeast hexokinase mRNA expression is primarily in the heart with lower level expression in lung of transgenic mice. Br, brain; Ht, heart; Kd, kidney; Li, liver; Lu, lung; Sk, skeletal muscle. The position of 18S RNA is shown. Fig. 2 Open in new tabDownload slide Yeast hexokinase mRNA expression is primarily in the heart with lower level expression in lung of transgenic mice. Br, brain; Ht, heart; Kd, kidney; Li, liver; Lu, lung; Sk, skeletal muscle. The position of 18S RNA is shown. Fig. 1 Open in new tabDownload slide Hexokinase activity in transgenic hearts is increased and resistant to g6p inhibition. Transgenic (MyHEX) and control hearts (FVB) were extracted and assayed as described in Methods under the indicated concentrations of g6p. The asterisks indicate that MyHEX and FVB are different at that g6p concentration: **P<0.001 by ANOVA and Bonferroni post hoc test. Values show the mean±S.E.M. for six hearts assayed in triplicate. Fig. 1 Open in new tabDownload slide Hexokinase activity in transgenic hearts is increased and resistant to g6p inhibition. Transgenic (MyHEX) and control hearts (FVB) were extracted and assayed as described in Methods under the indicated concentrations of g6p. The asterisks indicate that MyHEX and FVB are different at that g6p concentration: **P<0.001 by ANOVA and Bonferroni post hoc test. Values show the mean±S.E.M. for six hearts assayed in triplicate. 3.3 Effect of yeast hexokinase B on basal and insulin stimulated glycolysis The effect of the MyHEX transgene on cardiac glycolysis was assessed in Langendorff perfused hearts. Glycolysis was evaluated by determining the release of tritiated water from 5-tritiated glucose, which measures glycolytic steps through the triose phosphate isomerase reaction. As shown in Fig. 3A, glycolysis was greater in transgenic hearts, and this difference was significant at most time points before adding insulin and at all time points after the addition of insulin (P≤0.05). Before the addition of insulin the average glycolytic rate was greater by ∼250 pmol glucose/min per mg heart. Adding insulin stimulated glycolysis in all hearts and increased the average difference between transgenic and control hearts by an additional 100 pmol glucose/min per mg. Cardiac contractility and perfusion pressure were measured to determine whether these parameters influenced glycolysis. Throughout the assay, contractility (Fig. 3B) and perfusion pressure (Fig. 3C) were stable and essentially identical in control and transgenic hearts. Cardiac g6p, measured at the end of the insulin exposure in these hearts, was the same for FVB and MyHEX, 377±9.5 and 365±28 μM, respectively. An additional four hearts of each type were assayed for g6p after 20 min of perfusion without insulin. These hearts had g6p levels of 163±14 and 183±26 μM for FVB and MyHEX hearts, respectively. Fig. 3 Open in new tabDownload slide Yeast hexokinase increases glycolysis in perfused hearts in the presence or absence of insulin but has no effect on contractility. (A) Production of tritiated water from [5-3H]glucose. (B) Contractile force. (C) Perfusion pressure. Perfusion conditions and assay methods are described in the text. Each value shows the mean±S.E.M. for seven control or nine transgenic animals. *Indicates P<0.05 by two-way ANOVA for repeated measures and post-hoc analysis by Student-Newman-Keuls test. Fig. 3 Open in new tabDownload slide Yeast hexokinase increases glycolysis in perfused hearts in the presence or absence of insulin but has no effect on contractility. (A) Production of tritiated water from [5-3H]glucose. (B) Contractile force. (C) Perfusion pressure. Perfusion conditions and assay methods are described in the text. Each value shows the mean±S.E.M. for seven control or nine transgenic animals. *Indicates P<0.05 by two-way ANOVA for repeated measures and post-hoc analysis by Student-Newman-Keuls test. 3.4 Effect of yeast hexokinase B on basal, ischemic and reperfusion glycolysis Increased glycolysis may influence the cardiac response to ischemia, positively by providing a greater source of ATP or negatively by increasing lactate production and acidifying the heart. We examined the effect of the hexokinase transgene by assessing glycolysis, lactate production and contractility during ischemia and reperfusion (Fig. 4). The transgene appeared to increase glycolysis before and after ischemia, however unlike the results obtained for Fig. 3, this trend did not reach significance when assessed by two-way ANOVA for repeated measures. The difference in lactate production of transgenic and control hearts did reach significance. Post-hoc analysis revealed a significant increase in lactate production during the reperfusion phase. The failure to obtain a significant effect of the hexokinase transgene on glycolysis may have been due to the very similar rates of glycolysis in the two groups during the ischemic period. It is also worth noting that at most time points during the basal and reperfusion phase of perfusion, glycolysis and lactate production were greater in transgenic hearts than in control hearts by Student's t-test (P<0.05). In MyHEX hearts average rates of glycolysis and lactate production were greater during the reperfusion phase than during the basal phase (P<0.05). This is different from what was observed in FVB hearts where glycolysis and lactate production were almost the same before and after ischemia. During ischemia, neither glycolysis nor lactate production were clearly altered by the transgene. As shown in Fig. 4C, contractility was markedly affected by ischemia and reperfusion (P<0.05), but the hexokinase transgene had no impact on contractility during any phase of perfusion. Fig. 4 Open in new tabDownload slide Yeast hexokinase impact on glucose metabolism and contractility in perfused hearts subject to ischemia and reperfusion. (A) Production of tritiated water from [5-3H]glucose. (B) Production of lactate. (C) Contractile force. Each value is the mean obtained from eight control or nine transgenic animals. Perfusion conditions and assay methods are described in the text. Each value shows the mean±S.E.M. for eight control or nine transgenic animals. *Indicates P<0.05 by two-way ANOVA for repeated measures and post-hoc analysis by Student-Newman-Keuls test. Fig. 4 Open in new tabDownload slide Yeast hexokinase impact on glucose metabolism and contractility in perfused hearts subject to ischemia and reperfusion. (A) Production of tritiated water from [5-3H]glucose. (B) Production of lactate. (C) Contractile force. Each value is the mean obtained from eight control or nine transgenic animals. Perfusion conditions and assay methods are described in the text. Each value shows the mean±S.E.M. for eight control or nine transgenic animals. *Indicates P<0.05 by two-way ANOVA for repeated measures and post-hoc analysis by Student-Newman-Keuls test. 3.5 Effects of yeast hexokinase B on glycogen and glycogen synthase We measured cardiac glycogen content as an indicator of the transgene effect on in vivo cardiac metabolism. Yeast hexokinase produced a 2-fold increase in cardiac glycogen content. This was apparent in both free fed and fasted mice (Fig. 5). In mice, unlike rats, fasting does not increase cardiac glycogen content [26,27]. Important factors determining cardiac glycogen include glycogen synthase activity and g6p concentration. As shown in Fig. 6, g6p independent glycogen synthase activity was increased in MyHEX hearts (P<0.025). This is consistent with the greater g6p production in transgenic hearts, since g6p regulates the phosphorylation of glycogen synthase into the independent form. Fig. 6 Open in new tabDownload slide Glycogen synthase activity is increased in hearts of transgenic mice. Assays were performed in the presence (TOTAL) or absence (INDEPENDENT) of 2 mM g6p as described in Methods. Each value is the mean±S.E.M. obtained from ten or more animals. *Indicates that transgenic values were different from non-transgenic assayed at the same g6p concentration, P<0.025 by Student's t-test. Fig. 6 Open in new tabDownload slide Glycogen synthase activity is increased in hearts of transgenic mice. Assays were performed in the presence (TOTAL) or absence (INDEPENDENT) of 2 mM g6p as described in Methods. Each value is the mean±S.E.M. obtained from ten or more animals. *Indicates that transgenic values were different from non-transgenic assayed at the same g6p concentration, P<0.025 by Student's t-test. Fig. 5 Open in new tabDownload slide Glycogen content is increased in hearts of transgenic mice. Mice were either fed ad libitum or fasted for 16 h as indicated. Each value is the mean±S.E.M. obtained from five animals. *Indicates that transgenic values were different from non-transgenic values in the same feeding group, P<0.01 by Student's t-test. Fig. 5 Open in new tabDownload slide Glycogen content is increased in hearts of transgenic mice. Mice were either fed ad libitum or fasted for 16 h as indicated. Each value is the mean±S.E.M. obtained from five animals. *Indicates that transgenic values were different from non-transgenic values in the same feeding group, P<0.01 by Student's t-test. 4 Discussion To study the role of glucose phosphorylation in the regulation of cardiac fuel metabolism and pathology we produced transgenic mice that express yeast hexokinase B specifically in cardiac myocytes. The transgene increased hexokinase activity and produced no detectable cardiac pathology. In Langendorff perfused hearts overexpression of hexokinase increased glycolysis under oxidative conditions in the presence or absence of insulin, but glycolysis was not significantly altered under ischemic cardiac conditions. Accordingly, we found no protective effect of the transgene on ischemia-induced deficits in contractility. In vivo glycogen content of transgenic hearts was elevated 2-fold, which was in part due to increased g6p independent glycogen synthase activity. These results provide direct evidence in support of the contention that glucose phosphorylation under certain conditions can regulate the rate of cardiac glucose metabolism. In MyHEX mice cardiac hexokinase activity was increased by 680% in the presence of 3 mM g6p, 67% at 0.46 mM g6p and by 12% in the absence of g6p. Based on our measured values of g6p the percentage increase in our perfused hearts was close to 67% in the presence of insulin. This g6p dependent variation in percentage increase was expected since endogenous hexokinases are inhibited by g6p while yeast hexokinase is not. What was not expected was that the absolute difference between transgenic and control hexokinase activity would decrease from 26 down to 8 pmol glucose/μg tissue per h when the g6p level was reduced from 3 to 0 mM (Fig. 1). Additional experiments verified this finding (data not shown). These results imply either that the transgene was less active at low levels of g6p or that endogenous hexokinase activity was reduced in transgenic hearts. Since yeast hexokinase activity is not inhibited by lowering g6p levels [28], we favor the hypothesis that endogenous hexokinase activity is decreased in MyHEX hearts. A reduction by ∼25% in the g6p sensitive, endogenous hexokinase is consistent with the values we obtained in these assays. Autoregulation of hexokinase expression by hexokinase activity has not been examined in mammals but has been demonstrated in yeast [29]. More specific examinations of altered endogenous hexokinase RNA and protein expression in our transgenic hearts are clearly needed to confirm this hypothesis. If indeed endogenous hexokinase was reduced by transgenic yeast hexokinase expression then the functional impact of the transgene would be less. Values of g6p in transgenic and control hearts were not significantly different. This implies that the increased g6p generated by yeast hexokinase must have been rapidly incorporated into glycogen or utilized in glycolysis. This is consistent with the increase in glycogen synthase, glycogen content and glycolytic rate that we observed. Prior studies in skeletal muscle with hexokinase IV [15] have shown that overexpression increased g6p, however similar studies with hexokinase II did not result in increased g6p [30]. Glycolysis in Langendorff perfused MyHEX hearts was increased by 250 pmol glucose/min per mg heart, which was between 30 and 50% of the total glycolysis of control hearts. This demonstrated that elevated hexokinase activity enhanced glucose metabolism, at least in the absence of insulin. The effect of insulin was assessed by adding it to the perfusion buffer. Insulin widened the separation between control and transgenic glycolysis by an additional 100 pmol glucose/min per mg heart. This was probably due to increased glucose uptake, which put a greater substrate load on hexokinase. The effect of the transgene on glycolysis could not have been due to altered contractility since this parameter was indistinguishable in control and transgenic hearts. During the ischemic phase of perfusion, the difference between transgenic and control metabolism was minimal. The reduced impact of hexokinase during ischemia suggests a shift in regulation of glycolysis away from hexokinase. This is consistent with reports that glyceraldehyde 3-phosphate dehydrogenase [31] and 2-phosphofructokinase [32] are inhibited during severe cardiac ischemia. Since ischemia stimulates glycogen breakdown [33], the diminished difference in glycolysis may also be due to the larger contribution of glycogen-derived g6p to glycolysis and to the greater stores of glycogen in the transgenic heart. Glucose 6-phosphate derived from glycogen enters glycolysis independent of hexokinase and should compete with hexokinase-derived g6p for phosphorylation by phosphofructokinase. During the 60 min of ischemia, glycogen stores would have probably been depleted, as seen in other studies [34]. Oxidative phosphorylation is prevented by hypoxia, leaving glycolysis as the sole source of ATP in the ischemic heart. By overexpressing hexokinase we hoped to enhance glycolysis and thereby reduce the damaging impact of ischemia. However, we found that the transgene did not increase glycolysis during ischemia. Thus it was not surprising that ischemia was equally damaging to contractility in transgenic and control hearts. Lactate is the primary end product of glycolysis under hypoxic conditions. By acidifying the myocyte, lactate contributes to ischemic damage. Therefore we measured lactate production during ischemia and reperfusion assays. While lactate production was similar in transgenic and control hearts during ischemia, during reperfusion lactate production was significantly higher in MyHEX hearts and failed to return to basal levels. In transgenic hearts lactate production was 44% higher during reperfusion than during the basal period. This suggests that there may have been less recovery of oxidative metabolism in transgenic hearts. Alternatively, it may also have been due to the fact that glycolysis in transgenic hearts was 31% higher during reperfusion than during the basal period. A possible mechanism for increased glycolysis during reperfusion of MyHEX hearts is that ischemia stimulates recruitment of glucose transporters and increases glucose uptake [35]. The increased glucose uptake may increase the relative effect of the yeast hexokinase transgene, as we also saw during perfusion with insulin. Alterations in glycolysis are associated with several cardiac pathologies. In cardiac hypertrophy and congestive heart failure [1] glycolysis increases in the heart. Conversely, in diabetes there is a reduction in cardiac glycolysis, which may predispose the heart to diabetic cardiomyopathy. 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TI - Elevated hexokinase increases cardiac glycolysis in transgenic mice
JF - Cardiovascular Research
DO - 10.1016/S0008-6363(01)00495-3
DA - 2002-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/elevated-hexokinase-increases-cardiac-glycolysis-in-transgenic-mice-jJlK3OvH2Q
SP - 423
EP - 430
VL - 53
IS - 2
DP - DeepDyve
ER -