Insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. Synthesis of a high affinity component of transport.

Insulin and glucagon stimulate amino acid transport in freshly prepared suspensions of isolated rat hepatocytes. The kinetic properties of alpha-amino[1-14C]isobutyric acid (AIB) transport were investigated in isolated hepatocytes following stimulation by either hormone in vitro. In nonhormonally treated cells (i.e. basal state), saturable transport occurred mainly through a low affinity (Km approximately equal to 40 mM) component. In insulin or glucagon-treated hepatocytes, saturable transport occurred through both a low affinity component (similar to that observed in the basal state) and a high affinity (Km approximately equal to 1 mM) component. At low AIB concentrations (less than 0.5 mM), insulin and glucagon at maximally stimulating doses increased AIB uptake about 2-fold and 5-fold, respectively. The high affinity component induced by either hormone exhibited the properties of the A (alanine preferring) mediation of amino acid transport. This component required 2 to 3 h for maximal expression, and its emergence was completely prevented by cycloheximide. Half-maximal stimulation was elicited by insulin at about 3 nM and by glucagon at about 1 nM. Dibutyryl cyclic AMP mimicked the glucagon effect and was not additive to it at maximal stimulation. Maximal effects of insulin and glucagon, or insulin and dibutyryl cyclic AMP, were additive. We conclude that insulin and glucagon can modulate amino acid entry in hepatocytes through the synthesis of a high affinity transport component.     

Glucocorticoid regulation of amino acid transport in anucleate rat hepatoma (HTC) cells

The transport of alpha-aminoisobutyric acid (AIB) by rat hepatoma tissue culture (HTC) cells is rapidly and reversibly inhibited by dexamethasone and other glucocorticoids. To investigate the role of the nucleus in the regulation of transport and to determine whether steroid hormones or steroid-receptor complexes may have direct effects on cytoplasmic or membrane functions, we have examined the regulation of transport by dexamethasone in anucleate HTC cells. Cytoplasts prepared from suspension cultures of HTC cells fully retain active transport of AIB with the same kinetic properties as intact cells. However, the uptake of AIB is not inhibited by dexamethasone or other corticosteroids. Neither is the inhibited rate of transport, manifested by cytoplasts prepared from dexamethasone-treated cells, restored to normal upon removal of the hormone. Anucleate cells exhibit specific, saturable binding of [3H]dexamethasone; however, the binding is reduced compared with that of intact cells. The nucleus is thus required for the glucocorticoid regulation of amino acid transport in HTC cells.     

A Na+-DEPENDENT SYSTEM A AND ASC-INDEPENDENT AMINO ACID TRANSPORT SYSTEM STIMULATED BY GLUCAGON IN RAT HEPATOCYTES

The effects of glucagon on amino acid transport in rat hepatocytes are not fully understood. We examined the effect of this hormone on alanine, serine and cysteine preferring system (system ASC)-mediated amino acid transport in rat hepatocyte monolayers using 2-aminoisobutyric acid (AIB) and L -cysteine. Glucagon induced a time and protein synthesis-dependent stimulation of Na⁺-dependent alanine preferring system (system A)-independent AIB transport. The glucagon-induced increase in transport activity was not modified by substrate starvation and not related to changes in the intracellular pool of amino acids. Glucagon did not modify system ASC activity measured by L -cysteine. Therefore the transport activity of AIB independent of system A stimulated by glucagon cannot be attributed to system ASC. This suggests a Na⁺-dependent transport system in rat hepatocytes not identified until now.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2-amino [1-¹⁴C]isobutyric acid (AIB) and N-Methyl-2-amino [1-¹⁴C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium-dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane-1-carboxylic acid, 1- [carboxyl-¹⁴C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium-independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium-dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3-fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8-fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.     

Regulation of neutral amino acid transport in hepatocytes isolated from adrenalectomized rats

The present report shows that System A-mediated 2-aminoisobutyric acid (AIB) uptake is elevated in hepatocytes isolated from adrenalectomized rats when they are compared to control cells. Although System ASC activity also shows this perturbation, Systems N, beta, L1, and L2 are unaffected. Transport of AIB in both cell types is stimulated by dexamethasone, insulin, and glucagon, yet the hepatocytes from the adrenalectomized rats are much less responsive to these hormones. This apparent decrease in competence is seen for adaptive regulation of System A as well. The in vitro addition of dexamethasone to the hepatocytes from the adrenalectomized animals does not restore fully their ability to respond to hormones or amino acid deprivation. These effects are observed even after the cells have been held in primary culture for 24 hr. The simultaneous addition of glucagon and dexamethasone to either cell type resulted in stimulation of transport to rates significantly greater than the sum of the increases produced by the two hormones when added separately. In contrast, insulin and dexamethasone were additive in their effects rather than synergistic. These results suggest that hepatocytes from adrenalectomized rats are less competent than control cells with respect to regulation of neutral amino acid transport, including stimulation by insulin or amino acid starvation, two processes which appear not to depend on glucocorticoid for maximal response.     

Hormonal regulation of amino acid transport and gluconeogenesis in primary cultures of adult rat liver parenchymal cells.

Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum-free culture medium. These cells carried out gluconeogenesis from three carbon precursors (alanine, pyruvate, and lactate) in response to glucagon addition. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). Addition of insulin or glucagon to culture rat liver parenchymal cells resulted in an increased influx of AIB transport. The glucocorticoid, dexamethasone, when added alone to cultures did not affect AIB transport. However, prior or simultaneous addition of dexamethasone to glucagon-treated cells caused a strong potentiation of the glucagon induction of AIB transport. Kinetic analysis of the effects of insulin and glucagon demonstrated that insulin increased the Vmax for transport without changing the Km while glucagon primarily decreased the Km for AIB transport. The effect of dexamethasone was to increase the Vmax of the low Km system.     

Maintenance of glucagon-stimulated system A amino acid transport activity in rat liver plasma membrane vesicles

Plasma membrane vesicles prepared from intact rat liver or isolated hepatocytes retain transport activity by systems A, ASC, N, and Gly. Selective substrates for these systems showed a Na+-dependent overshoot indicative of energy-dependent transport, in this instance, driven by an artificially-imposed Na+ gradient. Greater than 85% of Na+-dependent 2-aminoisobutyric acid (AIB) uptake was blocked by an excess of 2-(methylamino)isobutyric acid (MeAIB) with an apparent Ki of 0.6 mM. Intact hepatocytes obtained from glucagon-treated rats exhibited a stimulation of system A activity and plasma membrane vesicles isolated from those same cells partially retained the elevated activity. Transport activity induced by substrate starvation of cultured hepatocytes was also evident in membrane vesicles prepared from those cells. The membrane-bound glucagon-stimulated system A activity decays rapidly during incubation of vesicles at 4 degrees C (t1/2 = 13 h), but not at -75 degrees C. Several different inhibitors of proteolysis were ineffective in blocking the decay of transport activity. Hepatic system N transport activity was also elevated in plasma membrane vesicles from glucagon-treated rats, whereas system ASC was essentially unchanged. The results indicate that both glucagon and adaptive regulation cause an induction of amino acid transport through a plasma membrane-associated protein.     

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.     

Effects of polyamines on cyclic AMP-mediated stimulation of amino acid transport in isolated rat hepatocytes

The effects of natural polyamines on cyclic AMP-mediated stimulation of amino acid transport in isolated rat hepatocytes were analyzed. Despite the fact that polyamines could directly compete with alpha-aminoisobutyric acid (AIB) for uptake, preincubation of hepatocytes with polyamines did not significantly alter basal AIB transport. The stimulatory effect of glucagon or cyclic AMP analogs was differently affected by polyamines, since it was reduced in the presence of spermine and, inversely, potentiated by spermidine, putrescine, and cadaverine. Dose-dependence analysis showed that half maximal and maximal effects occurred with 2-3 and 6-10 mM external concentrations, respectively. None of the polyamine effects could be ascribed to transstimulation or transinhibition of amino acid uptake. The inhibitory effect exerted by spermine correlated its capacity to inhibit [3H]-leucine incorporation into proteins partially. The potentiating effect of the other polyamines did not result from stabilization of newly synthesized carrier proteins. Instead, the increase in Vmax of the high affinity transport component suggested that more carriers became available, presumably because polyamines facilitated their synthesis by interacting directly with one or several steps controlled by cyclic AMP. Polyamines appear to represent a new class of factors capable of modulating the cyclic AMP-mediated stimulation of amino acid transport, in hepatocytes.     

The Na+ gradient hypothesis in cytoplasts derived from Ehrlich ascites tumor cells

The concentration gradients of Na⁺ and the non-metabolizable amino acid, α-aminoisobutyric acid, and the membrane potential were measured in cytoplasts derived from Ehrlich ascites tumor cells in order to test the Na⁺ gradient hypothesis for the active transport of neutral amino acids in animal cells. According to this hypothesis, the Na⁺ electrochemical gradient and the amino acid activity gradient should be equal at the steady state. It has been difficult to measure the Na⁺ electrochemical gradient in intact Ehrlich cells because Na⁺ may be sequestered in the nuclei of these cells. This problem is avoided with cytoplasts derived from Ehrlich cells because they do not contain internal compartments where Na⁺ could be sequestered. Since these cytoplasts also maintain steady state concentrations of Na⁺, K⁺, and α-aminoisobutyric acid similar to those found in whole Ehrlich cells, they are uniquely suited for testing the Na⁺ gradient hypothesis. Assuming the activity coefficients of external and cytoplasmic Na⁺ are equal, the energy in the Na⁺ electrochemical gradient of cytoplasts was 90% of that in the α-aminoisobutyric acid concentration gradient at the steady state. If the Na⁺ gradient hypothesis is correct, the 10% difference between these two gradients cannot be explained in terms of the sequestration of Na⁺ in the nucleus because cytoplasts do not contain internal compartments.     

Aminoisobutyric acid transport in primary cultures of normal adult rat hepatocytes.

In contrast to suspensions of freshly isolated hepatic parenchymal cells (HPC), short-term monolayer cultures of HPC displayed properties of active transport for the amino acid analog aminoisobutyric acid (AIB). The uptake of AIB was inhibited by KCN and iodoacetate, failed to occur at 4 degrees, and was stimulated by glucagon. The apparent Km for AIB uptake by cultured HPC was approximately 19 mM. Glucagon did not alter the apparent Km but did increase V.     

Hormonal regulation of hepatic amino acid transport

The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na⁺ dependent and the present finding that the uptake of 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na⁺ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

α-Aminoisobutyric Acid Transport into Human Glia and Glioma Cells in Culture

The AIB transport into human glia and glioma cells in culture has been studied. Because of the high affinity of AIB to the plastic culture dishes, a special washing technique had to be developed. With this technique, it was possible to perform transport experiments in a single plate containing about one million cells. The cells were viable, intact and adhered to the supporting medium throughout the experiment. The AIB transport into both types of cells was Na⁺-dependent and showed saturation kinetics when the small component of the transport due to diffusion had been subtracted. The AIB transport capacity of neoplastic glioma cells was 3.6 times higher than that of glia cells. This difference was related to the V_max-values for the two types of cells. The apparent K_m-values were the same. Inhibition experiments with other amino acids support the view that AIB is transported via System A in both glia and glioma cells. Sulfhydryl reagents (ethacrynic acid and NEM) and cytochalasin B clearly inhibited the AIB transport into glia cells whereas the effect on glioma cells was minimal.     

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.     

Membrane transport of sugars and amino acids in isolated protoplasts

A method has been developed for observing membrane transport in isolated protoplasts. Transport of sugars and amino acids has been studied in protoplasts isolated from the mesophyll of Pisum sativum L. That uptake was not due to passive diffusion through damaged membranes was demonstrated by supplying simultaneously two sugar stereoisomers, the one ³H-labeled and the other ¹⁴C-labeled. The protoplast membranes were sufficiently functional to discriminate strongly between these stereoisomers.

To characterize transport the nonmetabolized glucose analogue 3-O-methyl glucose (MeG) and amino acid analogue α-aminoisobutyric acid (AIB) were employed. When uptake was compared per unit of protein as between leaf strips and protoplasts prepared from the same tissue, it was estimated that the protoplasts had retained approximately 40 to 50% of the uptake ability of the whole cells. Uptake of neither MeG nor AIB by protoplasts was linear with time, but the tendency to flatten was more marked for AIB. Addition of Mg-ATP to buffered medium significantly promoted AIB uptake, an effect not ascribable to either chelation or pH. Transport of both MeG and AIB was markedly pH-dependent, uptake falling with rise in pH.

The stimulatory effect of Mg-ATP and the pH dependence confirm that uptake was not due to a diffusional inward “leak” but involved membrane function.

This work demonstrates the feasibility of using isolated protoplasts for membrane transport studies. The potential advantages of using protoplasts for such studies are pointed out.

     

Derepression of amino acid transport by amino acid starvation in rat hepatoma cells.

Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation.     

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin-like growth factor-I

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [³H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [³H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [³H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 ± 0.03 nmol/l and 34.8 ± 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 μmol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells. © 1993 Wiley-Liss, Inc.     

The effect of glucagon on hepatic respiratory capacity

Data from numerous laboratories show that mitochondria isolated from livers treated acutely with glucagon have higher rates of state 3 respiration than control mitochondria. The purpose of the present study was to learn whether this phenomenon is an isolation artifact resulting from a stabilization of the mitochondrial membrane or whether it represents a real increase in the maximal respiratory capacity of liver cells due to glucagon treatment. Electron transport was measured through different spans of the electron transport chain by using ferricyanide as an alternate electron acceptor to O2. With isolated intact liver mitochondria, pretreatment with glucagon was found to cause an increase in electron flow, through both Complex I and Complex III, suggesting that the effect of glucagon was not specific for a single site in the electron transport chain. Using intact isolated hepatocytes, different results are obtained. Respiration was measured in isolated hepatocytes after quantitation of the hepatocyte mitochondrial content by assay of citrate synthase. Hepatocyte respiration could therefore be reported per mg of mitochondrial protein. By providing durohydroquinone to the cells, it was possible to measure electron flow from coenzyme Q to O2 in the absence of the physiological regulation of substrate supply. Likewise, the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone released the in situ mitochondria from control by the cytosolic ATP/ADP ratio and it was possible to measure maximal electron flow rates through Complex III. In the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, electron flow was higher in mitochondria in the cell than in isolated mitochondria. Glucagon caused no increase in mitochondrial respiration in situ either in the presence of the physiological substrates or in the presence of durohydroquinone. The data obtained do not support a role for the electron transport chain as a target of glucagon action in hepatocytes.