II) Level of fructose-6-P

Fructose-6-P is another control point of the glycolytic/gluconeogenic pathways.  Its concentration is the result of balance of phosphoglucose isomerase, phosphofructose kinase 1 and fructose-1, 6-biphosphatase.  Fructokinase, which phosphorylates fructose at the C1 position, is potentially an important contributor in high fructose diet. (Henry and Crapo, 1992)Fructose-6-P is an important control point for another reason.  F-6-P is also a substrate of the non-oxidative branch of the pentose cycle.  Its concentration is supposed to be in equilibrium with that of triose-P through the transketolase/transaldolase reactions. Xylulose-5-P, an intermediate of the pentose cycle reactions, is known to regulate the production of F2, 6P, thereby regulating the direction of the glycolytic/gluconeogenic flux.  At the present time there are no tracer method for the study of these interconversions. However, U¹³C-fructose is a potent useful tracer of these pathways.  Methods will also be developed to assay for concentrations of these substrates.

3) Level of Triose-P

Triose phosphate is another key substrate pool for the regulation of glucose metabolism. It is both an intermediate of glycolysis and gluconeogenesis. In addition, it serves to provide glycerol for phosholipid and triglyceride synthesis.  Dihydroxyacetone-P and glyceraldehyde-3-P are two essential components of this pool and are in equilibrium with fructose 1, 6-diphosphate through the enzyme aldolase.  This equilibrium requires an equal amount of dihydroxyacetone-P and glyceraldehyde-3-P. (Katz et al., 1966) Thus triose-p isomerase which equalizes the concentrations of the triose-P is important to the process of gluconeogenesis. The removal of glyceraldehyde-3-P by the reactions glyceraldehyde-3-phosphate dehydrogenase is the first step for the formation of pyruvate and lactate, and the reversible reaction of glycerol-3-P dehydrogenase on dihydroxyacetone-P leads to the formation of glycerol-3-P for triglyceride and phospholipid synthesis.  Pyruvate kinase which converts phosphoenolpyruvate to pyruvate is the last and irreversible step of the glycolytic process. This reaction is probably a determining factor of glycolysis.  The metabolic pathways arising from triose-P can be studied using U¹³C-glycerol or U¹³C-lactate (3-carbon precursors).

B.  Oxidative and Non-Oxidative Pathways of the Pentose Cycle

attention because of their role in lipogenesis (in obesity) and nucleic acid synthesis (providing ribose and deoxyribose) (Box B of Figure 1).  Except for the oxidative pathway initiated by the glucose-6-phosphate dehydrogenase, the non-xidative reactions are all reversible and thought to be in equilibrium.  The pentose cycle produces a large number of intermediates of different lengths of carbons (ribulose-5-P, ribose-5-P, xylulose-5-P, erythrose-4-P sedoheptulose-7-P and glyceraldehyde-3-P).  The fact that xylulose-5-P is known to regulate the production of F2, 6P makes the role of the pentose cycle important in glucose homeostasis. (Casazza and Veech, 1986) The G6PDH reaction has been studied with 1, 2-¹³C-glucose. (Lee et al., 1998) The action of G6PDH creates 1-¹³C-ribulose, which can be recycle to 1-¹³C-glucose-6-P. The ratio of lactate from 1, 2-¹³C-glucose and 1-¹³C-glucose-6-P will either have 2 or 1 ¹³C label. (m2 and m1), such ratio can be used in a similar way to C1/C6 yield ratio (Katz and Wood, 1960) to calculate pentose cycle flux. 1, 2-¹³C-glucose are converted to a number of ribose isotopomers as shown in Figure 2 above. We have adapted the method of Rognstad and Katz (Katz and Rognstad, 1967) to calculate the relative flux of the transketolase and transaldolase reactions using mass isotopomer distribution in RNA ribose. (Lee et al., 1998)

(C) The Cataplerotic and Anaplerotic Fluxes of The Tricarboxylic Acid Cycle

There are two fates of the 3-carbon metabolic product of glucose (pyruvate) (Box B of Figure 3).  The anaplerotic process begins with the action of pyruvate carboxylase, which leads to the regeneration of 3-carbon and 6-carbon species. The cataplerotic process begins with the action of pyruvate dehydrogenase, which leads to the formation of acetyl-CoA.  Acetyl-CoA can either enter the TCA cycle to produce energy, or be converted to malonyl-CoA for fatty acid synthesis by the action of acetyl-CoA carboxylase (ACC).  The sequential loss and dilution of carbon of a-ketoglutarate provides the basis for the study of TCA cycle flux using tracers. Beginning with either 1, 2-¹³C-lactate or 1, 2-¹³C-acetate, m2 isotopomers of a-ketoglutarate are formed. These m2 isotopomers will sequentially become m1 and m0 isotopomers (An example of the sequential loss of

¹³C from U¹³C-lactate through the TCA cycle is shown in Figure 4 above). Assuming isotopic equilibrium between a-ketoglutarate and glutamate, these m1 and m2 isotopomers can be quantitated in glutamate. The knowledge of the carbon positions that are labeled, and of the ratio of m1 to m2 isotopomers, can be used to determine anaplerotic flux relative to the flux of the TCA cycle.  (Katz et al., 1989; Katz et al., 1991; Lee, 1993; Large et al., 1997) Similarly, the enrichment of 4, 5-¹³C a-ketoglutarate can be used to determine the contribution of PDH relative to b-oxidation of fatty acids. (Jeffrey et al., 1997)

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