Gluconeogenesis in trout (Oncorhynchus mykiss) white muscle: purification and characterization of fructose-1,6-bisphosphatase activity in vitro

Abstract

Studies of the enzyme fructose-1,6-bisphosphatase (FBPase) of rainbow trout (Oncorhynchus mykiss) have been undertaken in order to illuminate aspects of skeletal muscle gluconeogenesis in these animals. Maximal activities in crude homogenates of several organs suggest that the liver possesses the greatest FBPase activity on a unit g(-1) tissue basis but that the white muscle, owing to its bulk, contributes substantially to whole body FBPase activity. Studies of fructose-6-phosphate-1-kinase (PFK) and FBPase in crude homogenates of several organs suggests an important role for intracellular pH in regulating the relative carbon flux through the FBPase/PFK locus in vivo. Furthermore, a three-step purification scheme is described for trout white muscle FBPase by which a stable and homogeneous (by SDS PAGE) enzyme preparation (isoelectric point = 7.2; molecular weight = 37.6 kd) was obtained. Kinetic studies of the purified enzyme were undertaken at 20°C under conditions reflective of "rest" and "exercise/recovery" intramuscular pH in vivo. Affinity for substrate (F-1,6-P2) was increased (Km = 6.88 versus 2.44 μmol 1-(-1) as was enzyme activity when pH was lowered from 7.0 to 6.5. Various inhibitor metabolites are identified including F-2,6-P2 (mixed-type inhibitor, Ki = 0.201 μmol 1(-1), pH 7.0) and AMP (non-competitive inhibitor, Ki = 0.438 μmol 1(-1), pH 7.0). Inhibition by F-2,6-P2 was strongly alleviated by a reduction in pH from 7.0 to 6.5 (I50 increased from 0.14 to 0.32 μmol 1(-1)). AMP on the other hand was a more potent inhibitor at pH 6.5 but this inhibition was totally reversed under conditions of citrate, NH4 (+) and AMP typical of muscle during recovery from exercise in vivo. In purified white muscle enzyme preparations, FBPase demonstrated maximal activity at pH 6.5 whereas the optimal pH of PFK was 7.0 or greater. Indeed, it appears from these in vitro data that regulation by metabolite levels as well as pH are required for net FBPase flux in vivo. It is concluded, therefore that trout white muscle FBPase demonstrates the potential to play an important enzymatic role in the control of intramuscular gluconeogenesis in these animals. The results are discussed in relation to present knowledge regarding the metabolic responses of trout white muscle to, and its subsequent recovery from, exhaustive exercise.

Influence of exercise on the distribution of enzymes in trout white muscle and kinetic properties of AMP-deaminase from free and bound fractions

“…Table 2 (condition I) shows comparable values for AMPdeaminase. Activities of PFK and FBPase, as well as the PFK:FBPase ratio, were similar to those reported previously (Ferguson and Storey 1992) but AMP-deaminase values are difficult to compare with earlier results which were presented only as units/mg protein (Kaletha et al 1991). Burst swimming did not alter the maximal activity of any of the three enzymes.…”

“…Assays conditions for FPBase were 20 mM imidazole-HCI buffer (pH 7.2), 5 mM MgSO 4 , 0.1 mM Fl,6P 2 , 0.2 mM NADP, 1 U glucose-6-phosphate dehydrogenase, and 1 U phosphoglucoisomerase (Ferguson and Storey 1992). One unit of enzyme activity was defined as the amount producing 1 iimol F6P per min at 22 0 C.…”

Evolutionary history of glucose-6-phosphatase encoding genes in vertebrate lineages: towards a better understanding of the functions of multiple duplicates

“…In lampreys, available studies suggest that this tissue could be, in a certain extent, autonomous from glucose supplied through the blood as demonstrated by endogenous glucose production either from gluconeogenesis or glycogenolysis, a process in which glucose-6-phosphatase action is involved [26, 28, 30, 31] thus suggesting a possible function of g6pc . In teleosts, levels of glycogen in brain are considerably lower than in lamprey even though the enzymes involved in gluconeogenesis and glycogen metabolism are also present and functional as previously demonstrated in rainbow trout [37, 38, 43]. The role of gluconeogenesis in teleost brain is however still an enigma though it could relate to glucose production under conditions of limited glucose supply such as under food deprivation.…”

Nutritional regulation of glucose metabolism-related genes in the emerging teleost model Mexican tetra surface fish: a first exploration

“…Except the weak decrease in glut3a mRNA level in hypothalamus, the majority of changes observed on glucose metabolism-related genes products occurred in the telencephalon (table 3). Even if unexpectedly downregulated by the nutritional status, the detection of pfkla and pklr transcripts in the telencephalon signed for a glycolytic potential of the brain in the surface A. mexicanus, as previously described in several teleost species regardless of their nutritional preferences [50][51][52]. Surprisingly, no gck mRNA was detected in the brain, although it has been demonstrated to exist in several fish species [53][54][55].…”