David A. Okar

PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate

Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated gluconeogenesis in the liver. This discovery has led to the realization that this compound plays a significant role in directing carbohydrate fluxes in all eukaryotes. Biophysical studies of the enzyme that both synthesizes and degrades this biofactor have yielded insight into its molecular enzymology. Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential in the treatment of diabetes.

Overexpression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase in mouse liver lowers blood glucose by suppressing hepatic glucose production.

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is an important regulatory enzyme of glucose metabolism. By controlling the level of fructose-2,6-bisphosphate, an allosteric activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase regulates hepatic glucose output. We studied the effects of adenovirus-mediated overexpression of this enzyme on hepatic glucose metabolism in normal or diabetic mice. These animals were treated with virus encoding either wild-type or bisphosphatase activity-deficient 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase. Seven days after virus injection, hepatic fructose-2,6-bisphosphate levels increased significantly in both normal and diabetic mice, with larger increases observed in animals with overexpression of the mutant enzyme. Blood glucose levels in normal mice overexpressing either enzyme were lowered, accompanied by increased plasma lactate, triglycerides, and FFAs. Blood glucose levels were markedly reduced in diabetic mice overexpressing the wild-type enzyme, and still more so in mice overexpressing the mutant form of the enzyme. The lower blood glucose levels in diabetic mice were accompanied by partially normalized plasma triglycerides and FFAs, increased plasma lactate, and increased liver glycogen levels, relative to diabetic mice treated with a control adenovirus. Our findings underscore the critical role played by hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in control of fuel homeostasis and suggest that this enzyme may be considered as a therapeutic target in diabetes.

Reduction of hepatic glucose production as a therapeutic target in the treatment of diabetes.

There has been an alarming increase in the population diagnosed with diabetes worldwide. Although there is an ongoing debate as to the role of liver in the pathogenesis of diabetes, reduction of hepatic glucose production has been targeted as a strategy for diabetes treatment. Indeed, reduction of hepatic glucose production can be achieved through modulation of both hepatic and extra-hepatic targets. This review describes the role of the liver in the control of glucose homeostasis. Gluconeogenesis and glycogenolysis are pathways for glucose production, whereas glycolysis and glycogenesis are pathways for glucose utilization/storage. At the biochemical and molecular level, the metabolic and regulatory enzymes integrate hormonal and nutritional signals and regulate glucose flux in the liver. Modulating either activities of or gene expression of these metabolic enzymes can control hepatic glucose production. Dysfunction of one or several enzyme(s) due to insulin deficiency or resistance results in increases in fluxes of glycogenolysis and gluconeogenesis and/or decreases in fluxes of glycolysis and glycogenesis, which thereby lead to glucose generation exceeding glucose consumption/disposal, as well as dysregulation of lipid metabolism. Activation of enzymes that promote glucose utilization/storage and/or inhibition of enzymes that reduce glucose generation achieve reduction of hepatic glucose production, and hence lower levels of plasma glucose in diabetes. This is also beneficial for the correction of dyslipidemia. Therefore, many enzymes are viable therapeutic targets for diabetes.

Adenovirus-mediated overexpression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in gluconeogenic rat hepatoma cells. Paradoxical effect on Fru-2,6-P2 levels.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been postulated to be a metabolic signaling enzyme, which acts as a switch between glycolysis and gluconeogenesis in mammalian liver by regulating the level of fructose 2,6-bisphosphate. The effect of overexpressing the bifunctional enzyme was studied in FAO cells transduced with recombinant adenoviral constructs of either the wild-type enzyme or a double mutant that has no bisphosphatase activity or protein kinase phosphorylation site. With both constructs, the mRNA and protein were overexpressed by 150- and 40-fold, respectively. Addition of cAMP to cells overexpressing the wild-type enzyme increased the S0.5 for fructose 6-phosphate of the kinase by 1.5-fold but had no effect on the overexpressed double mutant. When the wild-type enzyme was overexpressed, there was a decrease in fructose 2,6-bisphosphate levels, even though 6-phosphofructo-2-kinase maximal activity increased more than 22-fold and was in excess of fructose-2,6-bisphosphatase maximal activity. The kinase:bisphosphatase maximal activity ratio was decreased, indicating that the overexpressed enzyme was phosphorylated by cAMP-dependent protein kinase. Overexpression of the double mutant resulted in a 28-fold increase in kinase maximal activity and a 3-4-fold increase in fructose 2,6-bisphosphate levels. Overexpression of this form inhibited the rate of glucose production from dihydroxyacetone by 90% and stimulated the rate of lactate plus pyruvate production by 200%. In contrast, overexpression of the wild-type enzyme enhanced glucose production and inhibited lactate plus pyruvate production. These results provide direct support for fructose 2,6-bisphosphate as a regulator of gluconeogenic/glycolytic pathway flux and suggest that regulation of bifunctional enzyme activities by covalent modification is more important than the amount of the protein.

Glutamate-Glutamine Cycle and Anaplerosis

Most excitatory neuronal activity in the brain is sustained by cycling the most prominent neurotransmitter, glutamate, and its metabolic product glutamine between neurons and astrocytes. Losses of these compounds during cycling are replenished by anaplerosis that primarily takes place in astrocytes. This chapter summarizes the methodological considerations in carbon tracer studies used to measure glutamate-glutamine cycling and anaplerosis, together with the estimated rates for these metabolic fluxes in the living brain. Questions regarding neuronal anaplerosis and the stoichiometry of the glutamate-glutamine cycle are examined. The possibility of a functional link between anaplerosis and the glutamate-glutamine cycle is discussed.

Hormonal FM: what's the frequency?

Recently, Peter Bergsten and co-workers measured slow oscillations (∼0.2 min−1) in the oxygen tension (pO2) and cytosolic calcium concentration {[Ca2+]} that correlated with the frequency of pulsatile insulin secretion into the portal vein [1xPrimary in vivo oscillations of metabolism in the pancreas. Bergsten, P. et al. Diabetes. 2002; 51: 699–703Crossref | PubMedSee all References[1]. Given the roles of [Ca2+] and pO2 in stimulating insulin secretion, these data provide strong evidence that secretion from individual islets, located at various distances from the portal vein, is coordinated throughout the pancreas. The frequency signature can only be maintained if the hormone moves through the pancreas quickly; the authors estimated the passage rate of insulin by measuring that of glucose. Although these studies targeted the portal vein, others have reported oscillations of approximately the same frequency in human plasma insulin [2xCyclic oscillations of basal plasma glucose and insulin concentrations in human beings. Lang, D.A. et al. N. Engl. J. Med. 1979; 301: 1023–1027Crossref | PubMedSee all References[2]. Similar to other blood-borne signaling molecules, the pulsatile nature of plasma insulin is important for promotion of the hormones action.Bergstens results raise many questions regarding the mechanisms, not only for coupling the fluctuating levels of cytosolic signals to the exocytosis of insulin, but also for coordinating secretion from individual islets. Citing the lack of synchronous electrical oscillations between islets within the same pancreas, the authors suggest that the rapid passage of glucose through the pancreas implies that coordination might be achieved by a blood-borne agent. The relationships between cellular carbohydrate metabolism, cytosolic [Ca2+] and secretion of insulin, are well studied. However, that these parameters also oscillate in response to glucose in cultured cells is less well appreciated. These in vivo findings suggest that the pulsatile nature of signaling molecules – be they blood-borne compounds (e.g. insulin and non-esterified fatty acids) or intracellular signals {e.g. [Ca2+] and ADP/ATP} – should be considered when describing the machinery that uses them to regulate fuel metabolism. The relevance of this work to the highly popular metabolic clamp approach, in which the blood and/or tissue content of select molecules is held constant, remains to be determined. Even so, the metabolic clamp has provided significant advances in our understanding of in vivo regulatory mechanisms.Although not discussed by the authors, the in vivo oscillation of insulin levels raises the possibility that some portion of the blood-borne insulin signal is carried by the frequency of the pulses, not simply by the level of insulin. The ability of extra-pancreatic tissues to receive this putative frequency-modulated (FM) signal has not been sufficiently investigated. Even so, oscillations of fructose-1,6-bisphosphate (F16P2) and ADP/ATP have been reported in skeletal muscle extracts [3xFructose-2,6-bisphosphate and glycolytic oscillations in skeletal muscle extracts. Tornheim, K. J. Biol. Chem. 1988; 263: 2619–2624PubMedSee all References[3]. If such an FM signaling system is actually functioning in vivo, then it might constitute another regulatory mechanism for coordinating whole-body fuel metabolism.

Feed a fever and starve a tumor

Genotoxic damage elicits a number of cellular responses that are essential for preserving the integrity of the genome, including apoptotic cell death, cell-cycle arrest and DNA repair. Aberrant responses appear to play a role in the development of cancers, as well as the acquisition of resistance to genotoxic treatments such as radio- and chemotherapies. The mechanisms by which DNA damage can elicit programmed cell death are not well understood, but it is clear that malfunction and adaptation of these intracellular signaling pathways are associated with tumorigenesis and therapeutic resistance, respectively.Zhou et al. have used subtractive suppressive hybridization to screen changes in lymphocyte (FL5.Bcl-xL) gene expression in response to exposure to cisplatin, a genotoxic chemotherapeutic [1xGenotoxic exposure is associated with alterations in glucose uptake and metabolism. Zhou, R. et al. Cancer Res. 2002; 62: 3515–3520PubMedSee all References][1]. They noted significant downregulation of the gene expression for cellular bioenergetic pathways, in particular, the glucose transporters (GLUT1 and GLUT3), hexokinases (HK1 and HK2), and 6-phosphofructo-1-kinase (PFK-1). The screening results were verified by northern blots and, in the case of GLUT 1 and 3, western blots as well. The authors then compared the effects of etoposide, another DNA-damaging agent, and vincristine, a non-genotoxic chemotherapeutic, on the expression of these metabolic enzymes. The results indicate that reduced expression was related to genotoxicity, as vincristine was much less effective at suppressing expression of these genes. Even so, all three compounds coordinately reduced the rates of glycolysis and oxygen consumption, suggesting a functional linkage between cytosolic and mitochondrial metabolism that can stimulate redistribution of cytochrome c, precipitating cell-cycle arrest and subsequent apoptosis. The authors conclude that changes in glucose metabolism are a common pathway for the action of genotoxic and non-genotoxic chemotherapeutics.Apparently, vincristine downregulates the bioenergetic pathways by a mechanism that does not involve the alteration of gene expression. Because of its central role in the regulation of PFK-1 activity, the authors suggested that 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBP-2) could be involved through control of the cellular content of fructose-2,6-bisphosphate (F2,6P), as the relative rates of the enzymes opposing activities are determined by post-translational modification (Ser/Thr phosphorylation). These speculations correlate well with the earlier report from Minchenko et al. [2xHypoxia-inducible factor-1-mediated expression of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) gene. Its possible role in the Warburg effect. Minchenko, A. et al. J. Biol. Chem. 2002; 277: 6183–6187Crossref | PubMed | Scopus (199)See all References][2] that transformation of tumor cells is associated with a change in the relative expression of isoforms of PFK-2/FBP-2, switching to an isozyme that can maintain higher levels of F2,6P and thus support higher rates of glycolysis.The work points to a fundamental role for energy metabolism in the control of cell cycling and eventual apoptosis. This crucial role of energy metabolism in the biochemical pathways involved in the development and progression of cancer could open up new therapeutic avenues, perhaps attempting to diminish cellular fuel metabolism by targeting metabolic regulators to elicit tumor-specific cell death.