Effects of ethanol on sodium, 3-O-methyl glucose, and L-alanine transport in the jejunum

Effects of ethanol on Na+, Cl-, 3-O-methyl glucose (3-O-MG), and L-alanine fluxes were studied in the isolated rattit jejunal mucosa. Ethanol (3% v/v present on both sides of the mucosa) decreased electrical potential difference (PD), short-circuit current (Isc) and inhibited active transport of Na+, 3-O-MG, and L-alanine. This concentration also increased the permeability of the mucosa for Cl-, 3-O-MG, and L-alanine. Ethanol at 5.4% potentiated the effects on PD, Isc, and the permeability for electrolytes and organic substances. These effects of ethanol could not be fully explained by an osmotic action.     

The effect of acute uraemia on gluconeogenesis in isolated perfused rat livers

Abstract. The effect of acute uraemia on glucose and urea formation by isolated perfused livers of fasting rats was investigated. The basal gluconeogenesis following nephrectomy was significantly increased as compared to normal and sham operated controls. Enhanced glucose formation was associated with an increase in both urea synthesis and output of the poorly metabolizable amino acids valine, leucine, and isoleucine. In the presence of a mixture of amino acids (serine, threonine, lysine, glutamic acid, ornithine, and citrulline) all added at near physiological concentrations, the stimulating effect of uraemia on gluconeogenesis was further enhanced. This was paralleled by an increased formation of urea and an increased uptake of the amino acids. It is concluded that acute uraemia may stimulate glucose synthesis by increased substrate supply. This seems to be achieved by at least two different mechanisms, namely increased protein degradation and accelerated amino acid utilization.     

Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin.

Primary cultured adult rat hepatocytes were used to study regulation of thyroid hormone deiodination. Control studies showed that these cells survived for at leas 4 d, during which time they actively deiodinated both the phenolic (5'-) and non-phenolic (5-) rings of L-thyroxine (T4),3,5,3'-triiodo-L-thyronine, and 3,3',5'-triiodothyronine. Increasing the substate concentration caused a decrease in fractional iodide release and a corresponding increase in conjugation with sulfate and glucuronide. Propylthiouracil strongly inhibited the 5'-deiodinase activity and caused only a slight decrease in 5-deiodinase activity. Thus, these monolayer-cultured cells preserved many of the properties of normal hepatocytes. Incubation with combinations of insulin, glucagon, and/or glucose for 5 h showed that insulin stimulated T4 5'-deiodination, whereas glucagon inhibited the insulin stimulation but had no effect in the absence of insulin. Glucose had no effect and did not alter the effect of the hormones. The insulin-enhanced deiodination increased between 1 and 5 h, which suggests that the previous inability to demonstrate an insulin effect was due to the short survival of the in vitro liver systems used in those studies. The present data suggest that the inhibition of T4 5'-deiodination observed during fasting, and its restoration by refeeding, may be related to the effects of feeding on insulin and glucagon release rather than on glucose per se.     

Mitochondrial function of isolated rat hepatocytes from normal and cirrhotic liver

Mitochondrial fractions were obtained from purely isolated hepatocytes of the normal and cirrhotic livers. Mitochondrial function of isolated hepatocytes was evaluated to compare between those from the normal and those from the cirrhotic livers in addition to the evaluation of the mitochondrial function of the normal and cirrhotic liver tissues. Respiratory control, ADP/O ratio and ATP synthesis were significantly lower in the cirrhotic liver tissue than those in the normal liver tissue. However, the mitochondrial function of isolated hepatocytes showed no difference between normal and cirrhotic groups. By electron microscopic examination, debris was more observed in the mitochondrial fractions from the normal and cirrhotic tissues compared to those from hepatocytes. However, no difference of the shape and size of the mitochondria was seen between the fraction from hepatocytes and that from the tissue in each group. These results suggest that as far as hepatocyte itself is concerned, the cirrhotic liver preserves the sufficient function as well as the normal liver.     

Effects of chronic sepsis on the voltage-gated sodium channel in isolated rat muscle fibers

OBJECTIVE: Physiopathology of critical illness polyneuromyopathy was investigated in several animal-based models. Electrophysiologic approach was achieved in denervated and corticosteroid-induced myopathy; other models based on sepsis or inflammatory factors (zymosan, cytokines) were also used but did not consider voltage-gated sodium channel implication in neuromuscular weakness. We have studied electrophysiologic effects of chronic sepsis on an intact neuromuscular rat model with special consideration to the subtypes of sodium channels involved. DESIGN: Experimental animal study. SETTING: University laboratory. SUBJECTS: Wistar rats. INTERVENTIONS: Chronic sepsis was achieved by a technique of cecal ligature and needle perforation. Ten days after surgery, the rats were killed. Fast-twitch flexor digitorum brevis was excised and dissociated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline supplemented with 3.0 mg/mL collagenase. Fast sodium currents were recorded by a macropatch clamp technique at room temperature (22+/-2 degrees C) in a cell-attached configuration. MEASUREMENTS AND MAIN RESULTS: A decrease in maximal sodium current and in conductance was evidenced without modification of the sodium Nernst potential. A shift of the voltage inactivation curve toward more negative potentials could explain the observed decrease in excitability. In parallel, we observed an up-regulation of NaV 1.5-type sodium channels. CONCLUSIONS: Chronic inflammation and sepsis induced modifications of sodium channel properties that could contribute to muscular inexcitability. This inexcitability can be elicited by a modification of properties or type of voltage-gated sodium channels. Our results lead us to explain this inexcitability by an up-regulation of NaV 1.5 sodium channel.     

Effects of chronic norepinephrine administration on sympathetic neurotransmission in the isolated perfused rat kidney

The effects of chronic NE administration (100 micrograms/kg/hr s.c., 6 days) on the stimulus-induced overflow of neurotransmitter from the isolated perfused rat kidney were examined. This treatment increased renal NE content and increased the absolute stimulus-induced overflow of NE. The increase in absolute overflow was not simply the result of the increase in renal NE content as fractional overflow was also increased slightly (20%). Alpha adrenoceptor blockade with phentolamine eliminated the NE treatment-induced difference in fractional overflow. However, the dose-response curves to phentolamine and the alpha-2 adrenoceptor agonist UK 14,304 on stimulus-induced overflow from the kidney were not significantly different after NE treatment. Chronic EPI treatment (same dose) produced an 80% increase in fractional stimulus-induced overflow of neurotransmitter but the dose-response curve to UK 14,304 was shifted only slight to the right (3-fold) of the control curve. No influence of prejunctional beta adrenoceptors on stimulus-induced overflow was observed in either the control of the NE-treated group. In conclusion, the data regarding fractional overflow and the effects of the phentolamine suggest that NE treatment produces minimal change whereas EPI treatment produces marked decreases in the influence in prejunctional alpha adrenoceptors. However, although the data with UK 14,304 after NE treatment support this conclusion, the failure of EPI treatment to alter the dose-response curve to UK 14,304 does not. The apparent contradiction of the results with the alpha adrenoceptor agonist in the EPI-treated group as well as the large differences between the effects of NE vs. EPI treatment on stimulus-induced overflow and prejunctional alpha adrenoceptor function cannot be explained at this time.     

Effects of fasting on insulin binding, glucose transport, and glucose oxidation in isolated rat adipocytes: relationships between insulin receptors and insulin action.

Insulin binding, glucose transport, and glucose oxidation were studied in isolated adipocytes obtained from fasting rats. Fasting led to an increase in the overall binding affinity for insulin, while the number of receptor sites per cell remained constant. Glucose oxidation was markedly attenuated during fasting. Basal rates of oxidation decreased by about 50%, while insulin-stimulated rates decreased 6 to 10-fold. Glucose transport was assessed by measuring initial uptake rate of 2-deoxy-glucose. Fasting led to a 40-50% decrease in the apparent maximal transport capacity (Vmax) of 2-deoxy-glucose uptake with no change in apparent Km. A progressive decrease in basal and insulin-stimulated rates of 2-deoxy-glucose uptake was seen from 24-72 h of starvation and a significant correlation (r=0.85, P less than 0.001) existed between basal and maximal insulin-stimulated uptake rates in individual animals. When 2-deoxy-glucose uptake was plotted as a function of insulin bound, due to the decrease in maximal uptake capacity, cells from fasting animals took up less hexose for any amount of insulin bound. When the insulin bound was plotted as a function of the percent insulin effect on uptake, control cells and cells from 24-h-fasted rats gave comparable results, while cells from 48- and 72-h-fasted animals still took up less hexose for any amount of bound insulin. The effects of fasting on 3-O-methyl glucose uptake were comparable to the 2-deoxy-glucose data. In conclusion: (a) insulin binding is increased during fasting due to an increased overall binding affinity with no change in receptor number; (b) glucose oxidation is severely impaired during fasting; (c) 2-deoxy-glucose uptake decreases with fasting due to a decrease in maximal transport capacity (Vmax) with no change in Km; (d) the decrease in glucose oxidation is much greater than the decrease in glucose transport, indicating impaired intracellular oxidative metabolism; and (e) coupling between insulin receptors and the glucose transport system is normal after 24 h of fasting but is impaired at 48 and 72 h.     

Effect of dexamethasone on insulin binding, glucose transport, and glucose oxidation of isolated rat adipocytes.

We have studied the in vitro effects of dexamethasone on isolated rat adipocytes at concentrations of dexamethasone therapeutically achieved in man. Glucose oxidation, glucose transport, and insulin binding were assessed. In dexamethasone-treated cells, glucose oxidation was decreased by 30-40% both in the absence of insulin (basal state) and at low insulin levels (less than 25 mu/ML). At maximally effective insulin levels (over 100 muU/ml) no differences existed between control and treated cells. If glucose transport were the rate-limiting step for glucose oxidation in the basal state and at low (submaximal) insulin levels, but not at maximally effective insulin concentrations, then these data could be explained by postulating that dexamethasone has a direct affect on glucose transport and does not affect intracellular oxidative pathways. We tested this hypothesis by directly assessing glucose transport in dexamethasone-treated cells. Glucose transport was assessed by measuring the uptake of [14C]2-deoxy glucose. These studies demonstrated a 30-40% decrease in 2-deoxy glucose uptake by treated cells both in the basal state and at all insulin concentrations. Thus, a direct glucocorticoid effect on the glucose transport system seems to account for the decreased ability of dexamethasone-treated cells to oxidize glucose. Since dexamethasone treatment leads to decreased insulin binding to adipocytes in vivo, we examined the possibility that the in vitro decreases in insulin-mediated glucose transport could be due to decreased insulin receptors. Insulin binding to control and treated adipocytes was measured, and no differences were found. Therefore, in cntrast to previously reported in vivo studies, adipocytes treated in vitro with dexamethasone retain a normal ability to bind insulin. Thus, these studies suggest that all of the in vitro effects of dexamethasone on glucose oxidation are due to direct inhibition of the glucose transport system.     

The effect of arginine vasopressin on ureagenesis in isolated rat hepatocytes

The effect of arginine vasopressin (AVP) on ureagenesis was measured in isolated rat hepatocytes with ammonium chloride and L(+)-lactate as substrates. AVP was found to stimulate urea synthesis and the dose-response curve suggests that such an effect is present at concentrations of the hormone as low as 25-50 pmol/l. Both the dose-response curve and the concentrations of NH+4 employed suggest that the effect observed could be of physiological significance.     

Ethanol inhibition of codeine and morphine metabolism in isolated rat hepatocytes

Suspensions of isolated hepatocytes from male Wistar rats were prepared according to a two step Ca++-free collagenase perfusion method. Codeine, morphine or norcodeine were incubated with hepatocytes at 37 degrees C for up to 90 min in the absence and presence of ethanol. The elimination rate constant (Kel) of codeine and morphine was reduced with approximately one-third and one-fourth, respectively, in the presence of 60 mM ethanol, whereas the presence of ethanol did not alter the Kel of norcodeine significantly. The inhibition of codeine metabolism was dose-dependent, extending from approximately 15% at 10 mM ethanol to 40 to 50% at 100 mM. A 3-fold increase in the ratio of morphine concentration (formed from codeine) to the amount of codeine metabolized was observed in the presence of ethanol as compared to control cells. The mean morphine concentration was 170% higher in the ethanol-treated suspensions than in the controls. The ratio of norcodeine concentration to codeine metabolized was unchanged. The inhibition of morphine metabolism was accompanied by a similar reduction of morphine-3-glucuronide formation. The accumulation of morphine observed in the cell medium in the presence of ethanol might be due to inhibition of other metabolic pathways from codeine, thus shunting to morphine formation, combined with the inhibitory effect of ethanol on morphine metabolism per se.     

Insulin resistance in uremia. Characterization of insulin action, binding, and processing in isolated hepatocytes from chronic uremic rats.

We have developed a model in the rat that leads to a predictable degree of severe uremia to study the role of the liver in the insulin-resistant state of uremia. The uremic animals were euglycemic and had increased serum immunoreactive insulin when compared with their pair-fed controls. Insulin action, binding, internalization, and degradation were characterized in freshly isolated hepatocytes from uremic animals, sham-operated pair-fed, and ad lib.-fed controls. The basal rate of aminoisobutyric acid (AIB) uptake was increased in hepatocytes from both uremic and pair-fed control rats. However, while hepatocytes from uremic animals were refractory to insulin with regard to AIB uptake, there was no significant difference in the absolute increment above basal AIB uptake by hepatocytes from pair-fed and fed ad lib. animals at any insulin concentration studied. 125I-Insulin binding at 24 degrees C was higher in hepatocytes from uremic rats at every insulin concentration studied when compared with fed ad lib. controls. The time course of 125I-insulin binding to the cell and to the fractions that were membrane bound or internalized were studied at 37 degrees C. An increase in membrane-bound 125I-insulin at 37 degrees C was present also in hepatocytes from uremic animals. The same fraction of membrane-bound 125I-insulin was internalized in hepatocytes from all groups of animals. Extracellular and receptor-mediated 125I-insulin degradation at the plasma membrane and after internalization was studied at 37 degrees C by gel chromatography. There was a delayed and decreased rate of 125I-insulin degradation in hepatocytes from uremic rats in the three compartments. We conclude: (a) In chronic uremia the liver is refractory to insulin with regard to AIB uptake. (b) Insulin resistance in uremic rat liver is not due to defects in insulin binding or internalization. (c) Despite the high level of circulating immunoreactive insulin, hepatocytes from uremic rats did not show the expected "down regulation" of their insulin receptors or an increased rate of insulin degradation. These studies further emphasize the primary role of postbinding events in the regulation of insulin binding and degradation. The mechanism as to how the coordinated steps of insulin metabolism in the liver are disrupted in a pathological state is presently unknown.     

The effects of spontaneous obesity on insulin binding, glucose transport, and glucose oxidation of isolated rat adipocytes.

We have studied insulin, binding, glucose transport, and glucose oxidation, using large adipocytes isolated from older, fatter rats (greater than 12-mo-old, greater than 550 g), and smaller cells obtained from younger, leaner animals (4-5-wk-old, 120-160 g). At media glucose levels less than 5 mM, basal (absence of insulin) rates of glucose oxidation are comparable in both groups of cells. However, in the presence of insulin, the increase in glucose oxidation is much greater in the smaller cells. Maximally effective insulin levels could not overcome the defect in glucose oxidation by larger cells, and thus, even though studies of insulin binding demonstrated a 30-40% decrease in insulin receptors on the larger cells, it is probable that the defect in glucose oxidation is distal to the insulin receptor. Glucose transport was assessed by direct measurement of 2-deoxy glucose uptake. Basal levels of uptake were greater for the larger cells, whereas at maximally effective insulin concentrations, rates of 2-deoxy glucose uptake were the same for both groups of cells. Thus, in the presence of maximally effective levels of insulin, the apparent Km (2.3-2.7 mM) and Vmax values (2.6 and 2.7 nmol/10(5) cells per min) of 2-deoxy glucose uptake were comparable, indicating that the glucose transport system of the larger cells was intact. However, at submaximal levels of insulin, small adipocytes took up more 2-deoxy glucose than larger cells. These findings represent a rightward shift in the insulin dose-response curve in the cells from the older, fatter animals, and this is the predicted functional sequelae of the observed decrease in insulin receptors. Finally, when the amount of insulin bound was plotted as a function of 2-deoxy glucose uptake, no difference was seen between both groups of cells. This indicates that coupling between insulin receptor complexes and the glucose transport system is intact in large adipocytes, and is further evidence that a defect(s) in intracellular glucose metabolism is responsible for the decrease in glucose oxidation of adipocytes from older, fatter rats. In conclusion: (a) insulin-mediated glucose oxidation is markedly decreased in large adipocytes from older, fatter rats, and since this decrease cannot be corrected by maximally effective insulin levels, the defect is probably distal to the insulin receptor; (b) the glucose transport system is basically normal in large adipocytes; (c) insulin binding to receptors is decreased in large cells and the functional sequelae of this decrease in insulin binding i.e., a rightward shift in the insulin dose-response curve for 2-deoxy glucose uptake, was observed, and (d) since the decreased rates of insulin-mediated glucose oxidation can not be attributed to changes in insulin receptors or to changes in glucose transport, an intracellular defect in glucose metabolism is suggested.     

Antinuclear antibodies: analysis of images and classification of immunofluorescence aspects on nuclei isolated from rat hepatocytes

Image analysis and Fourier transformation of the various nuclear immunofluorescence patterns observed while detecting antinuclear antibodies allow an objective and quantitative definition of the fluorescence. They also point out various IF types hidden by the main pattern, without having to dilute the test serum. They make obvious the difference between speckled and reticular patterns, and reveal the existence of intermediate states. The usual nuclear IF patterns (homogeneous, ring, nucleolar, reticulated, speckled and diffuse) may be grouped, according to their photo emission, into nuclear and subnuclear patterns. The first group includes homogeneous, annular and passive nucleolar IF. The second group is composed of speckled, reticulated, mixed, and active nucleolar IF. Alternatively, these aspects may be grouped into three types: homogeneous nuclear IF (homogeneous and ring), heterogeneous nuclear IF (speckled, reticulated and mixed) and nucleolar IF (active or passive). Diffusion can affect or not these aspects and does not apply to a special type or pattern. Image analysis and the study of the image spatial spectrum lead to automated recognition of the IF types, and later on, to the discrimination of antinuclear antibodies.     

Nonlinear elimination kinetics of 5-fluoro-2'-deoxyuridine in isolated perfused rat liver and isolated hepatocytes

The kinetic parameters of the cytostatic agent 5-fluoro-2'-deoxyluridine (FUDR) were studied in isolated rat hepatocytes and in the isolated perfused rat liver. In both experimental setups a dose dependency of the elimination parameters, half-life and clearance, was observed with a calculated turning point around 250 microM. In the medium of rat hepatocytes incubated at low (0.1 microM) to high (2000 microM) FUDR, the majority of the metabolites consisted of the catabolite alpha-fluoro-beta-alanine. The nucleobase metabolites, 5-fluorouracil and its primary product 5,6-dihydro-5-fluorouracil, approached apparent steady-state levels comprising 10 to 15% of the initial concentration. In the intracellular phase of hepatocytes incubated at 300 microM FUDR almost 90% of the FUDR-derived material was alpha-fluoro-beta-alanine, whereas essentially no unchanged FUDR could be detected. Similar results were obtained at extracellular FUDR concentrations exceeding 300 microM. In the isolated perfused rat liver, the clearance decreased to 15 to 20% of the corresponding values when the initial concentration was raised from 24 to 2400 microM. At the end of perfusion alpha-fluoro-beta-alanine comprised 90 to 95% of FUDR-derived total radioactivity in the tissue even at initially 2400 microM FUDR, although at this FUDR dosage 20% of the substrate remained unmetabolized in the medium. These results suggest that the limitation of hepatic FUDR elimination is not due to saturable hepatic metabolism but must be due to saturable uptake of these pyrimidine derivatives across the cellular membrane of parenchymal liver cells.     

Potentiation by piridoxilate of the synthesis of hippurate from benzoate in isolated rat hepatocytes. An approach to the determination of new pathways of nitrogen excretion in inborn errors of urea synthesis

The synthesis of hippurate from benzoate as compared to ureagenesis has been investigated in isolated rat hepatocytes. Half-maximal synthesis of hippurate was observed at 0.3 mmol/l benzoate. In the presence of 1 mmol/l benzoate, hippurate synthesis proceeded linearly with time at a rate of 40 +/- 10 mumol X h-1 X g-1 dry weight. This provided less than 10% of nitrogen epuration supported by concomitant urea synthesis (350 +/- 82 mumol X h-1 X g-1 dry weight). The incorporation of benzoate to hippurate was markedly limited by the availability of glycine. Half-maximal hippurate synthesis was observed at 2 mmol/l glycine. In the absence of glycine, piridoxilate, a glyoxylate derivative, markedly potentiated hippurate synthesis. Half-maximal stimulation was observed at 10 mmol/l piridoxilate. In the presence of 10 mmol/l piridoxilate, hippurate synthesis (420 +/- 35 mumol X h-1 X g-1 dry weight) provided more than 50% of nitrogen epuration supported by urea synthesis. It is concluded that supplementation with nitrogen-free analogues of glycine such as piridoxilate are required to potentiate hippurate synthesis in an attempt to replace ureagenesis as an alternative pathway of waste nitrogen excretion in inborn errors of urea synthesis.