Michael N. Berry

Techniques for pharmacological and toxicological studies with isolated hepatocyte suspensions

Since its introduction in 1969, the high-yield preparation of isolated hepatocytes has become a frequently used tool for the study of hepatic uptake, excretion, metabolism and toxicity of drugs and other xenobiotics. Basic preparative methods are now firmly established involving perfusion of the liver with a balanced-saline solution containing collagenase. Satisfactory procedures are available for determining cell yields, for expressing cellular activities and for establishing optimal incubation conditions. Gross cellular damage can be detected by means of trypan blue or by measuring enzyme leakage, and damaged cells can be removed from the preparation. Specialized techniques are available for preparing hepatocyte couplets and suspensions enriched with periportal or perivenous hepatocytes. The isolated hepatocyte preparation is particularly convenient for the study of the kinetics of hepatic drug uptake and excretion because the cells can be rapidly separated from the incubation medium. Isolated liver cells have also proved valuable for investigating drug metabolism since they show many of the features of the intact liver. However, they also show important differences such as losses of membrane specialization, some degree of cell polarity and the capacity to form bile. The many consequences of the hepatic toxicity of xenobiotics including lipid peroxidation, free radical formation, glutathione depletion, and covalent binding to macromolecules are also readily studied with the isolated liver cell preparation. A particular advantage is the ease with which morphological changes as a result of drug exposure can be observed in isolated hepatocytes. However, it must be remembered that the isolation procedure inevitably introduces changes that may make the cells more susceptible than the normal liver to damage by xenobiotic agents. Despite its limitations, the isolated hepatocyte preparation is now firmly established in the armamentarium of the investigator examining the interaction of the liver with xenobiotics.

Isolated hepatocytes - past, present and future

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca2+-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase.The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: Further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.

Linear relationships between mitochondrial forces and cytoplasmic flows argue for the organized energy‐coupled nature of cellular metabolism

We have studied rates of formation of glucose, urea and lactate by isolated hepatocytes incubated with a variety of inhibitors of energy transduction. Linear relationships have been found between these metabolic rates and mitochondrial forces (membrane, redox and phosphorylation potentials). The findings are suggestive of extensive enzyme organization within these metabolic pathways.

Effects of fatty acid oxidation on glucose utilization by isolated hepatocytes

We have studied the inhibitory action of long‐ and short‐chain fatty acids on hepatic glucose utilization in hepatocytes isolated from fasted rats. The rates of hepatic glucose phosphorylation and glycolysis were determined from the tritiated products of [2‐3H] and [6‐3H]glucose metabolism, respectively. The difference between these was taken as an estimate of the ‘cycling’ between glucose and glucose‐6‐phosphate. In the presence of 40 mM glucose this cycling was estimated at 0.68 μ;molmin/g wet wt. Glucose phosphorylation was unaffected during palmitate and hexanoate oxidation to ketone bodies but glycolysis was inhibited. The rate of glucose cycling was increased during this phase to 1.25 μmol/min > g. Following the complete metabolism of the fatty acids, glycolysis was reinstated and cycling rates returned to control levels. Hepatic glucose cycling appears to be an important component of the glucose/fatty acid cycle.

On the thyroid hormone-induced increase in respiratory capacity of isolated rat hepatocytes

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.

The administration of triiodothyronine to rats results in a lowering of the mitochondrial membrane potential in isolated hepatocytes.

Although thyroid status has been shown to influence the magnitude of the membrane potential in isolated rat-liver mitochondria, there is variation in the reported size and direction of the thyroid hormone-induced change relative to the normal state. Measurement of the mitochondrial membrane potential in intact hepatocytes isolated from hyperthyroid and euthyroid rats reveals that hyperthyroidism results in a decrease of approximately 30 mV in the magnitude of this potential relative to that in the euthyroid state. As well, the magnitude of the plasma membrane potential of hepatocytes from hyperthyroid rats is reduced by 6 mV compared with that in cells from euthyroid rats. The thyroid hormone-induced decrease in these potentials may reflect reported changes in the lipid composition of the membranes.

The characterization of perfluorosuccinate as an inhibitor of gluconeogenesis in isolated rat hepatocytes

The effects on metabolism of the fluorinated dicarboxylic acid, perfluorosuccinate, were examined in hepatocytes from fasted rats. Perfluorosuccinate (5 mM) inhibited gluconeogenesis from lactate by 80% and from pyruvate by 40%. Significant inhibition (up to 30%) occurred at a concentration of perfluorosuccinate of 50 microM. Cellular ATP levels were not affected by perfluorosuccinate, nor was the rate of formation of ketone bodies from palmitate, although the ratio [3-hydroxybutyrate]/[acetoacetate] was increased up to 5-fold relative to the control. An increased concentration of cellular L-malate was measured in the presence of perfluorosuccinate but this did not reflect inhibition of malate transport between the mitochondrial and cytoplasmic compartments. In addition, ethanol oxidation by hepatocytes was inhibited 25% by 1 mM perfluorosuccinate. Ureogenesis from ammonia was relatively insensitive to inhibition by perfluorosuccinate. In cytoplasmic extracts of rat liver, the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase were inhibited 40-50% and 23%, respectively, by 1 mM perfluorosuccinate. The observed metabolic effects of perfluorosuccinate are consistent with inhibition of the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase within the cytoplasm.

Evidence that stimulation of gluconeogenesis by fatty acid is mediated through thermodynamic mechanisms

We have studied the stimulatory effects of palmitate on the rate of glucose synthesis from lactate in isolated hepatocytes. Control of the metabolic flow was achieved by modulating the activity of enolase using graded concentrations of fluoride. Unexpectedly, palmitate stimulated gluconeogenesis even when enolase was rate‐limiting. This stimulation was also observed when the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase were modulated using graded concentrations of quinolinate and aminooxyacetate, respectively. Linear force‐flow relationships were found between the rate of gluconeogenesis and indicators of cellular energy status (i.e. mitochondrial membrane and redox potentials and cellular phosphorylation potential). These findings suggest that the fatty acid stimulation of glucose synthesis is in part mediated through thermodynamic mechanisms.

Energy-dependent regulation of the steady-state concentrations of the components of the lactate dehydrogenase reaction in liver

It has been known for some years that the components of the lactate dehydrogenase reaction in liver are maintained in a steady-state believed to be close to thermodynamic equilibrium [ 1,2]. The ratio, [lactate]/[pyruvate] has thus been considered to reflect the ‘redox state’ (the ratio of ‘free’ [NAD]/ [NADH]) within the cytoplasmic compartment of the hepatic cell [3]. Analogous investigations of mitochondrial dehydrogenase systems in liver have led to the conclusion that the mitochondrial redox state is -lOO-times more reduced than that of the cytoplasm [3]. It has been pointed out that because of these differences in redox state, the transfer of reducingequivalents from cytoplasmic to mitochondrial NAD(H) pools would be against the electrochemical potential gradient and therefore likely to be energydependent [4,5]. However, few relevant experimental observations using whole cell preparations have been described. The work presented here provides evidence for a direct involvement of energy in the maintenance of the steady-state [lactate]/[pyruvate] ratio in.isolated liver cells.