The effect of zinc ions on carbohydrate metabolism and intracellular Zn2+ was studied in hepatocytes from fed rats. The addition of ZnCl2 to the medium led to an almost 3-fold increase in lactate production and an increase in net glucose production of about 50%. Half-maximal rates occurred at about 40 microM ZnCl2. These effects were not seen with Mn2+, Co2+, or Ni2+ up to 80 microM, whereas Cu2+ at 80 microM and Cd2+ or Pb2+ at 8 microM exhibited similar effects as 80 microM ZnCl2. Changes in intracellular Zn2+ were followed by single cell epifluorescence using zinquin as a specific probe. Intracellular free Zn2+ in isolated hepatocytes was 1.26 +/- 0.27 microM, and the addition of ZnCl2 led to a concentration-dependent increase in epifluorescence. CdCl2 or PbCl2 at 8 microM was as potent as ZnCl2 at 20-80 microM, whereas NiCl2 at 80 microM was without effect. ZnCl2 completely abolished the inhibition of glycolysis by glucagon (cAMP). Glucagon led to a pronounced drop in cytosolic Zn2+. Both glucagon and zinc stimulated glycogenolysis by increasing the phosphorylation of glycogen phosphorylase but acted oppositely on glycolysis. Zinc overcame the inactivation of pyruvate kinase by glucagon without changing the hormone-induced protein phosphorylation. The antagonistic action of zinc and cAMP on glycolysis together with the rapid and marked decrease in free zinc concentration induced by glucagon (cAMP) may indicate an as yet unknown role of zinc as an important mediator of regulation of carbohydrate metabolism.

A boy and a girl born to a consanguineous Tunisian couple are suffering from a slowly progressive nervous disorder. Initially they both had normal psychomotor development with acquisition of gait and speech. First symptoms in the boy were athetoid movements during the second year of life. He later lost all motor and language skills and developed muscular rigidity and intention tremor. At the age of five years, he was completely bedridden while he appeared mentally much less affected. His younger sister followed a similar course. The major specific abnormality detected was a strikingly elevated excretion of 2-oxoglutaric acid, which was identified by gas liquid chromatography, mass spectrometry, and enzymatic analysis. 2-oxoglutarate dehydrogenase activity in homogenates of cultured skin fibroblasts was reduced to about 25% of control values in both children. Although the pathogenetic mechanisms leading to brain damage remain obscure, the finding strongly suggest an autosomal recessive neurometabolic disease with predominant involvement of the extrapyramidal system.

Because of differences in the pattern of enzyme activities involved in glycolysis and gluconeogenesis between rat and guinea pig liver, the regulation of gluconeogenesis in guinea pig liver was studied.The following results were obtained: 1. Starvation increases the capacity for gluconeogenesis from pyruvate in vivo. 2. Ghconeogenesis from L-lactate and from pyruvate in isolated perfused livers is greater in the guinea pig than in the rat, whereas gluconeogenesis from other precursors is similar in both species.3. Long chain and medium chain fatty acids inhibit gluconeogenesis from L-lactate in isolated perfused guinea pig liver but stimulate gluconeogenesis from L-lactate in isolated perfused rat liver. I n the guinea pig liver the difference does not result from a lower rate of fatty acid oxidation but from a greater reduction in the cytoplasmic NAD+/NADH system.During enhanced fatty acid oxidation in guinea pig liver and in rat liver, similar changes in the concentrations of both acetyl-S-CoA and CoA-XH are observed.With high concentrations of pyruvate as the gluconeogenic precursor, both fatty acids and ethanol lead to a slight stimulation of glucose formation by supplying hydrogen equivalents to the cytosolic NAD+/NADH system. 4. Both glucagon and dibutyryl-Ado-3' ; 5'-P stimulate ureogenesis and ketogenesis in isolated perfused guinea pig liver. However, the stimulation of gluconeogenesis by glucagon and Ado-3';5'-P normally observed in isolated perfused rat livers was not found in isolated perfused guinea pig livers.5. Partially purified pyruvate carboxylases from rat and guinea pig liver do not differ with respect to the apparent K , values for pyruvate and ATP, and also the apparent K , values for acetyl-S-CoA a t different pH values. At constant concentrations of MgATP2-or MnATP2-the pH-optima for the guinea pig enzyme are significantly lower.6. 5-Methoxyindole-2-carbonic acid inhibits gluconeogenesis in guinea pig liver in the same way as in rat liver. Quinolinate, on the other hand is far less inhibitory in guinea pig liver and does not inhibit a t all in isolated perfused pigeon livers.7. Major species differences are found when the activities of the rate limiting enzymes involved in gluconeogenesis are measured under V,,, conditions. Pyruvate carboxylase activity is in the same range in rat and guinea pig liver; pyruvate kinase activity is considerably lower, phosphoenolpyruvate carboxykinase activity is considerably higher in guinea pig liver. According to our results gluconeogenesis in guinea pig liver from lactate or pyruvate is mainly regulated by the concentration of pyruvate.The difference in the regulation of gluconeogenesis between rat and guinea pig liver probably results from a difference in the compartmentalization of phosphoenolpyruvate carboxykinase. A higher rate of futile cycling between phosphoenolpyruvate and pyruvate in rat liver, due to the higher activity of pyruvate kinase in relation to the activities of phosphoenolpyruvate carboxykinase and pyruvate carboxylase, may also cont...

Gluconeogenesis was studied in isolated perfused livers from pigeons, guinea pigs and rats in order to evaluate the role of intramitochondrial formation of phosphoerwlpyruvate and the rate and importance of "futile cycling" of carbon for the regulation of glucose formation. The net formation of glucose from lactate (20 mM) by pigeon liver is about twice as high as in guinea pig liver and about 3 to 4 times higher than in rat liver, although the intracellular ATP/ADP ratio in pigeon liver is extremely low (< 1) in the presence and absence of gluconeogenesis.I n contrast to experiments with rat livers, but in accordance with those with guinea pig livers, oleate (2 mM) failed to stimulate gluconeogenesis from lactate in pigeon liver.With pyruvate (20 mM) there is no net formation of glucose by pigeon livers in the absence or presence of hexanoate, oleate or xylitol. In the presence of ethanol the rate of glucose formation from pyruvate increased but did not exceed 30°/, of the rate observed with lactate as precursor.In contrast to the results obtained in rat liver experiments the transaminase inhibitor (aminooxy)acetate did not inhibit gluconeogenesis from lactate in pigeon liver although soluble and mitochondrial glutamate oxaloacetate aminotransferase from pigeon liver were strongly inhibited by (aminwxy)acetate. On the other hand, n-butylmalonate (5 mM) inhibited strongly gluconeogenesis from lactate in isolated perfused rat and pigeon livers. This inhibition was accompanied by an inhibition of oxygen uptake in pigeon but not in rat liver. Benzene 1,2,3-tricarboxylate, an inhibitor of the tricarboxylate translocator, had no effect on gluconeogenesis from lactate in isolated perfused rat and pigeon livers. Gluconeogenesis from propionate (I0 mM) decreased in the order guinea pig-rat-pigeon.The activity of phosphoenolpyruvate carboxykinase as well as the phosphoerwlpyruvate carboxykinase/pyruvate kinase ratio in pigeon liver were significantly higher than in rat and guinea pig liver. The activities of phosphoenolpyruvate carboxykinase as well as pyruvate carboxylase in pigeon liver were almost completely located intramitochondrially. Malic enzyme activity in pigeon liver was about 33 times higher than in rat liver and about 260 times higher than in guinea pig liver. However, the sum of the activities of the 4 NADPH-generating enzymes, malic enzyme + isocitrate dehydrogenase (NADP) + glucose-6-phosphate dehydrogenase + 6-phosphogluconate dehydrogenase was similar in pigeon and guinea pig liver, but somewhat lower in rat liver.Measurement of oxygen uptake during gluconeogenesis from intraportally infused lactatefpyruvate ( l O / l ) was performed with isolated perfused livers from pigeon, rats and guinea pigs. The ratio oxygen used/glucosc formed was 2.47 in pigeon liver, 2.42 in rat liver, and 2.35 in guinea pig liver.These results are very close to the theoretically expected value of 2. The following conclusions have been made : In pigeon liver phosphoenolpyruvate formed intramitochondrittlly is used for gluconeog...

The effects of intraportal infusion of 0.18-7.20 mmole/h of sodium caproate on gluconeogenesis were studied in isolated perfused livers of fed male rats.Even 0.18 mmole/h of sodium caproate enhanced the incorporation of carbon from the C-2 and the C-1 position of the pyruvate-lactate pool into glucose and glycogen, increased the specific activity of glucose and glycogen, and stimulated the net uptake of lactate and pyruvate as well as the net production of glucose. Net changes in liver glycogen Concentration were the same with and without the infusion of caproate.With 0.72 mmole/h of sodium caproate, the ratio of carbon incorporated into glucose to carbon incorporated into GO, had increased about 8 times for carbon from the C-2 position and about 4 times for carbon from the C-1 position of the lactate-pyruvate pool.An increase in the lactate/pyruvate and the 8-hydroxybutyratelacetoacetate ratio in the liver and in the perfusion medium occurred only with 0.72 mmole/h or more of sodium caproate, whereas the CoASH/CoASAc ratio had already been decreased significantly with 0.18 mmolelh.A similar decrease in the CoASH/CoASAc ratio was observed in vivo in livers of fat-fed rats.Fat-feeding led to a 300 Ol0 increase in the steady-state concentration of CoASH plus CoASAc i n vivo, whereas the steady-state concentration of CoASH plus CoASAc in perfused livers did not change either with or without caproate. I n contrast to this, the total concentration of ATPplus ADP was decreased by 40-500/0 after 30 min of perfusion with and without caproate. The ATP/ADP ratio was not changed by the intraportal infusion of caproate.From this it has been concluded, that: 1. The primary mechanism by which fatty acids enhance gluconeogenesis is a stimulation of the pyruvate carboxylase reaction and an inhibition of pyruvate oxidation. This is accomplished by an increase in the steady-state concentration of CoASAc and a decrease in the CoASH/ CoASAc ratio, rather than a stimulation of the triosephosphate dehydrogenase reaction by a decreased cytoplasmic NADf/NADH ratio.2. The chance that oxaloacetate formed by carboxylation of pyruvate will be converted to phosphoenolpyruvate is higher in liver than in kidney.3. Changes in the ATP/ADP ratio are not directly related to the regulation of gluconeogenesis by fatty acid oxidation.In previous experiments with isolated perfused rat livers we observed an increased uptake of lactate and pyruvate immediately after application of sodium caproate to the perfusion medium [ l, 21. We Non-Standard Abbreviations. Dimethyl-POPOP = 1,4-bis-2-(4,methyl-5-phenyloxazolyl)-benzcne; PPO = 2,5-diphenyloxazole.