Regulation of Amino Acid Transport in the Liver EMERGENCE OF A HIGH AFFINITY TRANSPORT SYSTEM IN ISOLATED HEPATOCYTES FROM FASTING RATS*

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The increased capability of the liver to extract glucogenic substrates during fasting was investigated at. the cellular level by measuring the transport of neutral amino acids in hepatocytes freshly isolated from fed and 4%h-fasted rats. The uptake of 2-a-amino[l-14C]isobutyric acid, a nonmetabolizable analog of alanine, was markedly increased in hepatocytes from fasting rats when measured at 0.1 mu. At the plateau a distribution ratio of 30 to 40 was achieved, compared to 10 in hepatocytes from fed rats. This marked increase was not observed when uptake was measured at 50 mM. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contributed to entry of a-aminoisobutyric acid in hepatocytes from fasting rats: 1) a low affinity component (Km E 50 to 70 mM) similar to that observed in cells from fed animals; 2) a high affinity component (K,,, E 0.6 to 0.8 mM) not observed in cells from fed rats. The transport of a-aminoisobutyric acid occurring through the high affinity component was dependent on Na+, and could be completely inhibited by a-(methylamino)isobutyric acid (Ki z 0.6 mM), the specific substrate for the "A" (alanine preferring) transport system. Ouabain, valinomycin, and gramicidin D partly inhibited cy-aminoisobutyric acid transport occurring through this high affinity component. Puromycin and cycloheximide administered in vivo prevented the emergence of the high affinity transport component induced by fasting. We conclude that the increase in amino acid active transport in hepatocytes from fasting rats results from the emergence of a high affinity transport component which has the properties of a pure "A" system. This system endows the hepatocyte with a high power for concentrating amino acids at low ambient levels, and thus may play an important role in the regulation of gluconeogenesis.
The liver plays a central role in the adaptation to fasting by its increased capacity to extract glucogenic substrates (l-4).
It is well established that alanine, predominantly synthesized from pyruvate in muscle (3)(4)(5) and released in plasma during the early stages of fasting (4,6,7), is the preferential glucogenic * This work was supported in part by Grants 77. 7 of liver to metabolize large amounts of amino acids is largely dependent upon its transport capability which is rate-limiting for subsequent metabolism. We have investigated this step at the cellular level by measuring the transport of neutral amino acids in hepatocytes isolated from fed and 4%h-fasted rats. In a previous study (8) isolated hepatocytes were shown to take up amino acids through the major transport systems described for eukaryotic cells, namely the "A," " ASC," and "L" systems (9). We show here that the capacity of hepatocytes to concentrate AIB,' a nonmetabolizable analog of alanine, is strongly increased after fasting. This results essentially from the emergence of a high affinity component of transport which has the properties of a pure "A" system.

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Amino Acid Transport in Hepatocytes from Fasting Rats were carried out as described previously (8). Experiments were repeated at least twice and each experimental condition was performed in triplicate.
Results have been corrected to account for the viability of the cell suspensions, which was estimated by the trypan blue exclusion method and phase contrast microscopy (8,10 atocytes from fed and 48-h-fasted rats was studied with AIB at 0.1 mM and 50 mM, which represent the two extreme concentrations used in subsequent kinetic analysis. Fig. 1 (left panel) shows that the uptake of 0.1 mM AIB by fasting rat hepatocytes increased linearly with time for only 4 min, to reach a plateau after 20 min (distribution ratio, R = 30 to 40). In hepatocytes from fed rats, the uptake increased linearly with time for about 20 min ( Fig. 1, left), and the plateau (R = 6 to 10) was not reached before 90 min (not shown). This marked increase in AIB uptake by hepatocytes from fasting rats was not observed with AIB at 50 mM ( Fig. 1, right).
Time Course of AIB Efflux-The fact that a shorter time was required to reach a plateau in hepatocytes from fasting rats suggested that the rate of exodus might also be altered. Fig. 2 shows the fractional efflux of AIB from hepatocytes of fed and fasting rats preloaded with AIB under conditions where a low intracellular concentration (~0.5 mM) was achieved. A semilogarithmic plot of the data yielded straight lines (Fig. 2), indicating that efflux processes followed first order kinetics for both types of cells. However, a higher rate of exodus was observed with hepatocytes from fasting rats (tlj2 z 8 min) than with hepatocytes from fed rats (tl12 = 50 min). Although not shown, no significant difference was observed in the rate of exodus when both types of cells contained a high intracellular concentration of AIB (25 to 30 mM). Therefore, it appears that the increase in AIB transport which is observed in hepatocytes from fasting rats at low substrate  concentration involves process(es) operative for influx and efflux.

Kinetic Analysis of Influx in Hepatocytes from Fed and
Fasting Rats-- Fig.  3 shows the dependence of transport on substrate concentration for AIB (Fig. 3, left) and N-Me-AIB, the specific substrate for the "A" (alanine preferring) transport system (9) (Fig. 3, right), in hepatocytes from fed and fasting rats. For fed controls, a plot of the initial velocity of the Na+-dependent transport, v, against v/S, was linear over the entire range of substrate concentrations tested for AIB and N-Me-AIB.
This indicates that amino acid transport in hepatocytes from fed rats is mediated either by a homogeneous family of carriers, or by several types of carriers that have a similar affinity for the substrate. In hepatocytes from fasting rats, in contrast, the plots were curvilinear (Fig. 3). This can be explained either by cooperative interactions or more likely by the contribution of several independent families of carriers with different affinities, as described for amino acid transport in embryonic heart cells (11). The goodness of the fit observed between the experimental data and a theoretical model which assumes that only two independent components participate in transport, supports the latter hypothesis. Table I gives the values of kinetic parameters of the transport components derived from plots of v against v/S (Fig. 3)  rat hepatocytes into two linear components revealed that a high affinity-low capacity and a low affinity-high capacity components contribute to the total, saturable transport. From the values of kinetic parameters, it can be calculated that at low substrate concentrations the high affinity component, that operates in fasting rat hepatocytes contributes 75% (at 1 mM AIB) to 85% (at 0.1 mM AIB) of amino acid entry, and thus accounts for the increased accumulation of AIB in hepatocytes from fasting rats compared to cells from fed controls. It should also be noted that, for both AIB and N-Me-AIB, the kinetic parameters of the low affinity transport component in fasting rat hepatocytes were similar to those of the single component that operates in fed rat hepatocytes ( Table I).
Effect of Osmolarity on the High Affinity Transport Component-To exclude the possibility that the adsorption of AIB to some cellular component(s) might have accounted for the increased accumulation observed in fasting rat hepatocytes at low substrate concentration, the influx of AIB (at 0.1 mM) was measured in media of varying osmolarities. Fig. 4 shows that increasing the osmolarity of the extracellular medium by addition of sucrose resulted in a decrease in AIB influx. Extrapolation of the experimental plot (Fig. 4, dotted line) indicated that uptake would be virtually abolished at about 2 osmol/liter. This indicates that the high affinity process actually reflects a transport into an osmotically reactive intracellular space and thus cannot, even partially, be accounted for by adsorption to cellular component(s).
Effect of Na' on AIB Influx-As previously observed in hepatocytes from fed rats (8), a Naf-independent component of AIB entry was also found in cells from fasting rats. However, this component accounted for only 10  (not shown). Fig. 5 shows the relationship between the external concentration of Nat and the influx of AIB measured at 0.1 mM, a condition in which AIB transport occurs predominantly through the high affinity component. The linear relationship found between v and v/[Na+] indicates a first order dependence of this component on Na+ concentration. The apparent K, for Na+ (~60 mM) was analogous to that of the low affinity component of transport present in cells from fed rats (8). Fig. 6  inset) showed that the K,,, values of the low and high affinity transport components were not modified by varying the external Na' concentration.
In contrast, the V,,, values of the two components were strongly increased when Na+ was raised from 40 to 120 mM. Such a specific effect on the V,,, has been generally interpreted as an effect of Na+ on the rate of translocation of a ternary complex (amino acid. Na' . carrier) across the cell membrane (12,13). It is worth noticing that both the high and low affinity components of transport in hepatocytes from fasting rats have similar properties with respect to Na' dependence; these properties are analogous to those previously described for the low affinity component operative in cells from fed rats (8).
Effect of Temperature on the High Affinity Component of Transport-The dependence of AIB influx on substrate concentration in hepatocytes from fasting rats was measured at different temperatures and the kinetic parameters of the high affinity transport component were determined. The apparent K, was not modified, as suggested by the parallelism of the experimental plots (Fig. 7). In contrast, the V,,, largely decreased when the temperature was lowered from 37-17°C. The activation energy = 16 kcal/mol, calculated from the Arrhenius plot (Fig. 7, inset), is within the range of the values reported for carrier-mediated, active transport processes (14) and is similar to that determined for the low affinity, saturable component of AIB transport operating in fed rat hepatocytes (8).

Inhibition of AIB Transport
Occurring through the High Affinity Component in Fasting Rat Hepatocytes-In hepatocytes from fed rats, AIB entry occurs through two Nafdependent saturable systems, the "A" and "ASC" systems (8). In hepatocytes from fasting rats, the Na+-dependent AIB influx through the high affinity component could be completely inhibited by N-Me-AIB (Fig. 8). Kinetic analysis of competition experiments following the method of Inui and Christensen (15)  closely agrees with the K,,, ( Table I). The D stereoisomer of alanine had no effect (Fig. 8).
The relative ability of natural amino acids to compete for AIB influx is shown in Table II. In hepatocytes from fasting rats, the most potent inhibitors of the high affinity component of AIB transport were alanine, methionine, serine, glycine, and proline. These amino acids were also found to be the most effective in inhibiting AIB influx in hepatocytes from fed rats. However, the extent of inhibition was consistently lower than that observed in hepatocytes from fasting rats (Table II). This is in keeping with the fact that AIB entry at low concentration occurs through a component with a much higher affinity in hepatocytes from fasting rats than in cells from fed controls.
Cycloleucine Transport in Hepatocytes from Fed and Fasting Rats-To investigate the effect of fasting on the "L" system, the influx of cycloleucine was measured under conditions where the transport of this nonmetabolizable amino acid   -AIB  38  11  Alanine  25  13  Methionine  53  19  Serine  63  21  Glycine  72  32  Proline  72  34  Phenylalanine  92  63  Arginine  75  63  Leucine  85  68  Lysine  89  70  Valine  95  82  Glutamic  acid  100  88 analog occurs through the "L" system (16), i.e. in a Nat-free medium, or in a Nat-containing medium in the presence of a large excess of AIB. Table  III shows that, in either condition, fasting did not alter the transport of cycloleucine through "L" system.

Effect of Ouabain,
Valinomycin, and Gramicidin D on Active Transport in Hepatocytes from Fed and Fasting Rats-The high affinity transport component induced by fasting and the low affinity component operative in hepatocytes from fed and fasting rats are strongly concentrative (Fig.  1). To investigate the possible contribution of alkali-metal ion gradients in the energization of transport, the effects of ouabain, valinomycin, and gramicidin D were tested (Table IV) under conditions where cell viability was not affected. The high affinity component in hepatocytes from fasting rats was found to be more sensitive to all three agents than the low affinity component operating in hepatocytes from fed and fasting rats. Valinomycin and, to a lesser extent, ouabain and gramicidin D, decreased the intracellular ATP content in both types of cells (Table IV). It should be noted that even a large decrease in ATP content, as obtained with valinomycin, was still compatible with a transport which remained largely unaltered. These results suggest that the high affinity component of transport induced by fasting is energized, partly at least, through transmembrane cationic gradients, and that the in- Effect of Puromycin and Cycloheximide on the Emergence of the High Affinity Transport Component in Hepatocytes from Fasting Rats-When rats were deprived of food overnight, the high affinity transport component was already detectable by kinetic studies, even though its relative contribution to total AIB transport was reduced (Fig. 9). Puromycin (100 mg/kg) and to a greater extent cycloheximide (1 mg/kg) largely prevented the appearance of the high affinity transport component.
These results suggest that the emergence of the high affinity transport component in hepatocytes from fasting rats is dependent on new protein synthesis.

DISCUSSION
Fasting is accompanied by an increased hepatic extraction of glucogenic substrates and particularly amino acids (l-4). This suggests that the transport of amino acids into the hepatocyte is enhanced during fasting. The present study was designed to investigate this process at the cellular level, using suspensions of freshly isolated hepatocytes from fasting and fed rats.
The fact that an increase in the concentrative uptake of AIB was observed only at low concentrations of substrate in hepatocytes from fasting rats, strongly suggested that a change in carrier affinity may have occurred following fasting. This was confirmed by the analysis of the relationship between initial velocity of transport and substrate concentration, which revealed that more than one Michaelis-Menten component contributes to AIB (and N-Me-AIB) transport in hepatocytes from fasting rats. Quantitative treatment of the data using a curve-fitting method and a computer analysis allowed for the characterization of a high affinity transport component in hepatocytes from fasting rats, in addition to a low affinity component analogous to that present in hepatocytes from fed rats. It should be pointed out that, as stressed elsewhere (9), detection of heterogeneity in transport and characterization of its components require measurements of initial velocities over a very broad range of substrate concentrations and a suitable quantitative analysis. The high affinity component has the properties of a pure "A" system, as demonstrated by the following observations: 1) AIB transport through this component was completely inhibited by N-Me-AIB, the specific substrate of the "A" system (9); 2) transport through this component was strictly dependent on the presence of Na+. Furthermore, the high affinity transport component displayed stereospecificity toward L-alanine, and the most potent amino acids which competed for AIB transport through this component (alanine, methionine, serine, glycine and proline) were those reported to have the greatest reactivity toward the "A" system (9).
Several modes of energization of active amino acid transport have been proposed in eukaryotic cells (17). It was observed here that AIB transport through the high affinity system induced by fasting was partially inhibited following treatment of hepatocytes by ouabain, valinomycin, and gramicidin D. This inhibitory effect was obtained irrespective of the degree of alteration in cellular ATP content, suggesting that the high affinity transport is partially energized through transmembrane ionic gradients.
The results obtained with puromycin-and cycloheximidetreated rats suggest that new protein synthesis is required for the emergence of the high affinity transport system. However, it is presently unknown whether the emergence of this transport system results from new synthesis of the carrier itself, or from a mechanism involving a protein synthesis-dependent activation of a pre-existing carrier of the "A" type.
The results of efflux experiments deserve comment. It has been suggested that the net entry of amino acids into cells occurs mainly through the "A" transport system, whereas exodus involves the "L" system (17,18). Our results in fasting rat hepatocytes appear to be inconsistent with this assumption since we observed that the rate of exodus was enhanced despite the fact that the activity of system "L" was not altered by fasting. Thus it seems that the carriers of the "A" transport system might be operative for both upward and downward transport.
Fasting represents a situation where a marked decrease in plasma levels of most amino acids (3)(4)(5) is concomitant with an important requirement for amino acids in liver. Accordingly, the emergence of a high affinity transport system endows the hepatocyte with a high power for concentrating amino acids at low ambient levels. In addition to their physiological implications, our findings stress the critical importance of the nutritional state of the animal for the study of amino acid transport processes in hepatocytes. It should be pointed out that even a relatively short period of fasting (24 h) can induce the emergence of a high affinity transport system (see Fig. 9) which may be underestimated or even not detected by a classical analysis of the data.
The entry of amino acids into the liver has been shown to be hormonally dependent in contrast to that of other glucogenie precursors (19). It has previously been shown that in isolated hepatocytes only the "A" system of transport is subjected to hormonal regulation (16,(20)(21)(22). Fasting is accompanied by changes in the circulating levels of many hormones (for review see Ref. 23). Thus, the plasma levels of glucagon, glucocorticoids, and growth hormone increase whereas that of insulin decreases. These changes may be involved in the emergence of the high affinity system of the "A" type observed in hepatocytes from fasting rats. It is also possible that an adaptive regulation involving a derepression mechanism (24) induced by the low concentration of circulating amino acids in the fasting state might be implicated in the emergence of the high affinity transport system.