The shunt pathway of mevalonate metabolism in the isolated perfused rat liver

The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated livers from fed rats perfused with physiological concentrations of variously labeled [14C]mevalonates. The measured rates of 14CO2 production were converted to rates of mitochondrial acetyl-CoA production from mevalonate by methods which take into account underestimations of metabolic rates derived from 14CO2 production. Our data confirm that the shunt pathway leads to mitochondrial acetyl-CoA. The apparent negligible rate of mevalonate shunting in liver, previously reported by others, stems from the very low contribution (congruent to 0.1%) of plasma mevalonate to total mevalonate metabolism in the liver. This contribution was assessed from the relative incorporations of 3H2O and [5-14C]mevalonate into sterols. In livers from fed rats, the shunt diverts about 5% of the production of mevalonate. The total rate of mevalonate shunting in the liver is about 200 times greater than in two kidneys. The liver is therefore the main site of mevalonate shunting in the rat.     

Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

Cortisol metabolism and excretion in the isolated perfused rat kidney

The isolated perfused rat kidney allows a simultaneous kinetic study of both the renal metabolism and the urinary excretion of cortisol and its metabolites in the rat. In this system, cortisol was completely metabolized within 120 minutes. The main renal metabolites of cortisol (cortisone, 20 reduced cortisol and 20 reduced cortisone) were found in the recirculating perfusate and in urine. The formation of these metabolites was quantitatively evaluated and compared to a theoretical model.     

Glutamine metabolism in isolated perfused rat liver. The transamination pathway

In isolated perfused rat liver, added 4-methyl-thio-2-oxobutyrate and phenylpyruvate are rapidly transaminated to the corresponding amino acids with glutamine, the latter being supplied via the portal vein or by endogenous synthesis. With portal glutamine concentrations below 5mM and in the presence of a oxo-acid acceptor, the flux through glutamine transaminases exceeded the ammonium ion-stimulated glutaminase flux. 4-Methylthio-2-oxobutyrate-induced extra glutamine uptake was not dependent on the perfusate pH in the range of pH 7 to 8. During glutamine/4-methylthio-2-oxobutyrate transamination, the amide nitrogen of glutamine is fully recovered as glutamate, ammonia, urea and alanine. Oxoglutarate formed by omega-amidase activity is released as glutamate or oxidized by oxoglutarate dehydrogenase. alpha-Cyanocinnamate, the inhibitor of the monocarboxylate translocator in the mitochondrial membrane inhibited 4-methylthio-2-oxobutyrate-induced glutamine uptake and methionine release by about 30%. This might indicate that about 2/3 of glutamine transaminase flux is cytosolic. alpha-Cyanocinnamate inhibited 4-methylthio-2-oxobutyrate-induced glutamate efflux by about 90%. Stimulation of flux through glutamine transaminases is accompanied by a 70-80% inhibition of glutaminase flux. This is not explained by a direct inhibition of glutaminase by 4-methylthio-2-oxobutyrate but by a substrate competition between glutaminase and glutamine transaminases. 4-Methylthio-2-oxobutyrate decreases glutamine release by the liver due to withdrawal by transamination. The oxo acid itself is without effect on glutamine synthetase flux. With respect to hepatocyte heterogeneity there is no evidence for a zonal distribution of glutamine transaminase activities, as it has been shown for glutamine synthetase and glutaminase activities.     

Contribution of the shunt pathway of mevalonate metabolism to the regulation of cholesterol synthesis in rat liver

The modulation of the shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in livers from fed, starved, and diabetic rats perfused with a physiological concentration (300 nM) of [5-14C] + [5-3H]mevalonate. Shunt activity was measured by (i) production of 14CO2 (corrected for loss of label by exchange reactions) and (ii) production of 3H2O. Contribution of exogenous mevalonate to total mevalonate production (0.06-0.11%) was assessed in parallel experiments by the incorporation of 3H2O into sterols. Inhibition of non-saponifiable lipid synthesis by starvation and diabetes is not associated with an inhibition of mevalonate production but with a major increase in shunting (7-34%) of sterol-bound mevalonate. The shunt pathway of mevalonate metabolism appears to participate in the regulation of cholesterol synthesis.     

The fuels of respiration of the isolated perfused rat kidney

The oxidation of ten substrates: monosaccharides, fatty acids and amino acids, was studied in the isolated perfused rat kidney. Glucose, when offered 3.75 mM, contributed to tissue respiration by a rate equivalent to 18% of the total O₂-consumption of the preparation. The corresponding data for the other nine substrates, each offered in the presence of 3.75 mM glucose, were as follows: pyruvate: 66 %, lactate: 45 %, acetate: 34 %, palmitate: 30 %, glutamate: 25 %, fructose: 18 %, propionate: 12 %, alanine: 10 %, and tyrosine: < 1 %. Under the conditions used less than 2.2 % of the metabolized glucose, pyruvate, lactate and acetate respectively were recovered in the lipid fraction of the kidney, indicating direct oxidation of the respiratory fuels offered and a rather low turnover rate of the endogeneous lipid pool.     

Effects of estrogen and testosterone on the metabolism of mevalonate by the shunt pathway

Mevalonate is metabolized by a sterol-forming and a non-sterol-forming, also called the "shunt", pathway. Effects of estrogen and testosterone administration on the shunt activity were examined in male and female Wistar and Sprague-Dawley rats. Shunt activity was determined in vivo from the yield of expired 14CO2 following [5-14C]mevalonate injection. Total mevalonate utilized was determined from the yield of expired 14CO2 following [1-14C]mevalonate injection. In the female, about 45% of mevalonate appears to be metabolized via the shunt, and in the male about 20%. This difference between male and female rats is dependent on both testosterone and estrogen, and apparently on testosterone to a greater extent. Thus estrogen treatment produced a 20-35% increase in shunt activity over castrated controls, while castration of males without hormonal treatment resulted in about a 50% increase in shunt activity, and testosterone administration returned castrated male and female shunt activity to that of intact males, or nearly so. Light/dark cycle had no effect in vivo on shunt activity, but may be critical in demonstrating sex differences in shunt activity in kidney slices.     

Xylitol metabolism in the isolated perfused rat liver

1. Loading the isolated perfused liver from well-fed rats with xylitol (20mm) caused a depletion of adenine nucleotides and P_i and an accumulation of α-glycerophosphate. The ATP content fell to 66% of the control value after 10min and to 32% after 80min. The ADP and AMP contents also fell. After 80min 63% of the total adenine nucleotides and 59% of the P_i had been lost. 2. The α-glycerophosphate content rose from 0.13 to 4.74μmol/g at 10min and reached 8.02μmol/g at 40min. 3. Xylitol was rapidly metabolized, the main products being glucose, lactate and pyruvate. 4. The [lactate]/[pyruvate] ratio in the presence of xylitol rose to 30–40. 5. On perfusion of livers from starved animals the main product of xylitol metabolism was glucose and the mean ratio xylitol removed/glucose formed was 1.29 (corrected for endogenous glucose and lactate production). This is close to the predicted value of 1.2. 6. Evidence is presented indicating that the loss of adenine nucleotides caused by xylitol is not due to the increased ATP consumption but to the accumulation of α-glycerophosphate and depletion of P_i. 7. The loss of adenine nucleotides accounts for the hyperuricaemia which can occur after xylitol infusion in man. 8. The relevance of the findings to the clinical use of xylitol as an energy source is discussed.     

Metabolic activities of the isolated perfused rat kidney

1. A technique for perfusing the isolated rat kidney is described. It is primarily designed for the study of renal metabolism but is also suitable for studying some aspects of the secretory function; this was normal with respect to minimal glucosuria. The glomerular filtration rate as measured by creatinine clearance was lower than in vivo and slowly decreased with time. 2. Gluconeogenesis from a variety of precursors was rapid and similar to that in kidney-cortex slices, in contrast with liver where the perfused organ is more effective than slices. Whereas the maximal rates of gluconeogenesis from glycerol and pyruvate were similar in liver and kidney, the rates from succinate, malate and fumarate were 14–20 times, and those from glutamate and aspartate about three times, as high in the kidney. 3. The oxygen consumption of the perfused organ was about twice that of cortex slices, presumably because of the secretory work done in the perfused organ but not in slices. 4. The rate of acetoacetate oxidation was about the same in the perfused organ and in slices but, because of the higher rate of oxygen consumption, the percentage contribution of acetoacetate to the fuel of respiration was lower in the perfused organ. The results suggest that acetoacetate can supply energy for the basal requirements and for gluconeogenesis but not for the secretory work. 5. Glutamine was formed at a high rate from glutamate and at a lower rate from aspartate. The high rates indicate that, in the rat, the kidney is a major source of body glutamine.     

Alanine transport by the isolated perfused rat kidney

L-alanine transport kinetics were examined in the isolated perfused rat kidney (1) using different perfusate concentrations of alanine (PAla) to obtain different filtered loads and (2) under conditions of osmotic diuresis. The transport maximum for alanine (TmAla) was found to be very high relative to $\text{in}$$\text{vivo}$ filtered loads of alanine. The apparent TmAla was dependent on glomerular filtration rate (GFR) and it could be modified by osmotic diuresis. It is suggested that the variation of TmAla with changes in GFR may be the consequence of variations in fractional volume flow through the proximal tubule.     

19-nor-DOC biosynthesis in the isolated perfused rat kidney

19-nor-deoxycorticosterone (19-nor-DOC) is a potent salt retaining and hypertensinogenic mineralocorticoid that is excreted in the urine. While the precursor of 19-nor-DOC, 19-oxo-DOC, is produced by the adrenal cortex, conversion to 19-nor-DOC does not occur in the adrenal gland. We have examined the hypothesis that 19-nor-DOC is synthesized from precursors in the kidney. 19-oxo-DOC was added to the perfusate of isolated rat kidney preparations (n = 5) at a concentration of 10 μM. During 1 h of perfusion following addition of 19-oxo-DOC, 71 ± 6% of the precursor was converted to 19-oic-DOC, an immediate precursor of 19-nor-DOC, and 8.3 ± 1.8% was converted to 19-nor-DOC. This represents the first definitive evidence that 19-nor-DOC is produced in the kidney from adrenal precursors.     

Dopamine production by the isolated perfused rat kidney

We used isolated perfused rat kidneys to examine dopamine (DA) production and its relation to renal function. Both innervated and chronically surgically denervated kidneys perfused with a solution containing neither albumin nor tyrosine, excreted 0.2 +/- 0.1 ng DA X min-1 X g wet weight-1 during the 10-min collection period between 30 and 40 min after starting perfusion. When perfused with 6.7% albumin, without tyrosine, innervated kidneys excreted 1.0 +/- 0.06 ng DA X min-1 X g-1 and denervated kidneys excreted 1.0 +/- 0.07 DA X min-1 X g-1. When 0.03 mM tyrosine was included in the albumin perfusate, innervated kidneys excreted 1.2 +/- 0.1 ng DA X min-1 X g-1 (p less than 0.1). Under these conditions DA excretion continued for at least 100 min at which time it was 0.6 ng X min-1 X g-1 and 86 ng/g kidney weight had been excreted. Denervated kidneys perfused with albumin + tyrosine excreted 0.9 +/- 0.13 ng DA X min-1 X g-1. Renal stores of free DA, conjugated DA, and dihydroxyphenylalanine (DOPA) could have provided at the most 30 ng/g of DA. Carbidopa inhibited DA excretion completely. DA excretion did not correlate with renal vascular resistance, inulin clearance, or fractional sodium excretion. In summary, nonneural tissue in isolated perfused kidneys produced DA at the same rate as denervated kidneys in vivo. Less than one-third of the DA produced by isolated kidneys could have come from intrarenal stores of DOPA, free DA, and conjugated DA; the rest was synthesized from unknown precursors.(ABSTRACT TRUNCATED AT 250 WORDS)