Integration of flux measurements to resolve changes in anabolic and catabolic metabolism in cardiac myocytes

Abstract

Although ancillary pathways of glucose metabolism are critical for synthesizing cellular building blocks and modulating stress responses, how they are regulated remains unclear. In the present study, we used radiometric glycolysis assays, [13C6]-glucose isotope tracing, and extracellular flux analysis to understand how phosphofructokinase (PFK)-mediated changes in glycolysis regulate glucose carbon partitioning into catabolic and anabolic pathways. Expression of kinase-deficient or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat neonatal cardiomyocytes co-ordinately regulated glycolytic rate and lactate production. Nevertheless, in all groups, >40% of glucose consumed by the cells was unaccounted for via catabolism to pyruvate, which suggests entry of glucose carbons into ancillary pathways branching from metabolites formed in the preparatory phase of glycolysis. Analysis of 13C fractional enrichment patterns suggests that PFK activity regulates glucose carbon incorporation directly into the ribose and the glycerol moieties of purines and phospholipids, respectively. Pyrimidines, UDP-N-acetylhexosamine, and the fatty acyl chains of phosphatidylinositol and triglycerides showed lower 13C incorporation under conditions of high PFK activity; the isotopologue 13C enrichment pattern of each metabolite indicated limitations in mitochondria-engendered aspartate, acetyl CoA and fatty acids. Consistent with this notion, high glycolytic rate diminished mitochondrial activity and the coupling of glycolysis to glucose oxidation. These findings suggest that a major portion of intracellular glucose in cardiac myocytes is apportioned for ancillary biosynthetic reactions and that PFK co-ordinates the activities of the pentose phosphate, hexosamine biosynthetic, and glycerolipid synthesis pathways by directly modulating glycolytic intermediate entry into auxiliary glucose metabolism pathways and by indirectly regulating mitochondrial cataplerosis.

Cardiac Insulin Signaling Regulates Glycolysis Through Phosphofructokinase 2 Content and Activity

“…In the heart, phosphofructokinase 1 (PFK‐1) is the rate‐limiting enzyme of glycolysis 12, 13. The bifunctional enzyme, 6‐phosphofructo‐2‐kinase/fructose 2,6‐bisphosphatase (PFK‐2), either produces or consumes the potent allosteric activator of PFK‐1, fructose 2,6‐bisphosphate.…”

Cardiac mesenchymal cells from diabetic mice are ineffective for cell therapy-mediated myocardial repair

“…Glycolytic flux was determined by analyzing the conversion of [5- 3 H]-glucose to [ 3 H] 2 O, as described previously [19, 56, 57]. …”

“…Lipid and nucleotide spectra were acquired using a hybrid linear ion trap–FT-ICR mass spectrometer (Finnigan LTQ FT, Thermo Electron), as described previously [19, 57]. The FT-ICR MS spectra were exported as exact mass lists into a spreadsheet file using QualBrowser 2.0 (Thermo Electron).…”

Metabolic regulation of myocardial adaptation to exercise.

“…The three salient findings from our studies, related to the heart, are: (1) that periodic decreases in myocardial glycolytic activity are sufficient to promote physiologic cardiac growth 304 ; (2) that maintenance of metabolic flexibility in the heart is essential for mitochondrial health 304 ; and (3) that glycolytic activity in cardiomyocytes coordinates the biosynthesis of nucleotides, nucleotide sugars, and glycero(phospho)lipids. 305 Our findings have several implications, germane to both the basic understanding of cardiac remodeling as well as future targets for therapy.…”

“…We found that the synthesis of abundant phospholipids in the cardiomyocyte, such as phosphatidylcholine, phosphotidylethanolamine, and phosphatidylinositol, 308 appear to be regulated strongly by PFK-mediated changes in glycolytic activity. 305 Higher levels of these phospholipids would be required for de novo expansion of cellular membranes as well as for maintaining larger myocytes, in response to stressors such as exercise.…”