Robert Rognstad

A method is proposed to detect whether a given enzyme catalyzes a rate-limiting step in a metabolic pathway. With the use of a range of concentrations of specific inhibitors of an enzyme, the finding of a biphasic response with an initial null effect indicates the non-rate-limiting nature of the enzyme. With this method, phosphoenolpyruvate carboxykinase is indicated to catalyze a rate-limiting step in lactate gluconeogenesis in hepatocytes from fasted rats.

1. Rat kidney-cortex slices incubated with d-malate alone formed very little glucose. d-Malate, however, augmented gluconeogenesis from l-lactate and inhibited gluconeogenesis from pyruvate and l-malate. 2. d-Malate had little effect on the rate of the tricarboxylic acid cycle with or without other substrates added. 3. d-Malate inhibited the activity of the l-malate dehydrogenase in a high-speed-supernatant fraction from kidney cortex. 4. It was concluded that d-malate inhibited either the operation of the cytoplasmic l-malate dehydrogenase or malate outflow from the mitochondria in the intact kidney-cortex cell. This supports the hypothesis of Lardy, Paetkau & Walter (1965) and Krebs, Gascoyne & Notton (1967) on the role of malate as carrier for carbon and reducing equivalents in gluconeogenesis. 5. Gluconeogenesis from l-lactate in kidney-cortex slices was strongly inhibited by a low concentration (0.1mm) of amino-oxyacetate, whereas glucose formation from pyruvate, malate, aspartate and several other compounds was only slightly affected. 6. High concentrations of l-aspartate largely reversed the inhibition of gluconeogenesis from l-lactate caused by amino-oxyacetate. 7. Amino-oxyacetate inhibited strongly the glutamate-oxaloacetate transaminase in the 30000g supernatant fraction of a kidney-cortex homogenate. The presence of l-aspartate decreased the inhibition of the transaminase by amino-oxyacetate. 8. Detritiation of l-[2-(3)H]aspartate was inhibited by 90% during an incubation of kidney-cortex slices with l-lactate and amino-oxyacetate. 9. Low concentrations (10mum) of artificial electron acceptors such as Methylene Blue and phenazine methosulphate abolished most of the inhibition of gluconeogenesis from l-lactate by amino-oxyacetate. This is interpreted as an activation of net malate outflow from the mitochondria by-passing the inhibited transfer of oxaloacetate. 10. These findings support the concept that transamination to aspartate is involved in the transfer of oxaloacetate from mitochondria to cytosol required in gluconeogenesis from l-lactate.

Abstract Hepatocytes, prepared by collagenase treatment of livers of ad libitum- and meal-fed rats, were incubated with glucose, fructose, lactate, and a number of other substrates, labeled uniformly with 14C and in the presence of 3HOH. The uptake of lactate and the incorporation of 14C into CO2 and glucose equals or exceeds rates reported in perfused liver. The 14C yield in glucose from labeled fructose, glycerol, and dihydroxyacetone exceeds considerably that in CO2 and greatly that in lipids. The 14C yield in CO2 from pyruvate, lactate, alanine, and acetate is equal to or exceeds that in glucose, and the fraction incorporated into lipids is greater than that from the neutral compounds. Fatty acid synthesis is greatly increased in meal-fed as compared to ad libitum-fed animals. Incorporation of glucose and of lactate carbon into fatty acids increases with concentration up to concentrations of 100 mm. Lactate carbon is a much better fatty acid precursor than glucose carbon. When both substrates are present at physiological concentrations (10 mm in glucose and 2 mm in lactate), the incorporation of lactate exceeds several times that of glucose carbon. At concentrations below 10 mm fructose is a better precursor of fatty acids than glucose, but the reverse is true at higher concentrations. Fructose and glycerol at concentrations above 5 mm inhibit the incorporation of 3H from 3HOH into fatty acids. In cells from meal-fed rats considerable incorporation of 3H from 3HOH into fatty acids occurs in the absence of added substrate. The ratio, microatoms of 14C from substrate to microatoms of 3H from water (14C:3H ratio), in the presence of low glucose concentrations is below 0.1 and increases at 100 mm glucose up to 0.5 to 0.7. Lactate stimulates 3H incorporation up to a concentration of 20 mm, but incorporation of lactate carbon continues to increase. The 14C:3H ratio attains values close to 1.0 at about 20 mm and increases to 1.5 to 2.0 at 100 mm lactate. The incorporation of tritium from water into fatty acids by hepatocytes of meal-fed rats in the presence of 10 to 20 mm lactate ranged in most experiments from 200 to 600 µatoms per g dry weight per hour, but in a number of experiments rates above 1000 µatoms per g per hour were obtained. This corresponds to an incorporation of over 120 acetyl equivalents per g wet weight of tissue per hour. Our results indicate that free glucose has a minor role as direct precursor of fatty acids in liver, and the major substrates are glycogen and lactate. Due to the presence of glycogen and the reversibility of glycogen and glucose synthesis and glycolysis in liver, the quantitative interpretation of 14C incorporation from labeled glucose is difficult.

Glucose, labeled with 14C uniformly or in positions 1 or 6, and with tritium in positions 1, 3, 4, or 6, was incubated with rat epididymal fat tissue in the presence of insulin or epinephrine. Incorporation of the label into CO2, fatty acids, glycerol, lactate, and water was determined. From the 14C yields, the contribution of the pentose cycle to glucose metabolism was calculated. A carbon balance and a balance of formation and utilization of reduced pyridine nucleotides in the cytoplasm were established. Tritium from glucose-3-T was recovered in water and fatty acids, and a little in the α position of glycerol. Only trace amounts from this position were found in the α position of lactate and the β position of glycerol. The fatty acid yield of tritium from this sugar equaled the 14CO2 yield from glucose-1-14C via the pentose cycle. The yield of fatty acids from glucose-3-T was twice that of the yield of fatty acids via tritium-labeled reduced triphosphopyridine nucleotide from glucose-1-T. In glycerol formed from glucose-1- and -6-T, all the tritium was recovered in the α position. In the presence of insulin, the T:14C ratio in glycerol from glucose-6-T ,6-14C was about 1.0; that of glycerol from glucose-1-T ,1-14C was 1.2 to 1.3. The T:14C ratios in lactate from these two sugars were less than 1, but the T:14C ratio from glucose-1-T ,1-14C was about 1.2 times that from glucose-6-T ,6-14C. In the presence of epinephrine the ratios in glycerol and lactate were the same for these two labeled sugars. Nearly 90% of tritium from glucose-4-T appeared in water in the presence of insulin, and 60% in the presence of epinephrine. The rest was recovered in lactate, fatty acids, and the α and β positions of glycerol. The T:14C ratio in the α position of glycerol from glucose-4-T labeled uniformly with 14C indicated extensive loss of the tritium from this sugar prior to the oxidation of triose phosphate. The T:14C ratios of position 2 of glycerol and of lactate indicated further loss of tritium. The contribution of TPNH and DPNH to fatty acid synthesis was calculated independently from 14C and tritium data. In the presence of insulin, it appears from 14C data that with rats on high carbohydrate diet 50% of the hydrogen equivalents are provided by TPNH, and that with rats on a regular diet 75% are provided by TPNH. From tritium data it appears that, with both diets, about 75% of the hydrogen equivalents are provided by TPNH and 25% by DPNH. With epinephrine, when fatty acid synthesis is low, DPNH may be the major source of reducing hydrogen.