Matilde S. Ayuso-Parrilla

Cellular redistribution of metabolites during glucagon and insulin control of gluconeogenesis in the isolated perfused rat liver.

Abstract Livers isolated from fasted rats were perfused in a blood-free recirculating system using alanine (10 m m ) as the carbon source. Glucagon at a concentration of 2.1 × 10 −9 m enhanced gluconeogenesis, ureogenesis, and ketogenesis. The proportion of alanine utilized to glucose formed remained rather constant in all the situations studied, suggesting that the contribution of glycogen breakdown to the total glucose output was negligible. The glucagon stimulation of gluconeogenesis was accompanied by a decrease in the [ATP]/[ADP]ratio and a rise in the reduction state of the cytosolic and mitochondrial NAD systems. The calculation of the intracellular distribution of metabolites indicates that glucagon increases the intramitochondrial oxaloacetate concentration. This finding seems to support the hypothesis of pyruvate carboxylation, the first nonequilibrium enzymic step in the gluconeogenic sequence, as one of the main sites of glucagon action. The rise in the mitochondrial:cytosolic concentration gradient of malate suggests that glucagon may also act by facilitating the transfer of three-carbon units from the mitochondria to the cytosol. The fact that insulin reversed virtually all the glucagon-induced changes strongly suggests that both hormones act on common steps. It is remarkable that these insulin effects occur at glucagon/insulin ratios similar to those normally found in the portal vein of the intact animal.

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

Abstract The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the [ATP] [ADP] ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the [NADH] [NAD + ] ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD+ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the [ATP] [ADP] ratio brought about by glucagon may be secondary to the increase in the [NADH] [NAD + ] ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the [NADH] [NAD + ] and [ATP] [ADP] ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD+ system.

Role of the state of reduction of the nad system on the regulation of hepatic protein synthesis in the rat in vivo

1. The administration of octanoate to rats in vivo increased the state of reduction of the hepatic NAD system and decreased the phosphorylation potential. This effect was accompanied by a 20% inhibition of protein synthesis. 2. The acute administration of ethanol produced similar reduction of the hepatic NAD system; however, in contrast to octanoate no effect on the phosphorylation potential was detected and rates of protein synthesis were unaffected. 3. It is concluded that a rise in the state of reduction of the NAD system is not effective in decreasing hepatic protein synthesis in vivo unless it is accompanied by a decrease in the phosphorylation potential.

Use of endogenous triglycerides to support gluconeogenesis in the perfused isolated rat liver

SummaryThe carbon balances in isolated perfused rat liver during gluconeogenesis froml-alanine and sodiuml-lactate indicate that assuming the substrate unaccounted for were fully oxidized the energy yielded was not sufficient to support the observed rates of glucose synthesis. This observation indicates that endogenous substrates must also be oxidized. The possibility that endogenous fatty acid oxidation was the source of the energy needed to support glucose synthesis was investigated by measuring the rate of14CO2 formation from tracer quantities of added [U-14C] palmitate. Short pulses ofl-alanine or sodiuml-lactate infusion produced an increased rate of14CO2 production paralleled by increases in oxygen uptake indicating that more endogenous fuel is being mobilized.That the rate of14CO2 output is an expression of fatty acid mobilization was supported by experiments demonstrating that the addition of octanoate to dilute the fatty acid pool produced an immediate fall in the rate of14CO2 output. On the other hand, the administration of glucose produced no changes in oxygen uptake or14CO2 output. However, lactate even in the presence of glucose induced a rise in14CO2 production which occurred in parallel with the enhancement in oxygen uptake.It is concluded that mobilization of hepatic endogenous fatty acid is a metabolic event intimately associated with enhancement of gluconeogenesis. Consequently the control of the different steps of this process may indirectly control gluconeogenesis.

Preparation and metabolic characterization of isolated rat lung cells.

Collagenase digestion of minced lung tissue yielded isolated cells, functionally viable as judged by several metabolic and morphological criteria, representative of all the cell species normally present in the tissue. The efficiency of the isolation procedure was about 25 per cent. Aerobic metabolism was not affected by most of the substrates tested except by succinate which increased oxygen utilization, and glucose, fructose and octanoate which significantly decreased oxygen uptake. Since no significant changes have been observed in the cellular adenine nucleotide content during glucose depression of aerobic metabolism it is concluded that the glycolytic flux had to be sufficient as to account for the decrease in the mitochondrial energy production. The mechanism responsible for these effects as well as their physiological significance are discussed herewith.

Glucagon effect on rat liver protein synthesis in vivo.

Abstract The in vivo effect of glucagon administration on hepatic polyribosomal profiles has been studied. Glucagon did not change significantly total, free or bound polyribosomal fractions 30–45 minutes after its administration. The combined administration of glucagon plus antiinsulin serum failed to show any significant effect of glucagon over the antiinsulin serum treated control. Glucagon increased valine production in the perfused isolated liver. These results suggest that the well known amino acid catabolic action of glucagon may be preferentially mediated through an increased proteolysis. Since it is known that glucagon increases considerably in vivo the liver cyclic AMP levels then its lack of effect on polyribosomal profiles might indicate that the postulated role for the cyclic nucleotide on liver protein synthesis must be taken cautiously.

Relationship between cellular energy production and rates of glucose utilization by lung cells.

SummaryThe relationship between the cellular energetic state and glucose uptake by isolated lung cells has been studied by perturbing the oxidative metabolism by anoxia, treatment with uncouplers of the oxidative phosphorylation, inhibitors of the respiratory chain or inhibitors of the adenine nucleotide translocation across the mitochondrial membrane. Anoxia did not produce a significant decrease in the cellular ATP content or total adenine nucleotides. Inhibitors of the respiratory chain produced a fall of 60% in ATP content and a significant decrease in the [ATP]/[ADP] ratio. Dinitrophenol or atractyloside decreased ATP by 30% and produced also a milder decrease in the [ATP]/[ADP] ratio. Under none of these situations the glucose uptake was significantly enhanced, however, the percentage of glucose accounted for as lactate was increased. These observations, together with the fact that the intracellular glucose concentration was undetectable, seem to indicate that the glucose transport into cells, not the glycolytic flux as previously suggested, is the ratelimiting factor for its utilization, and that both events, glucose transport into the cells and glucose breakdown, are not under the same control mechanism. Anoxia, although it does not accelerate significantly the glucose uptake when a saturating dose of glucose was used, acts on lung cells lowering the apparent “Km” for glucose utilization. This effect, nevertheless, may not be very important from a physiological point of view since the “Km” for glucose uptake is well below the plasma glucose concentration.

Cellular metabolite distribution and the control of gluconeogenesis in the perfused isolated rat liver

SummaryGlucose production was measured in isolated rat livers perfused with 100 ml of blood-free recirculating medium. The gluconeogenic rate usingl-alanine as substrate was only 55% of that obtained withl-lactate.The steady-state concentration of gluconeogenic and tricarboxylic acid cycle intermediates were measured in freeze clamped biopsies. Livers perfused withl-lactate displayed higher concentrations of malate, α-glycerophosphate and β-hydroxybutyrate probably as a result of a higher state of reduction of the nicotinamide system. Hexose-phosphate intermediates were also increased whenl-lactate was the substrate. Phosphoenolpyruvate and 3-phosphoglycerate were considerably elevated whenl-alanine was the glucose precursor.Livers perfused withl-lactate displayed higher cytosolic concentration of all the tricarboxylic acid cycle intermediates except oxaloacetate while glutamate was slightly and aspartare considerably higher when alanine was the substrate. In the mitochondrial compartment the pattern of distribution tended to be the opposite; that is, livers perfused withl-lactate showed lower concentrations of all the intermediates except α-ketoglutarate. The mitochondrial: cytosolic metabolite gradients of all the intermediates whose distribution was studied were higher in livers perfused withl-alanine. The relevance of these findings to the observed differences in the gluconeogenic fluxes are discussed.

Factors controlling the metabolic response to glucose in isolated rat lung cells.

Abstract Glucose decreases the oxygen utilization by isolated rat lung cells. Its effect displays saturation type kinetics with a “Ki” of 2.2. mM. The similarity of this value with the reported “Km” of 2.4 mM described for glucose uptake by these cells, suggests that both processes may be intimately related and both of them are under the control of the same rate limiting step. Several arguments point to glucose transport into these cells as the most important rate limiting step for its utilization: 1) Phloridzin prevented glucose inhibition of oxygen uptake while mannoheptulose did not; 2) The activity of hexokinase which is the least active glycolytic enzyme in these cells far exceeded the observed rates of glucose utilization and a decrease of 45 per cent in its activity in starved animals did not affect the rate of glucose uptake; 3) The “Km” of hexokinase for glucose is two orders of magnitude below the observed “Km” for glucose uptake and the “Ki” for glucose inhibition of respiration.

On the mechanism of glucagon stimulation of hepatic gluconeogenesis

SummaryThe addition of L-alanine as substrate to a perfused rat liver preparation produced a five-fold increase in the rate of glucose production. This enhancement of the gluconeogenic flux seems to be a consequence of a rise in the steady-state levels of pyruvate and oxaloacetate subsequent to the rise in alanine concentration.Glucagon (2×10−9 M) increased the gluconeogenic flux from alanine (10 mM) by 50 percent, even though the concentration of the substrate in the perfusion fluid was at saturation. This effect was accompanied by a rise in the intracellular concentrations of alanine. However, the steady-state concentrations of pyruvate and oxaloacetate were decreased, probably as a consequence of a more reduced state of the nicotinamide-nucleotide system. In vivo, the intraperitoneal administration of glucagon to starved rats was accompanied by a decrease in the hepatic alanine and pyruvate concentrations despite the striking effects raising the plasma glucose levels. These observations seem to indicate that the effect of the hormone increasing the hepatic glucose output must be mediated through some other mechanism(s) independent of the intracellular variations in the hepatic amino acids levels.