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A study of regulation of gluconeogenesis and the supply of cytosolic reducing equivalents for lactate formation in rat kidney-cortical-tubule fragments incubated with pyruvate.

ABSTRACT: 1. Tubule fragments were isolated after treatment of rat kidney cortex with collagenase. The formation of glucose and lactate on incubation with 5mM-pyruvate was then measured under various conditions. 2. When tubule fragments were isolated from fed rats in the absence of Ca2+ and then incubated with various Ca2+ concentrations, an incubation period of 15--30 min was necessary to establish a metabolic steady state. Under these conditions glucose formation was increased by Ca2+, adrenaline or 3':5'-cyclic AMP to a greater extent than was lactate formation. Data show that appreciable lactate formation could not have resulted from glycolytic metabolism of glucose formed by gluconeogenesis during incubation. 3. When tubule fragments were isolated from fed rats in the presence of 1.27 mM-Ca2+ and adjustments made to the Ca2+ concentration at the commencement of incubation, metabolic steady state was rapidly established. Under these conditions lactate formation was almost insensitive to Ca2+ concentration (0.16--4.5 mM), whereas glucose formation varied with Ca2+ concentration in a sigmoidal manner. 3':5'-Cyclic AMP decreased this sigmoidicity. 4. Ca2+ depletion of the tissue before incubation appeared to change permanently the relationship between extracellular Ca2+ concentration and the measured rates of metabolic processes. 5. Under conditions of metabolic steady state, glucose formation by tubule fragments from fed rats was less sensitive than lactate formation to inhibition by 3-mercaptopicolinate or 2-n-butylmalonate. Lactate formation by tubule fragments prepared from 48 h-starved rats was more sensitive to these inhibitors. 6. Estimates were made of the rate of futile cycling of C3 species through pyruvate kinase. This was greater in the starved than in the fed state, was decreased by 3':5'-cyclic AMP in both the fed and the starved state, but was unaffected by Ca2+. 7. These results suggested that formation of lactate and glucose is less tightly linked in kidney cortex than in liver. A considerable amount of the supply of reducing equivalents for lactate formation did not appear to be associated with an energy-dependent translocation from mitochondria to cytosol involving a pyruvate leads to oxaloacetate leads to phosphoenolpyruvate leads to pyruvate cycle.

SUBMITTER: Saggerson ED

PROVIDER: S-EPMC1185893 | biostudies-other | 1978 Jul

REPOSITORIES: biostudies-other

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Project description:In order to obtain a global view of energy metabolism pathways of the sulfate-reducer Desulfovibrio vulgaris Hildenborough and the proteins involved therein whole-genome microarrays were used to compare the transcriptional response of cells grown with hydrogen/sulfate, pyruvate/sulfate, lactate/thiosulfate, and pyruvate with limiting sulfate, relative to growth in standard lactate/sulfate condition. Growth with hydrogen/sulfate showed the largest number of differently expressed genes and the largest changes in expression levels. The most up-regulated energy metabolism genes were those coding for the periplasmic [NiFeSe] hydrogenase, followed by the Ech hydrogenase, and the most down-regulated were genes coding for the Coo hydrogenase. The results point to the involvement of formate cycling and the ethanol pathway during growth on hydrogen, whereas there is evidence for CO cycling during growth on lactate and pyruvate, but not on H2. Growth with thiosulfate showed the hallmarks of a reduced energy status of the cells with down regulation of the ATP synthase and the Qmo and Dsr complexes., whereas growth with pyruvate showed the smallest differences but an increased role for the Ech hydrogenase.that in this case functions in reverse from the case of growth with H2. The multiple periplasmic hydrogenases and formate dehydrogenases, do not display the same regulation pattern showing that their metabolic roles are not totally interchangeable. This result together with the observation that several genes coding for proteins that have not been biochemically characterised were considerably affected in this study, reveals a more complex energy metabolism than previously considered and provides guidance for further studies. Keywords: Growth protocol Desulfovibrio vulgaris from our laboratory culture collection was cultured at 37°C with pyruvate as the only substrate (without sulfate as the electron acceptor) to mid-log phase. Cultures were also cultivated similarly in Lactate-Sulfate medium to mid-log phase. Gene expression profiles of cultures grown under pyruvate fermentation were compared with those of the cultures grown via sulfate reduction. Total RNA was harvested from four replicate cultures for microarray analysis. RNA extraction, purification, and labeling were performed independently on each cell sample.Two samples of each total RNA preparation were labeled, one with Cy3-dUTP and another with Cy5-dUTP for microarray hybridization.

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A study of regulation of gluconeogenesis and the supply of cytosolic reducing equivalents for lactate formation in rat kidney-cortical-tubule fragments incubated with pyruvate.

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