Cardiac Expression of Kinase-deficient 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase Inhibits Glycolysis, Promotes Hypertrophy, Impairs Myocyte Function, and Reduces Insulin Sensitivity

Abstract

Glycolysis is important to cardiac metabolism and reduced glycolysis may contribute to diabetic cardiomyopathy. To understand its role independent of diabetes or hypoxic injury, we modulated glycolysis by cardiac-specific overexpression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (kd-PFK-2). PFK-2 controls the level of fructose 2,6-bisphosphate (Fru-2,6-P2), an important regulator of glycolysis. Transgenic mice had over 2-fold reduced levels of Fru-2,6-P2. Heart weight/body weight ratio indicated mild hypertrophy. Sirius red staining for collagen was significantly increased. We observed a 2-fold elevation in glucose 6-phosphate and fructose 6-phosphate levels, whereas fructose 1,6-bisphosphate was reduced 2-fold. Pathways branching off of glycolysis above phosphofructokinase were activated as indicated by over 2-fold elevated UDP-N-acetylglucosamine and glycogen. The kd-PFK-2 transgene significantly inhibited glycolysis in perfused hearts. Insulin stimulation of metabolism and Akt phosphorylation were sharply reduced. In addition, contractility of isolated cardiomyocytes was impaired during basal and hypoxic incubations. The present study shows that cardiac overexpression of kinase-deficient PFK-2 reduces cardiac glycolysis that produced negative consequences to the heart including hypertrophy, fibrosis, and reduced cardiomyocyte function. In addition, metabolic and signaling responses to insulin were significantly decreased. Glycolysis is important to cardiac metabolism and reduced glycolysis may contribute to diabetic cardiomyopathy. To understand its role independent of diabetes or hypoxic injury, we modulated glycolysis by cardiac-specific overexpression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (kd-PFK-2). PFK-2 controls the level of fructose 2,6-bisphosphate (Fru-2,6-P2), an important regulator of glycolysis. Transgenic mice had over 2-fold reduced levels of Fru-2,6-P2. Heart weight/body weight ratio indicated mild hypertrophy. Sirius red staining for collagen was significantly increased. We observed a 2-fold elevation in glucose 6-phosphate and fructose 6-phosphate levels, whereas fructose 1,6-bisphosphate was reduced 2-fold. Pathways branching off of glycolysis above phosphofructokinase were activated as indicated by over 2-fold elevated UDP-N-acetylglucosamine and glycogen. The kd-PFK-2 transgene significantly inhibited glycolysis in perfused hearts. Insulin stimulation of metabolism and Akt phosphorylation were sharply reduced. In addition, contractility of isolated cardiomyocytes was impaired during basal and hypoxic incubations. The present study shows that cardiac overexpression of kinase-deficient PFK-2 reduces cardiac glycolysis that produced negative consequences to the heart including hypertrophy, fibrosis, and reduced cardiomyocyte function. In addition, metabolic and signaling responses to insulin were significantly decreased. An important role of glycolysis in heart failure has been appreciated for over 35 years because Bishop and Altrud (1Bishop S.P. Altschuld R.A. Am. J. Physiol. 1970; 218: 153-159Crossref PubMed Scopus (185) Google Scholar) reported that glycolytic metabolism is increased in cardiac hypertrophy and congestive heart failure. Glycolysis is also critical to cardiac survival in hypoxia (2Neely J.R. Rovetto M.J. Methods Enzymol. 1975; 39: 43-60Crossref PubMed Scopus (135) Google Scholar), because glycolysis is the sole source of ATP production in the absence of oxygen. In diabetes there is a chronic reduction in cardiac glycolytic capacity (3Avogaro A. Nosadini R. Doria A. Fioretto P. Velussi M. Vigorito C. Sacca L. Toffolo G. Cobelli C. Trevisan R. Am. J. Physiol. 1990; 258: E606-E618PubMed Google Scholar, 4Stanley W.C. Lopaschuk G.D. McCormack J.G. Cardiovasc. Res. 1997; 34: 25-33Crossref PubMed Scopus (407) Google Scholar). Diabetics are prone to heart failure (5Rubler S. Dlugash J. Yuceoglu Y.Z. Kumral T. Branwood A.W. Grishman A. Am. J. Cardiol. 1972; 30: 595-602Abstract Full Text PDF PubMed Scopus (1371) Google Scholar) and they suffer more damage following an infarction (6Soler N.G. Pentecost B.L. Bennett M.A. FitzGerald M.G. Lamb P. Malins J.M. Lancet. 1974; 1: 475-477Abstract PubMed Scopus (114) Google Scholar). It has been proposed that reduced cardiac glycolysis contributes to the heart failure and cardiac damage often suffered by diabetics (7Belke D.D. Larsen T.S. Gibbs E.M. Severson D.L. Am. J. Physiol. 2000; 279: E1104-E1113Crossref PubMed Google Scholar, 8Abel E.D. Kaulbach H.C. Tian R. Hopkins J.C. Duffy J. Doetschman T. Minnemann T. Boers M.E. Hadro E. Oberste-Berghaus C. Quist W. Lowell B.B. Ingwall J.S. Kahn B.B. J. Clin. Investig. 1999; 104: 1703-1714Crossref PubMed Scopus (272) Google Scholar). The significance of cardiac glycolysis has led to careful analysis of its regulation. Metabolic flux analysis (9Kashiwaya Y. Sato K. Tsuchiya N. Thomas S. Fell D.A. Veech R.L. Passonneau J.V. J. Biol. Chem. 1994; 269: 25502-25514Abstract Full Text PDF PubMed Google Scholar) demonstrates that control is shared by several reactions. One of the most critical reactions is carried out by 6-phosphofructo-1-kinase (PFK-1) 1The abbreviations used are: PKF-1, 6-phosphofructo-1-kinase; PFK-2, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid; ANOVA, analysis of variance. (10Newsholme E.A. Randle P.J. Biochem. J. 1964; 93: 641-651Crossref PubMed Scopus (104) Google Scholar, 11Hue L. Beauloye C. Marsin A.S. Bertrand L. Horman S. Rider M.H. J. Mol. Cell Cardiol. 2002; 34: 1091-1097Abstract Full Text PDF PubMed Google Scholar), which catalyzes the phosphorylation of fructose 6-phosphate (Fru-6-P) to fructose 1,6-bisphosphate (Fru-1,6-P2). Fru-6-P is in equilibrium with glucose 6-phosphate (Glc-6-P) and these two sugars initiate glycogen synthesis, the hexosamine pathway, and the hexose monophosphate shunt. Thus PFK-1 not only has an important role in regulating glycolysis but by controlling the metabolism of Fru-6-P, PFK-1 can also modulate several important reactions branching off of glycolysis. PFK-1 activity is tightly controlled and the most important positive regulator is fructose 2,6-bisphosphate (Fru-2,6-P2). Until recently the sole function of Fru-2,6-P2 was thought to be regulation of PFK-1. However, Wu et al. (12Wu C. Okar D.A. Stoeckman A.K. Peng L.J. Herrera A.H. Herrera J.E. Towle H.C. Lange A.J. Endocrinology. 2004; 145: 650-658Crossref PubMed Scopus (39) Google Scholar) has recently provided evidence that liver Fru-2,6-P2 may directly or indirectly modulate gene expression and insulin signaling. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) catalyzes the synthesis and degradation of Fru-2,6-P2. PFK-2 is in turn regulated by a complex network of kinases, phosphatases, and metabolites (13Okar D.A. Manzano A. Navarro-Sabate A. Riera L. Bartrons R. Lange A.J. Trends Biochem. Sci. 2001; 26: 30-35Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). In this study we have used a cardiac-specific transgene for a mutant form of PFK-2 to directly reduce cardiac Fru-2,6-P2 and cardiac glycolysis. Our results demonstrate that impaired glycolysis produces several indicators of cardiac pathology, which are reminiscent of the cardiomyopathy seen in diabetes. In addition, reduced Fru-2,6-P2 produces a state of cardiac insulin resistance. The bifunctional enzyme PFK-2 controls levels of Fru-2,6-P2 by catalyzing two opposing reactions: Fru-6-P + ATP → Fru-2,6-P2 + ADP and Fru-2,6-P2 → Fru-6-P + Pi. Development and in vivo analysis of the kinase-deficient PFK-2 (kd-PFK-2) mutant has been previously described (12Wu C. Okar D.A. Stoeckman A.K. Peng L.J. Herrera A.H. Herrera J.E. Towle H.C. Lange A.J. Endocrinology. 2004; 145: 650-658Crossref PubMed Scopus (39) Google Scholar, 14Wu C.D. Okar D.A. Peng L. Lange A.J. Diabetes. 2002; 51: A319Google Scholar, 15Kurland I.J. El Maghrabi M.R. Correia J.J. Pilkis S.J. J. Biol. Chem. 1992; 267: 4416-4423Abstract Full Text PDF PubMed Google Scholar). Cardiac-specific expression was obtained by ligating a 1.6-kb KpnI/HindIII fragment of kd-PFK-2 (12Wu C. Okar D.A. Stoeckman A.K. Peng L.J. Herrera A.H. Herrera J.E. Towle H.C. Lange A.J. Endocrinology. 2004; 145: 650-658Crossref PubMed Scopus (39) Google Scholar, 14Wu C.D. Okar D.A. Peng L. Lange A.J. Diabetes. 2002; 51: A319Google Scholar) behind the α-myosin heavy chain promoter (16Gulick J. Subramaniam A. Neumann J. Robbins J. J. Biol. Chem. 1991; 266: 9180-9185Abstract Full Text PDF PubMed Google Scholar). The transgene was designated Mb (for the myosin heavy chain promoter and the bisphosphatase active kd-PFK-2 gene). Transgenic mice were produced on the FVB background and maintained as heterozygotes by breeding to FVB mice. Standard embryo microinjection procedures were used for producing transgenic animals. All procedures conformed to the Guide for the Care and Use of Laboratory Animals (46National Institutes of Health Guide for the Care and Use of Laboratory Animals. National Institutes of Health, Bethesda, MD1996Google Scholar) and were approved by the USDA certified institutional animal care committee. Mice aged 90-120 days were used in the following studies. Cardiac Fru-2,6-P2 Content—Fru-2,6-P2 was extracted from fresh heart tissue in 10-20 volumes of 50 mm NaOH and kept at 80 °C for 5 min. The extract was cooled and neutralized at 0 °C by addition of ice-cold 1 m acetic acid in the presence of 20 mm HEPES. After centrifugation at 8,000 × g for 10 min, supernatant was collected and assayed for Fru-2,6-P2 by the 6-phosphofructo-1-kinase activation method (17Van Schaftingen E. Lederer B. Bartrons R. Hers H.G. Eur.J. Biochem. 1982; 129: 191-195Crossref PubMed Scopus (544) Google Scholar). Northern Blots—Expression and tissue specificity of the transgene were determined by measuring RNA. RNA for Northern blots was prepared with RNAzol as we have previously described from heart, liver, lung, kidney, skeletal muscle, and brain of transgenic and normal mice (18Liang Q. Donthi R.V. Kralik P.M. Epstein P.N. Cardiovasc. Res. 2002; 53: 423-430Crossref PubMed Scopus (20) Google Scholar). A 1.7-kb HindIII/SalI fragment of the transgene was labeled with 32P by random oligonucleotide priming and used as probe. This probe had little detectable cross-reactivity with any other mouse messenger RNA. PFK-2 Expression Analysis—PFK-2 protein was analyzed by Western blot as reported earlier (19Wu C. Okar D.A. Newgard C.B. Lange A.J. J. Clin. Investig. 2001; 107: 91-98Crossref PubMed Scopus (70) Google Scholar). In brief, PFK-2 was extracted from fresh hearts by homogenizing in buffer containing 20 mm TES, 1 mm dithiothreitol, 100 mm KCl, 5 mm EDTA, 5 mm EGTA (pH 7.8), 1.2 mm phenylmethylsulfonyl fluoride, 2.5 mg/liter leupeptin. After precipitation with 40% PEG, the pellet was dissolved in buffer containing 20 mm TES, 1 mm dithiothreitol, 100 mm KCl, 0.1 mm EDTA (pH 7.5), 0.5 mm phenylmethylsulfonyl fluoride, 2.5 mg/liter leupeptin. The concentration of extracted protein was measured by the BCA method (Pierce Chemical Co.). Equal amounts of extracted protein (100 μg) were used for Western blots. Rabbit anti-rat liver PFK-2 serum was used as primary antibody at a 1:1,000 dilution. The bands were visualized using horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and ECL (Amersham Biosciences). Hearts were homogenized in 1 m ice-cold perchloric acid and centrifuged. The supernatants were neutralized with 2 m KHCO3. The supernatant from neutralized tissue extracts was used for estimation of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate by the fluorometric method (20Lowry O.H. Passonneau J.V. A Flexible System of Enzymatic Analysis. Academic Press, New York1993: 111-228Google Scholar). The hearts were quickly removed, blotted dry, and snap frozen in liquid nitrogen. The frozen tissue was weighed, homogenized in 9 volumes of 30% KOH at 0 and at °C for min. The glycogen was with and and in 0.1 m buffer (pH 10 of glycogen was to glucose by the addition of 50 of in 100 m was measured by fluorometric using and as we reported earlier (18Liang Q. Donthi R.V. Kralik P.M. Epstein P.N. Cardiovasc. Res. 2002; 53: 423-430Crossref PubMed Scopus (20) Google Scholar). of UDP-N-acetylglucosamine were measured as described previously G. J. E.D. D.A. Diabetes. 2000; PubMed Scopus Google Scholar). Hearts from control and transgenic mice was homogenized at °C in volumes of mm perchloric acid and × g for at was extracted from the supernatants with 2 volumes of The was and liquid was on a × with a from 80 mm (pH to mm (pH over at a of 1 levels were by at with and to protein were carried out as previously described by (18Liang Q. Donthi R.V. Kralik P.M. Epstein P.N. Cardiovasc. Res. 2002; 53: 423-430Crossref PubMed Scopus (20) Google Scholar, Q. Donthi R.V. Kralik P.M. Epstein P.N. Diabetes. 2002; 51: PubMed Scopus Google Scholar, in Scholar). The heart was from the and the was to the The heart was perfused at 2 with buffer of mm 20 mm mm KCl, 1.2 mm 1.2 mm mm 5 mm the buffer was with that maintained a of and was maintained at The heart was the at on the of glycolysis was determined for the by 50 in the presence of of was maintained at 2 and contractility was during the as described previously (18Liang Q. Donthi R.V. Kralik P.M. Epstein P.N. Cardiovasc. Res. 2002; 53: 423-430Crossref PubMed Scopus (20) Google Scholar, Q. Donthi R.V. Kralik P.M. Epstein P.N. Diabetes. 2002; 51: PubMed Scopus Google Scholar). Glycolysis was measured using as the as we have previously reported Q. Donthi R.V. Kralik P.M. Epstein P.N. Diabetes. 2002; 51: PubMed Scopus Google Scholar). produced was measured as we have described (18Liang Q. Donthi R.V. Kralik P.M. Epstein P.N. Cardiovasc. Res. 2002; 53: 423-430Crossref PubMed Scopus (20) Google Scholar). produced from during was determined by A of cardiac and of were to that were containing 2 of were for at The was and was for from was assayed in background were determined by the on buffer that had not the was also measured in using concentration was measured in using (18Liang Q. Donthi R.V. Kralik P.M. Epstein P.N. Cardiovasc. Res. 2002; 53: 423-430Crossref PubMed Scopus (20) Google Scholar). in the heart was determined as described previously G. R. A. J. C. 2001; PubMed Scopus Google Scholar). heart were on and with a of acid containing Sirius red for staining collagen and which were kept in the and for min. were with and with The were and the was by a using a of on the of the collagen The was a of 0 for mild 1 for and 2 for were isolated and contractility was measured as we have described G. J. Epstein P.N. Diabetes. PubMed Scopus Google Scholar). the were in an in buffer for 2 cardiomyocytes were perfused with buffer for and maintained and assayed in a for 2 were at 1 using to the of were using a to the G. J. Epstein P.N. Diabetes. PubMed Scopus Google Scholar). Cell and were using the following to and of and Hearts were frozen in liquid the basal or following the of insulin extracts were prepared in buffer containing 20 mm (pH 7.5), mm 1 mm EDTA, 1 mm 2.5 mm 1 mm 1 mm 1 and 1 mm phenylmethylsulfonyl The were and protein concentration of the supernatants was measured by the BCA method to 20 were analyzed by Western blot as previously R.V. B. A. Cardiovasc. 2000; PubMed Scopus Google Scholar, B. Donthi R.V. A. Cardiovasc. 2001; PubMed Scopus Google Scholar). In brief, protein were on and The was with and with antibody or Akt antibody from Cell The were and with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and bands were visualized using ECL (Amersham Biosciences). of were by for and were by The level of significance was Transgenic designated Mb was for cardiac expression of a kinase-deficient mutant of PFK-2 (kd-PFK-2). transgenic were produced and these were designated as Mb of and produced The Northern blot in that the transgene was produced in the heart and was in FVB mice. of kd-PFK-2 protein was by Western blot with PFK-2 antibody indicated that transgenic and had levels of which were to and that of control mice. and were most and were used as for Hearts from and mice had only 40% as Fru-2,6-P2 as measured in control FVB hearts to have kd-PFK-2 protein was used for is reduced in transgenic that the of and are significantly from FVB The of used are: and Fru-2,6-P2 be to reduce activity and have a on of cardiac of several metabolites were measured in hearts of FVB and transgenic mice maintained in results are in and Fru-6-P levels were elevated more whereas the fructose 1,6-bisphosphate level was 2-fold in transgenic mice. with the elevation in the levels, we observed that glycogen was increased by more 2-fold in the transgenic mice. The of the hexosamine is in we a 2-fold elevation in This to the increased of Fru-6-P, which may the hexosamine of glycolytic metabolites in FVB and hearts The are for at mice in The levels of glucose 6-phosphate, fructose 6-phosphate, and were significantly elevated fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were significantly in in a of on Cardiac and Insulin of the Mb transgene on cardiac glycolysis was in hearts by measuring of glucose and production of is from the metabolism of by and of the glycolytic R.L. Lopaschuk G.D. J. 2000; PubMed Scopus Google Scholar, in Scholar). in from and were significantly reduced in transgenic hearts. was at mm and the was and in and FVB hearts. We a reduction in contractility in the mice hearts of not but this not significance in more 10 mice After addition of insulin to the the in metabolism transgenic and control hearts because of a to insulin in transgenic hearts and the of the with glucose was increased by in FVB hearts but only by in transgenic hearts was by in FVB hearts but only by in hearts The in insulin were To the in insulin glucose we measured phosphorylation of Hearts were perfused with or and protein was analyzed by Western The results are in In the absence of insulin there was in the expression levels of Akt any seen in the form of insulin phosphorylation of Akt was increased by in FVB hearts but only by in transgenic hearts. indicated that the was for FVB but not significance for hearts results that the kd-PFK-2 protein reduced insulin in other glucose in Transgenic hearts of mice of a but in heart weight of as in However, were not from control FVB mice. The heart weight/body weight ratio was significantly increased in and mice. The increased heart weight was by a in cardiac in mice as in and weight of and FVB mice The are hearts mild for for for for for for FVB in a To the of impaired glycolysis on cardiomyocyte were isolated from and FVB hearts and with or contractility was measured and the results are in significantly reduced contractility with FVB control as by and and hypoxic In addition, more to FVB in glucose metabolism cardiac hypertrophy but is not this in metabolism is an or To directly the role of glucose metabolism in cardiac function we transgenic mice with reduced cardiac glycolysis. of mice were produced with the kinase-deficient PFK-2 transgene which reduced cardiac levels of a regulator of PFK-1. This in in cardiac of which were and glycolysis was reduced and in the concentration of glycolytic and metabolites of that off from glycolysis. The transgene produced cardiac as by increased cardiac increased fibrosis, and cardiomyocyte the transgene cardiac to Cardiac metabolites and of PFK-1 were in transgenic hearts because of of PFK-1. The of and Fru-6-P were increased and is the for glycogen production and glycogen E.A. Physiol. PubMed Scopus Google Scholar). in a in glycogen of transgenic hearts. Fru-6-P is the of the hexosamine and the of this pathway, was increased 2-fold. in were not a of of the transgene to the reduction in the glycolytic flux by the Mb We that because we previously observed in cardiac glycogen we in the heart (18Liang Q. Donthi R.V. Kralik P.M. Epstein P.N. Cardiovasc. Res. 2002; 53: 423-430Crossref PubMed Scopus (20) Google Scholar). the kd-PFK-2 also produced a 2-fold in A. and P. N. The transgene had the on glycolysis as the Mb increased glycolysis (18Liang Q. Donthi R.V. Kralik P.M. Epstein P.N. Cardiovasc. Res. 2002; 53: 423-430Crossref PubMed Scopus (20) Google Scholar). However, production and the kd-PFK-2 protein increased results that important that from glycolysis are more directly by in the concentration of glycolytic they are by in glycolytic is the in which there is a chronic of cardiac glycolysis W.C. Lopaschuk G.D. McCormack J.G. Cardiovasc. Res. 1997; 34: 25-33Crossref PubMed Scopus (407) Google Scholar). In diabetes glycolysis is reduced because insulin in the of glucose metabolism including glucose and Our transgenic mice also had a reduction in glycolysis and they most of the of diabetic cardiomyopathy. hypertrophy M. Am. J. Cardiol. 1991; Full Text PDF PubMed Scopus Google Scholar) and increased A. M. B. G. 1994; PubMed Scopus Google Scholar) are in diabetic hearts. were seen in transgenic mice as a or in heart to weight in and was as increased Sirius red were not a function of cardiac overexpression of a because we have not seen these with any of the other cardiac-specific we have described (18Liang Q. Donthi R.V. Kralik P.M. 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All of these that impaired glycolysis contributes to the cardiac in Insulin was an but an of expression of the kd-PFK-2 was as a reduction in glycolytic to It was also seen in a reduction in phosphorylation of Akt produced by insulin Fru-2,6-P2 has not been to have a on cardiac insulin because Fru-2,6-P2 has not been directly However, Wu et al. (12Wu C. Okar D.A. Stoeckman A.K. Peng L.J. Herrera A.H. Herrera J.E. Towle H.C. Lange A.J. Endocrinology. 2004; 145: 650-658Crossref PubMed Scopus (39) Google Scholar) has recently that of liver Fru-2,6-P2 produces in the level of Akt phosphorylation is a to the metabolic of insulin this may the to insulin for glycolysis. The primary of insulin and reduced Akt phosphorylation is not One is increased hexosamine flux M. R. N. L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, D.A. E.D. Diabetes. PubMed Google Scholar), which was indicated in this by the 2-fold in levels of are to by the levels of of and by K. L. Sci. S. A. 2002; PubMed Scopus Google Scholar, D.A. M. Sci. S. A. 2002; PubMed Scopus Google Scholar, P.M. E. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). has been recently that elevated by can activation of Akt K. L. Sci. S. A. 2002; PubMed Scopus Google Scholar). Thus for insulin in hearts from the of glycolysis at to increased flux the hexosamine or of Fru-2,6-P2 may contribute to the of insulin In we have in the present study that cardiac Fru-2,6-P2 produces in cardiac metabolism and In perfused hearts glycolysis and insulin were decreased. In vivo transgenic hearts an elevation of glycolytic of which led to elevation of glycogen and The elevation in may have to the of insulin resistance. we that reduced glycolysis was by mild hypertrophy and of In with impaired cardiomyocyte contractility these results that a in glycolysis is an important of diabetic cardiomyopathy.