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.