Leif Sestoft

Regulation of fructose metabolism in the perfused rat liver. Interrelation with inorganic phosphate, glucose, ketone body and ethanol metabolism.

1. The regulation of fructose metabolism was investigated in rat liver using a non-recirculating perfussion system. 2. 1. At 25 mM fructose the rate of fructose uptake was 6.6 μmoles·min−1·g−1. Calculated per whole liver the rate of fructose uptake was independent of the nutritional state, whereas the rate of fructose uptake calculated per g liver increased 25% by 48 h fasting. 3. 2. The rate of fructose uptake was diminished in the first (20 min) period after addition of fructose. Glucose up to 25 mM did not significantly decrease the rate of fructose uptake indicating a minor contribution of hexokinase (ATP: d-hexone 6-phosphotransferase, EC 2.7.1.1) in phosphorylating fructose. The rate of fructose uptake was increased 25% by increasing the Pi concentration from 1 to 5 mM in the affluent medium. Ethanol caused a decreased in the rate of fructose phosphorylation. This was counterpoised by an increased rate of sorbitol production. 4. 3. 5 mM Pi caused an increased rate of O2 uptake with and without fructose, and the concentration of ATP during steady-state fructose metabolism was increased 25% which was enough to account for the increase in the rate of fructose uptake (ketohexokinase (ATP: d-fructose 1-phosphotransferase, EC 2.7.1.3) Km with ATP , 0.9 mM). 5. 4. At a high NAD redox level which was seen in the initial 10-min period after addition of fructose and after addition of ethanol, glycerol and sorbitol were released from the liver. The kinetics of sorbitol release reflected the intracellular fructose concentration. 6. 5. In livers of fed but not of fasted animals the release of glucose was inhibited in the initial 10-min period after addition of fructose. The inhibition is related to an increased inhibition of fructose 1-phosphate aldolase and fructose diphosphatase in the fed animal.

Kinetics of glycerol uptake by the perfused rat liver. Membrane transport, phosphorylation and effect on NAD redox level.

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by L-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K-m of the glycerol phosphorylation was 10 muM and the K-i of the L-glycerol 3-phosphate inhibition was 50 muM. The maximum activity (V) was 3.70 mumoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K-i was found to be somewhat lower in the intact organ. At low glycerol concentrations, a steep concentration gradient exists across the liver cell membrane. The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of L-glycerol 3-phosphate, not to the rate of glycerol uptake.

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver.

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high Km (67 mM) and V (30 μmoles · min−1 ·g−1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). Km with fructose 1.0 mM (0.7 mM), Km with ATP 0.54 mM (0.37 mM), V 10.3 μmoles · min−1 · g−1 (10.1 μmoles · min−1 · g−1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.

2Biochemistry and differential diagnosis of metabolic acidoses

Summary This paper reviews the biochemical background of metabolic acidoses. The rate of development is judged from production and/or elimination rate of organic acids, particularly carboxylic acids, namely lactate, ketoacids, acetate, formate and glycollate. Further, acid production from changes in the chemical state of phosphate in tissue is evaluated. The main conclusion is that pathological conditions with acidoses are always accompanied by changes in the rate of elimination of the carboxylic acids, whereas changes in the chemical state of phosphate is of quantitatively minor importance. Further, metabolic effects of metabolic acidoses are described withspecial reference to the effect of low pH in the extra cellular fluid on glycolysis, gluconeogenesis, lipolysis and ketogenesis. A short outline of the differential diagnostic problems in metabolic acidoses due to changes in carbohydrate and lipid metabolism or intoxication is given.

The influence of thyroid state on glycerol-induced hyperpolarization of the cell membranes in isolated, perfused rat liver

Abstract Intracellular electric potentials were measured in isolated livers from starved rats of different thyroid states. The potentials were identical in hypo-, eu- and hyperthyroid livers being −28.9, −29.1, and −29.8 mV, respectively. During a 15 min period with 2 mM glycerol in the perfusate, the intracellular negativity rose to approximately −36 mV in hypothyroid as well as in euthyroid livers, but no significant change occurred in hyperthyroid livers although these showed the highest rate of glycerol uptake. In the same period, hypo- and euthyroid livers accumulated respectively 9.0 and 4.3 μmol/g of l -glycerol 3-phosphate. The accumulation of this substance was only 0.6 μmol/g in hyperthyroid livers. The P i trapped in l -glycerol 3-phosphate balanced with the sum of P i taken up from the medium and P i made available from a decrease in the concentrations of P i and ATP in biopsies. The uptake of P i was closely accompanied by an uptake of K + which was 5.8 μequiv./g in hypothyroid livers, 3.3 μequiv./g in euthyroid livers, and 0.3 μequiv./g in hyperthyroid livers. The results seem to exclude that the glycerol-induced hyperpolarization of liver cells is an effect related to the transport of glycerol across the cell membranes. A correlation with metabolic events distinguished by their small prominence or absence in the hyperthyroid state appears more likely. However, the exact mechanism remains to be clarified since in the present experiments only a minor part of the hyperpolarization could be accounted for by the uptake of K + connected with l -glycerol 3-phosphate accumulation.

The Use of Tritium and 14C Labelled Ethanol in Studies of Ethanol Metabolism at High Ethanol Concentrations

Measurement of the rate of ethanol metabolism of the liver at concentrations at which the pharmacological actions of ethanol become manifest, i.e. 50–80 mM are of considerable importance. Such measurements can, however, not be performed with acceptable accuracy by determination of the decrease in ethanol concentration in preparations such as slices, isolated hepatocytes, or perfused liver, as the concentration differences are small compared to the absolute level. In the intact organism the overall metabolism of ethanol at high concentrations can be determined by measurement of the blood alcohol concentration at suitable intervals, but in this case we cannot decide in which organ the metabolism takes place. Extra-hepatic metabolism may play a more important role at high than at low ethanol concentrations. The obvious solution to this problem is to measure the products formed from ethanol, not the disappearance of the substrate.

Possibilities for Prediction of Blood Glucose and Improvement of Diabetes Control

Time series of blood glucose in diabetic patients and normal subjects measured at regular intervals over several days can be described adequately by autoregressive models of order 1 or 2 containing a 24 hour seasonal component. This opens new possibilities for characterization of blood glucose control, which can be used in clinical trials and for assessment of diabetic instability like the specially developed measures such as: M-value, MAGE, MODD and MBG. The time series models do, however, also enable a prediction of future blood glucose levels from present and near past measured values. Analysis of data from 10 normals, 8 diabetics treated with insulin injection, 9 treated with pump s.c. infused insulin and 5 non insulin dependent diabetics showed a large variation in the time series parameters between as well as within the groups. This emphasizes the need for individually adapted estimation and prediction. Using the model estimated from the first part of the individual time series the predicted and measured values were compared for the last few hours. It was found that a substantial part of the mean squared variation in blood glucose concentration could be predicted up to 12 hours ahead for most subjects.