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 Ca(2+)-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.

The effects of phosphate- and substrate-free incubation conditions on glycolysis in Ehrlich ascites tumour cells.

The influence of phosphate-free medium buffered with synthetic organic buffers, and of a preliminary incubation of cells in medium lacking added substrate ('pre-incubation') was investigated with mouse-cultured Ehrlich ascites tumour cells. In comparison to phosphate-containing bicarbonate-buffered balanced-salts medium, organic-buffered medium, without a preliminary substrate-free pre-incubation, was associated with 20-30% reduction in the rate of glycolysis, the 3- to 4-fold accumulation of fructose 1,6-bisphosphate and the halving of both ATP and total adenine nucleotide levels. These perturbations were reversed by the inclusion of 5 mM sodium phosphate in the organic-buffered medium. Pre-incubation for up to 90 min, before inclusion of glucose, resulted in greater depression of the glycolytic rate and concentrations of adenine nucleotides. This occurred in both the balanced-salts medium and the organic-buffered medium. During pre-incubation cells were lysed, releasing lactate dehydrogenase, when physically agitated too vigorously. It was concluded that the use of phosphate-free medium and pre-incubation are not advisable procedures for routine metabolic investigations with this cell line.

Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells.

The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes.

The capacity of reducing-equivalent shuttles limits glycolysis during ethanol oxidation.

The inhibition of glycolysis during ethanol oxidation has been examined in isolated hepatocytes from fasted rats. Glycolytic flux was measured by determining the rate of release of tritium from [6-3H]glucose. During ethanol oxidation, the rate of glycolysis was inhibited 80% in freshly prepared hepatocytes, in which shuttle intermediates are depleted, but was depressed only about 20% in the presence of asparagine, a condition under which activity of the malate/aspartate shuttle was restored to normal levels. The inhibition of glycolysis was also partially released by addition of pyruvate and when alcohol dehydrogenase activity was depressed by 4-methylpyrazole. Titrations with this inhibitor revealed inverse linear relationships between the rates of glycolysis and ethanol oxidation. For any given rate of ethanol oxidation, glycolytic flux was lowest and the [lactate]/[pyruvate] ratio highest in the presence of aminooxyacetate, an inhibitor of the malate/aspartate shuttle, whereas flux was highest and the ratio lowest in the presence of asparagine. During these titrations with 4-methylpyrazole the inhibition of ethanol oxidation and concomitant restoration of glycolysis were accompanied by a decline in the [lactate]/[pyruvate] ratio, a substantial fall in the rate of reducing-equivalent transfer from cytoplasm to mitochondria and an increase in lactate accumulation. These findings imply that the reducing equivalents generated during ethanol oxidation compete with those arising in glycolysis for transfer to the mitochondria. This competition leads to an inhibition of aerobic glycolysis, and at the same time contributes to a rise in cytoplasmic NADH and fall in NAD+ that results in depression of anaerobic glycolysis. Allosteric inhibition of 6-phosphofructo-1-kinase due to a decrease in the concentration of fructose 2,6-bisphosphate did not appear to play a primary role in the inhibition of glycolysis by ethanol. Ethanol oxidation had no effect on glucose phosphorylation as measured with [2-3H]glucose, but induced a substantial increase in cycling between glucose and glucose 6-phosphate.

Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes.

The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in isolated hepatocytes of fasted rats. Both lactate removal and glucose synthesis were inhibited by butylmalonate, aminooxyacetate or cycloserine confirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butylmalonate was considerably reduced, yet the transfer of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for butylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulatory effect on uptake of sorbitol, a substrate whose oxidation principally involves the alpha-glycerophosphate shuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potential (delta psi) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate oxidation was strongly inhibited when delta psi fell from 130 to 105 mV whereas O2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by delta psi. In the presence of added ammonium ions the removal of lactate was much less impaired by valinomycin, suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochondrial matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the butylmalonate-sensitive, energy-dependent, malate-aspartate shuttle.

Interactions between mitochondria and cytoplasm in isolated hepatocytes.

Results from a wide variety of metabolic studies have provided indirect support for conclusions derived from enzymological approaches that the enzymes of the so-called soluble cytoplasm (and the mitochondrial matrix) exist within the cell and function in the form of multienzyme complexes and that metabolite channeling takes place between the enzymes of each complex. Our studies support the possibility that the enzymes of glycolysis in liver are segregated from those of gluconeogenesis. Thus, the segregation and aggregation of Krebs cycle enzymes in the mitochondrial matrix, elucidated by Paul Srere, may be an example of a general pattern of enzyme organization pertaining to all metabolic pathways.

Role of mitochondria in hepatic fructose metabolism.

During metabolism of fructose at concentrations exceeding 5 mM, isolated liver cells accumulate fructose 1-phosphate and lose ATP. At added bicarbonate concentrations below 10 mM in the incubation medium, the addition of atractyloside (or carboxyatractyloside) causes a significant net accumulation of 2-phosphoglycerate, resulting in an increase in the ratio 2-phosphoglycerate: 3-phosphoglycerate from below 1 to greater than 5. Digitonin fractionation revealed that virtually all this 2-phosphoglycerate is associated with the mitochondrial fraction, where it achieves a concentration estimated to be about 40 mM. The amount of 2-phosphoglycerate that accumulates is directly related to the initial concentration of fructose. With DL-glyceraldehyde in place of fructose, an even greater accumulation of 2-phosphoglycerate occurs, and this is also dependent upon both the presence of atractyloside and low bicarbonate. Formation of 2-phosphoglycerate is also observed when isolated mitochondria from rat liver are incubated together with glyceraldehyde and an energy source. The obligatory role of atractyloside for the accumulation of 2-phosphoglycerate within intact cells indicates the involvement of the mitochondrial adenylate translocator in this process, possibly as a carrier directly responsible for 2-phosphoglycerate egress from the mitochondrial matrix. If this is so, competition between 2-phosphoglycerate and ATP for egress from the matrix would be predicted to further exaggerate the fructose-induced depletion of cytosolic ATP.

The cost of intensive and special care of the newborn.

The cost of providing intensive (level-3) and special (level-2) care for newborn infants in a tertiary perinatal service was determined prospectively and was expressed in 1984 Australian dollars. Direct costs that were expressed per occupied bed-day were $690 for level-3, high-dependency care; $421 for level-3, low-dependency care; $544 for over-all level-3 care; $242 for level-2, high-dependency care; $170 for level-2, low-dependency care; and $201 for over-all level-2 care. Each level of care generated additional costs of $42 per occupied bed-day. Taking these additional costs into account, the over-all occupied bed-day cost of level-3 and level-2 neonatal care was $339. The major components of this over-all cost were: nursing staff members, 50%; medical staff members, 11%; consumable and recyclable items, 12%; and diagnostic services, 8%.

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.

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.

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.

Preparation of cytoplasmic bodies (cytospheres) from isolated hepatocytes and their biochemical properties.

The controlled centrifugation of isolated rat hepatocytes at 260 000 g results in the formation of membrane-bounded cell fragments that we have termed 'cytospheres'. A method is described for the isolation of these cytospheres. Cytospheres are spherical, have a mean diameter of 9.2 +/- 3.2 microns (SD) and a protein content of 225 +/- 12 mg/g wet wt. About 3% of the protein from the original isolated hepatocyte suspension is recoverable. Transmission electron microscopy (TEM) shows cytospheres to possess a trilaminar membrane, and a finely granular hyaloplasm generally devoid of organelles, filaments and microtubules. Freeze-fracture studies reveal a membrane structure typical of a plasma membrane. Ouabain and wheat germ agglutinin (WGA)-binding studies indicate that the original orientation of the plasma membrane is maintained throughout the formation of the cytospheres. The cytospheres have also been characterized biochemically. Cytospheres are enriched in the enzymes normally associated with the hyaloplasm, whereas the activities of enzymes localized in organelles are greatly diminished. Lipid analysis of the cytosphere membrane indicates that it is derived from the plasma membrane of the hepatocyte. Cytospheres are sensitive to changes in the osmolarity and ionic composition of their environment. Cytospheres should therefore prove a useful preparation for the study of hyaloplasm metabolism and of plasma membrane receptor and permeability properties.

The contribution of hepatic metabolism to diet-induced thermogenesis.

In order to examine the possible contribution of the liver to diet-induced thermogenesis, we examined the metabolism of hepatocytes from rats that had been fed a varied choice of highly palatable human food items ("cafeteria feeding"). Liver cells derived from cafeteria-fed rats that had been fasted for 20 hours showed marked increases in rates of respiration and gluconeogenesis in the presence of glycerol or sorbitol. These cells were also much less sensitive to the inhibitory effects of rotenone than were hepatocytes of control animals. hepatocytes from fasted cafeteria-fed rats also demonstrated a substantially enhanced rate of fatty acid oxidation and ketogenesis, which did not appear to be correlated with cellular demands for adenosine triphosphate (ATP). This apparent fall in metabolic efficiency was confirmed by calorimetric studies, which indicated augmented cellular heat production. These changes in hepatic metabolism, associated with cafeteria-feeding, suggest that the liver may have a significant role in diet-induced thermogenesis.

Some unexplained features of hepatic ethanol oxidation.

Ethanol is an excellent substrate for the liver, competing effectively for oxidation with other substrates such as fatty acids. Using isolated liver cells from fed and starved rats, we have found that ethanol strongly inhibits Krebs cycle oxidations, so that the combustion through the cycle of acetyl CoA, derived from fatty acids, is reduced more than 50%. In contrast, fatty acid beta-oxidation to acetyl CoA is inhibited only 20% in fed and fasted states. Ethanol was not antiketogenic. In the fed state, octanoate but not palmitate inhibited ethanol oxidation whereas in cells from fasted rats palmitate inhibited ethanol oxidation. Gluconeogenesis from lactate was reduced 50% in hepatocytes from fasted rats but oxygen consumption was unaffected. This paradoxical maintenance of oxygen consumption in a state where the only overt need for ATP synthesis is depressed, suggests that ethanol oxidation may not be exclusively coupled to ATP synthesis but also can be linked to other energy transducing processes.

Biological significance of compartmentation of hepatic ethanol oxidation.

Intact cell preparations, isolated from the livers of fasted rats and treated with rotenone or antimycin A, retain a limited ability to oxidize long chain fatty acids but not short chain species or other usual substrates. Cells prepared from livers of animals treated with clofibrate to induce peroxisomal proliferation have even greater long chain fatty acid oxidizing capacity in the presence of these inhibitors. We infer that the peroxisomes of intact cells are capable of catabolizing long chain fatty acids by a superoxide-generating pathway. In normal cells or those from clofibrate-treated rats, reducing equivalents generated within peroxisomes compete with those originating in the cytosol for mitochondrial disposition. Because of this, peroxisomal fatty acid metabolism inhibits ethanol oxidation. Peroxisomal oxidations appear to be coupled to conversation of free energy and mitochondrial-peroxisomal relationships are regulated by interaction of free-energy transducing processes. Hence, uncoupling agents release the inhibition of ethanol oxidation induced by long chain fatty acid. When mitochondrial metabolism is impaired, reducing equivalents may flow from mitochondria to peroxisomes for reaction with O2. Thus, there exists a two-way interaction between these organelles. The biological and pathological implications of these relationships for ethanol oxidation and overall energy metabolism are discussed.

League tables of biochemical laboratory costs. An attempt to modify requesting patterns.

"League tables", ordered by potential cost of biochemical laboratory tests requested, were distributed at four-week intervals to all medical specialists of a 500-bed tertiary care teaching hospital, Flinders Medical Centre, Adelaide, in an attempt to reduce the workload of the laboratory. This information feedback did not appear to have any effect on over-all laboratory utilisation or on any individual requesting pattern. However, much useful information on laboratory use was collated and summarised. It appears that more active intervention is required to eliminate unnecessary requesting of laboratory tests.

Effect of feedback to clinical staff of information on clinical biochemistry requesting patterns.

Attempting to reduce the number of unnecessary tests requested, we initiated a program providing continuous feedback to clinicians concerning their requesting patterns of clinical biochemistry laboratory tests. At four-week intervals each medical specialist received data showing the number and costs of tests requested by all members of his team, the number of patients who had tests, and various related information. Comparative summaries, in frequency-histogram format, of the number of specimens submitted and the costs involved allowed each medical specialist to rate his own performance against that of his peers. The program appeared to have no effect on laboratory use, although reliable information concerning workload patterns was accrued. The degree of education of medical students in clinical biochemistry did not appear to influence the use they made of the laboratory after qualification. The specimens per bed-day ratio is suggested as a useful parameter in the identification of inappropriate laboratory use and as a standard parameter for comparing laboratory use in different hospitals.