Wolfgang Tarnowski

Coordinate control of intermediary metabolism in rat liver by the insulin/glucagon ratio during starvation and after glucose refeeding: Regulatory significance of long-chain acyl-CoA and cyclic AMP

Abstract The levels of serum insulin, glucagon, and free fatty acids (FFA) and the tissue concentrations of hepatic cyclic AMP, long-chain acyl-CoA (LCA), adenine nucleotides, inorganic phosphate, the intermediates of the Embden-Meyerhof pathway, the citric acid cycle (including acetyl-CoA and free CoA), and the cytoplasmic and mitochondrial redox couples were determined in the rat 12, 24, and 48 h after food withdrawal and 5, 10, 20, 40, 60, and 120 min after the refeeding of glucose. Using the measured metabolite contents in the liver, the alterations in the concentration of malate, oxaloacetate, citrate, and α-ketoglutarate and the changes in the energy state of the adenine nucleotide system and the redox state of the NAD system were attributed to the cytoplasmic and mitochondrial compartments by applying established calculation methods. Glucose refeeding provoked significant alterations in all parameters investigated. These changes occurred within minutes, reversing the hormone and metabolite pattern which had developed within 24 h in response to food withdrawal. Particularly, glucose refeeding resulted in a drastic increase in the insulin/glucagon ratio. Simultaneously, the level of serum FFA and the concentration of LCA in the liver declined. The latter alteration was accompanied by an increase in the cytoplasmic and a decrease in the mitochondrial ATP ADP × P ratios. Moreover, the redox state of the cytoplasmic NAD system was shifted toward the oxidized state. When the appropriate data were plotted against each other, highly significant correlations were obtained (i) between the insulin/glucagon ratio and the serum FFA concentration, (ii) between the serum FFA concentration and the concentration of hepatic LCA, (iii) between the hepatic LCA concentration and the cytoplasmic energy state, and (iv) between the cytoplasmic energy state and the redox state of the cytoplasmic NAD system. These findings are interpreted to support the hypothesis derived from experiments carried out in vitro that the insulin/glucagon ratio via the FFA-dependent concentration of hepatic LCA might affect the translocation of adenine nucleotides between the cytoplasmic and the mitochondrial compartment, thereby regulating the cytoplasmic energy state and the redox state of the cytoplasmic NAD system, consequently. Glucose refeeding provoked rapid coordinate changes in the concentration of the intermediates of both the citric acid cycle and the Embden-Meyerhof chain, indicating the altered substrate flow through these pathways. Those metabolites, known to modulate the activity of key regulatory enzymes in vitro, were analyzed with respect to their suggested regulatory function. As to the established shift from pyruvate carboxylation to pyruvate decarboxylation after glucose refeeding, the data revealed that the decrease in pyruvate carboxylase activity can be attributed to the decrease in the intramitochondrial ATP ADP ratio and the simultaneous fall in acetyl-CoA concentration, while the coordinate increase in pyruvate dehydrogenase activity can be ascribed to the decline in the concentration of LCA and, consequently, in the ratios of ATP ADP , NADH NAD , and acetyl- CoA CoA within the mitochondria. As for the citric acid cycle, increased citrate synthesis from acetyl-CoA and oxaloacetate was supported by the rapid drop in the concentration of the established inhibitor of citrate synthesis, LCA. In contrast, the concentration of succinyl-CoA, an inhibitor of the enzyme in vitro, remained practically constant, questioning its regulatory function under the present experimental conditions. In addition to the activation of citrate synthase, the coordinate activation of isocitrate dehydrogenase was indicated by the LCA-mediated decline in both the mitochondrial ATP ADP and the NADH NAD ratios. Glucose refeeding immediately reduced urea excretion to basal values. This alteration was preceded by a drastic fall in the tissue concentration of cyclic AMP, supporting the physiological role of the nucleotide in the control of hepatic gluconeogenesis. In contrast, the observed changes in the concentration of the effectory acting metabolites (ATP, AMP, fructose 1,6-diphosphate, citrate, and alanine) were incompatible with the suggested function of these intermediates in switching over the substrate flow through the Embden-Meyerhof pathway from gluconeogenesis to glycolysis. The results are discussed in reference to the known rapid stimulation of fatty acid biosynthesis in the liver and to the transfer of reducing equivalents by the different shuttles of the inner mitochondrial membrane. In summary, it can be concluded that the insulin/glucagon ratio in a moment-to-moment fashion controls the glucose balance across the liver by regulating hepatic intermediary metabolism via the concentration of both LCA and cyclic AMP.

Rapid rise in plasma glucagon induced by acute cold exposure in man and rat

The effect of acute cold exposure on the concentration of glucagon in the blood was investigated in man and in intact and adrenalectomized rats.In man fasted overnight acute cold exposure, which caused a twofold increase in O2-consumption resulted in a rapid rise in plasma glucagon. The levels of insulin and blood glucose remained unaltered, while the concentration of serum free fatty acids and β-hydroxybutyrate increased.In fasted intact rats acute cold exposure lead to similar effects. A close parallelism between the rise in plasma glucagon and the concentration of hepatic cycloAMP was observed. Adrenalectomy did not impair the cold induced rise in plasma glucagon and hepatic cycloAMP.It is concluded that acute cold exposure caused a rapid rise in the concentration of plasma glucagon leading to an increase in the concentration of hepatic cycloAMP, thus enhancing the rate of hepatic gluconeogenesis and ketogenesis. As these alterations were similar in the absence of glucocorticoids and medulla-derived catecholamines, it is suggested that glucagon may play a role in the metabolic adaptation to acute cold exposure.

Long-term perfusion of the isolated rat liver maintenance of its functional state by use of a fluorocarbon emulsion

Abstract In order to establish a long-term perfusion system a fluorocarbon emulsion was developed and employed for the perfusion of isolated rat liver up to 20 h. Its suitability for maintaining some specific organ functions was compared with that of a commonly used red cell-containing medium. All livers perfused with the fluorocarbon medium released phosphoglucose isomerase, glutamate-oxaloacetate transaminase and glutamate dehydrogenase almost linearly at a low basal rate, glutamate dehydrogenase release beginning after 5 h perfusion. In contrast to that, a certain percentage of the livers perfused with the red cell-containing medium showed an exponential enzyme release which was over two standard deviations above the mean of the livers perfused with fluorocarbon medium, the values being 25% for phosphoglucose isomerase, 38% for glutamate-oxaloacetate transmiinase and 87% for glutamate dehydrogenase after 10 h of perfusion. In each case the exponential release of phosphoglucose isomerase signaled the functional impairment of the preparation. Thus, defining those livers as “intact” only if their phosphoglucose isomerase release was within two standard deviations of the means of the fluorocarbon-perfused livers, the following liver functions were examined in fluorocarbon-perfused and, for comparison, in “intact” cell-perfused livers during a 10-h period: Metabolite state, galactose elimination from the perfusate, induction of tyrosine aminotransferase by dexamethasone, and gluconeogenesis from lactate and bile production. It was found that the fluorocarbon medium provided at least the same or an even better hepatic function than did the red cell-containing medium. However, while in red cell-perfused livers functional impairment always occurred at various percentages under the conditions mentioned above, this was never observed with the fluorocarbon medium. Electron microscopic examination of the livers perfused with the fluorocarbon medium showed no disturbance of the mitochondrial matrix and cristae after a 10 h perfusion. While within a large number of liver cells the ergastoplasm was seen in normal appearance, in other liver cells the cisternae of rough endoplasmic reticulum were vacuolated. Some important physicochemical data of the fluorocarbon medium such as O 2 capacity, viscosity and particle size are reported, and the technique and the problems of its preparation are described. The advantages of the fluorocarbon medium for long as well as short term perfusion experiments are discussed.

Physiologic significance of glucocorticoids and insulin in the regulation of hepatic gluconeogenesis during starvation in rats.

The physiologic significance of glucocorticoids and insulin in the regulation of hepatic gluconeogenesis was investigated during a 48-hr starvation period by studying the factors presumed to control the rate of glucose synthesis in the final gluconeogenetic pathway. Rats were used, in which glucorticoids were removed by adrenalectomy before starvation, and in which serum insulin was kept constant before and after food withdrawal by pre-feeding with a proteinfree diet. It was found that adrenalectomized rats at constantly low serum insulin levels responded to starvation as rapidly, and to the same degree, as intact control subjects (1) by a significant increase in plasma glucagon and, consequently, in hepatic cAMP concentration; (2) by a coordinate elevation of the activities of hepatic pyruvate carboxylase, P-enolpyruvate carboxykinase, and fructose-1,6-diphosphatase; (3) by systematic alterations in the concentration of effectors of gluconeogenetic key enzymes; (4) by a shifting of the cytoplasmic NAD system towards the reduced state; (5) by a decrease in the intrahepatic concentration of glycogenic precursor substrates. These results suggest that the hepatic gluconeogenic response to starvation with respect to the regulatory factors 1-5 occurs independently from changes in the concentration of plasma glucocorticoids and insulin. The crossing over of the gluconeogenetic intermediates between pyruvate and P-enolpyruvate (PEP), which was observed in intact but not in adrenalectomized rats, supports the assumption that during starvation, glucocorticoids enhance the rate of glucose production by the liver predominantly by permitting hepatic cAMP to stimulate the yet undefined mechanism, which has been demonstrated in the isolated perfused rat liver to control the substrate flow between pyruvate and PEP.

Influence of different types of narcosis and of neck fracture on the concentration of glycolytic intermediates and related compounds in rat liver

Abstract The consequences of some often-used methods of producing unconsciousness in experimental animals were investigated by comparing the concentrations of nine glycolytic intermediates and eight related compounds in the liver of normal, well-fed rats, anaesthetized with ether, halothane, urethan, evipan, nembutal, luminal, or killed by neck fracture, with the physiological values, obtained from unanaesthetized, unstressed animals by our double hatchet method. All types of narcosis as well as neck fracture severely disturbed the physiological metabolite pattern. It is concluded that, in experiments in which the physiological metabolite state of a tissue shall be determined, narcosis or neck fracture must be avoided.

Regulation of rat liver glucokinase activity in vivo: predominant role of hepatic cyclic AMP and glucocorticoids.

Abstract The role of insulin, glucocorticoids, and hepatic cyclic AMP in the regulation of the activity of rat liver glucokinase was investigated in vivo . The following results were found: (i) Refeeding of starved rats with glucose or injection of diabetic animals with insulin resulted in a dramatic increase in the concentration of serum insulin and a decrease in the concentration of hepatic cyclic AMP followed by a marked rise in the glucokinase activity. While refeeding with protein or fat also led to a drastic rise in the level of serum insulin, the hepatic cyclic AMP concentration remained unchanged as did glucokinase activity, (ii) Feeding of rats with a carbohydrate-rich, protein-free diet had previously been shown to be characterized by very low concentrations of serum insulin and hepatic cyclic AMP (1). Subsequent starvation did not alter the concentrations of insulin, but gradually increased the level of hepatic cyclic AMP. This was followed by a decrease in the highly elevated glucokinase activity, (iii) Refeeding of adrenalectomized, starved rats with glucose resulted in an impaired response of serum insulin and only a slight elevation of glucokinase activity. Injection of insulin in these animals, which led to similar concentrations of the hormone to intact rats refed glucose, did not accelerate the rise in glucokinase activity. Restoration of glucocorticoids prior to refeeding with glucose resulted in an increase in glucokinase activity, identical to that observed in intact controls, while this steroid per se had no effect on enzyme activity. Since under all conditions examined there was a close inverse correlation between the hepatic cyclic AMP concentration and glucokinase activity, but not between the insulin level and the enzyme activity, it is concluded that hepatic cyclic AMP and not serum insulin predominantly regulates the activity of glucokinase in rat liver. It is suggested that glucocorticoids exert a “permissive” action on glucokinase induction.

Regulation of hepatic phosphoenolpyruvate carboxykinase (GTP) Role of dietary proteins and amino acids in vivo and in the isolated perfused rat liver

The effect of protein feeding and the addition of amino acids on the activity of hepatic phosphoenolpyruvate carboxykinase (GTP : oxalacetate carboxylyase (transphosphorylating), EC 4.1.1.32) was investigated in vivo and in the isolated perfused rat liver. Protein feeding resulted in a considerable increase in phosphoenolpyruvate carboxykinase activity within 6 h. This rise was independent of the presence of glucocorticoids. In the isolated perfused liver system amino acids per se had a small effect on phosphoenolpyruvate carboxykinase activity and led to an increase by 20% when glucocorticoids were present, but resulted in a rise by 100% when glucocorticoids plus dibutyryl cyclic AMP were added to the perfusion medium. The effect of amino acids in the presence of dibutyryl cyclic AMP could also be observed in the liver of glucocorticoid-deprived rats. Cycloheximide, a translational inhibitor, totally blocked all effects of amino acids on enzyme activity. These results indicate that the concentration of amino acids in the portal vein modify the regulation of phosphoenolpyruvate carboxykinase by cyclic AMP.

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.