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Biochimica et Biophysica Acta, 496 (1977) 182--191 © Elsevier/North-Holland Biomedical Press

BBA 28123 PROTEIN-CATABOLIC STATE OF ISOLATED RAT HEPATOCYTES

PER O. SEGLEN Department o f Tissue Culture, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo 3 (Norway)

(Received July 9th, 1976)

Summary Isolated rat hepatocytes in suspension are in a protein-catabolic state {negative nitrogen balance), as measured by the continuous release of nitrogen in the form of amino acids and urea. The nitrogen loss corresponds to a protein degradation rate of 3--4% per h, while the rate of protein synthesis is negligible. Cells prepared from fasted, fed ot regenerating livers are all highly proteincatabolic. The nitrogen balance is unaffected by insulin or amino acids (physiological mixture), and various metabolites and sera have only moderate effects. However, incubation of the cells for 2--4 h in a tissue culture medium {Dulbecco's) reduces the nitrogen loss dramatically, suggesting the formation of an anticatabolic factor under these conditions.

Introduction

Renewal of cellular protein takes place at a very rapid rate in the rat liver. In vivo, renewal rates ranging from 1 to 4% per h have been estimated, depending on the age and dietary condition of the animal, and on the m e t h o d of measurem e n t [1--6]. On a balanced diet, liver protein synthesis and degradation proceed at the same rate, maintaining a constant size and protein content of the organ. This "futile cycle", i.e. the simultaneous operation of the opposing processes of protein synthesis and degradation, creates a sensitive control system, where relatively small changes in the absolute rates can cause large changes in the net output, i.e. in the overall protein balance (cf. analogous cycling in carbohydrate metabolism, refs. 7 and 8). When starvation for calories or protein is initiated, the rate of protein degradation increases rapidly while the rate of protein synthesis is unchanged, resulting in a rapid fall in hepatic p,~otein c o n t e n t [6]. On the other hand, after partial h e p a t e c t o m y [4] or protein feeding to starved animals [6], protein degradation is strongly inhibited, resulting in a rapid increase in the liver pro-

183 tein mass. Thus, the regulation of nitrogen (protein) balance and growth in the liver is primarily exerted at the level of protein degradation. The liver is subject to regulation by an enormous number of hormones, metabolites and other "differones" [9,10] and a few of these, such as amino acids [ 1 1 - - 1 3 ] , thyroid hormone [ 1 3 ] , insulin [ 1 1 - - 1 4 ] , glucagon [15] and the cyclic nucleotides [15,16] have been implicated in the control of liver growth and protein synthesis. However, the lack of satisfactory in vitro systems and techniques has precluded a precise analysis of h o w the various hepatotropic growth-regulatory factors (known and unknown) interact. The recent development of techniques for the preparation of intact isolated rat liver cells in high yield [ 17] n o w makes it possible to study protein metabolism under controlled conditions. In the present paper, the general protein-metabolic state of freshly isolated rat hepatocytes is described. A preliminary report of these results was presented at a recent meeting [ 1 8 ] . Materials and Methods Male Wistar rats (250--300 g) were maintained on an "8 + 1 6 " controlled feeding and illumination schedule [ 1 9 ] , and used for liver cell preparation at the end of the 16 h fasting period. Isolated hepatocytes were prepared by the two-step m e t h o d of collagenase perfusion [17,20]. The hepatocytes were incubated at 37°C in buffered salt solution (4.0 g NaC1, 0.4 g KC1, 0.15 g KH~PO~, 0.1 g Na2SO4, 0.13 g MgC12 • 6 H20, 0.18 g CaCI~ • 2 H20, 7.2 g HEPES, 6.9 g TES, 6.5 g Tricine, 2.1 g NaOH and H:O to 1000 ml, pH 7.6 at 37°C) as previously described [ 1 7 , 2 1 ] , at a cell concentration of 50--100 mg/ml (wet weight). Perchloric acid extracts of the incubated cell samples were used for analysis of the total amino acids (ninhydrin method, ref. 22), urea and ammonia (nesslerization according to Sigma urea nitrogen kit No. 14). Ammonia was usually present at a very low level, and was routinely included in the urea analysis. The total loss of nitrogen from the cells was estimated as the sum of amino acid and urea formation. When amino acid consumption exceeded amino acid formation, the nitrogen loss corresponded to urea formation minus amino acid consumption. Glucose and lactate were measured by enzymatic methods as previously described [ 21]. The amino acid concentration of the incubation medium was varied by adding an amino acid mixture of the composition previously given [23]. The mixture contained all the 20 amino acids found in protein, plus ornithine and citrulline, at relative concentrations corresponding roughly to the equilibrium levels established in perfused livers [24--30] and found in normal rat plasma [13,25,26,28,31--35]. The a m o u n t of amino acids added is expressed in multiples of the " n o r m a l " plasma or perfusate concentration as previously defined [ 2 3 ] . Incorporation of amino acids into protein was measured by adding a tracer dose (75 nCi/ml) of a l~C-labelled protein hydrolysate (Amersham CFB. 25) to the medium and precipitating the protein as previously described [ 2 3 ] . Controlled cell damage was produced by subjecting hepatocyte suspensions to the rapid vibrations of a Vortex mixer for defined periods of time; the resulting viability was determined by the trypan blue exclusion test [17]. Com-

184

plete cell damage (0% viability) was produced by freezing the cell suspension in liquid nitrogen. Dulbecco's medium was purchased from GIBCO (powdered medium); the various biochemicals from Sigma. Results and Discussion

Autoregulation o f amino acid levels When incubated at 37°C in amino acid-free suspension buffer [ 2 0 ] , isolated rat hepatocytes release amino acids into the medium (Fig. 1). This does not represent leakage of intracellular amino acids, because the intracellular amino acid level is maintained constant throughout the incubation [ 1 8 , 2 3 l . The amino acids released are a mixture of all the 20 amino acids found in protein (unpublished observations), and can only be derived from the degradation of endogenous protein. A similar "conditioning" of the medium was found by Miller [24] and by Schimassek and Gerok [25] to occur during perfusion of the isolated rat liver, also with maintenance of a constant intracellular amino acid level [ 2 5 ] . In the liver perfusion system, the amino acid concentrations in the perfusate eventually reach equilibrium levels very close to the normal plasma concentrations, except for the non-metabolizable amino acids leucine, isoleucine and valine, which accumulate continuously [ 2 4 - - 3 0 ] . Fig. 1 shows that isolated hepatocytes, like the perfused liver [ 2 4 ] , adjust the extracellular amino acid levels by consuming amino acids when these are added at a high concentration. The rate of urea formation is greatly increased in this situation (Fig. 2), indicating amino acid catabolism (transamination and i

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