THE EFFECT OF INSULIN ON THE GROWTH AND METABOLISM OF THE HUMAN DIPLOID CELL, WI-38

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1 J. Cell Sci. 7, (i970) 575 Printed in Great Britain THE EFFECT OF INSULIN ON THE GROWTH AND METABOLISM OF THE HUMAN DIPLOID CELL, WI-38 J.B.GRIFFITHS Microbiological Research Establishment, Porton, Salisbury, Wiltshire, England SUMMARY In a confluent culture of WI-38 cells the membrane area available for nutrient uptake is greatly reduced and the possibility exists that this reduction in uptake capacity of the cell is a contributory factor in contact inhibition. Insulin has been reported by many authors to facilitate glucose uptake and also to stimulate protein, DNA and RNA synthesis, glycolysis, pinocytosis and growth in cultured cells. The effect of insulin on WI-38 cells was determined, therefore, to find out whether it enabled the cell to escape from contact inhibition of growth. The action of insulin was found to be dependent upon medium composition. Growth and protein synthesis were stimulated in Eagle's minimal essential medium, but not when this medium was supplemented with glucose and glutamine. Apparently insulin is only effective when high-energy compounds become limiting. Whilst insulin did not induce any postconfluent division, the protein content of cells was increased by 30%, and this was correlated with an increased rate of protein synthesis. Despite this increased activity in protein metabolism, the utilization of amino acids was less in the presence of insulin indicating that a control mechanism for more economical utilization of amino acids for protein synthesis was activated by insulin. Insulin had no effect on RNA synthesis, and only a slight inhibitory effect on DNA synthesis. Evidence was produced suggesting that insulin blocked cell division and encouraged differentiation. Glucose uptake and incorporation into the cell was stimulated by insulin, and this was especially noticeable after the cell sheet became confluent. The turnover of labelled glucose and derivatives was also enhanced by insulin and this was accompanied by a much higher rate of lactic acid production. It is concluded that insulin does not overcome contact inhibition and permit post-confluent division, but that it does enable the cell to take up and utilize nutrients more efficiently in confluent cultures with a resultant increase in metabolic activity and cell size. INTRODUCTION Observations on the effect of insulin in cell cultures have not shown consistent results. In vivo, insulin acts by facilitating the entry of glucose into the cell and by controlling the blood sugar level. In cell cultures it has been reported to stimulate growth (Leslie, Fulton & Sinclair, 1957; Lieberman & Ove, 1959; Lockwood, Voytovitch, Stockdale & Topper, 1967; Temin, 1967), protein synthesis (Krahl, 1962; Schwartz & Amos, 1968), RNA and DNA synthesis (Leslie & Davidson, 1951; Leslie & Paul, 1954; Eboue-Bonis, Chambaut, Volfin & Clauser, 1963; Holly & Kiernan, 1968), the rate of glycolysis and pinocytosis (Paul & Pearson, i960). However, the primary role of insulin is considered to be that of increasing the uptake of glucose into the cell (Randle & Smith, 1958; Paul & Pearson, i960; Krahl, 1962), and

2 576 J. B. Griffiths the subsequent increases in glycolysis, protein and nucleic acid synthesis, and growth are all secondary in nature. In a previous paper (Griffiths, 1970) it was suggested that human diploid cells have a reduced efficiency for the uptake of nutrients, especially when the culture becomes crowded and the cell surface in contact with the growth medium is greatly decreased. This explanation was put forward as a contributory factor in contact inhibition of growth (see also Castor, 1969). The effect of insulin on human diploid cells was of great interest, therefore, in view of the stimulatory action insulin has on glucose uptake and on amino acid incorporation into protein (Krahl, 1962). Furthermore, the observation made by Paul & Pearson (i960) that insulin caused pinocytosis was of great interest following Carter's proposition that cells can only grow if the plasma membrane is not stabilized (Carter, 1968). This report describes the effect of insulin in cultures of the human diploid cell, WI-38, on the uptake and incorporation of [ 14 C]glucose, the rates of protein, RNA and DNA synthesis, the utilization of amino acids and glucose, cell growth and the protein content of cells. MATERIALS AND METHODS Cell culture The human diploid line, WI-38, derived by Hayflick from foetal lung tissue, was used. This line was cultured by the recommended procedure of Hayflick & Moorhead (1961) and only cells between passages 21 and 29 were used. The cells were at least 98% diploid as revealed by chromosome analysis. Cells were grown in Eagle's Minimal Essential Medium (MEM) (Gibco) supplemented with 10% foetal calf serum (Flow Laboratories) and 100/tg/ml Kannamycin sulphate (Bayer). In some experiments the medium was enriched with a supplement of glutamine (300 /tg/ml) and glucose (500 //.g/ml) and is designated MEM(E). Crystalline insulin (BDH) was used at 1 i.u./ml. Experimental cultures were grown in 5-cm NUNC plastic dishes with 5 ml medium and incubated at 37 C in gassed containers. Growth was measured by nuclei counts. Cultures were considered to be confluent when the cell density reached 1 x io 5 cells/cm 2. Analyses Cell protein was measured by the modified Lowry method (Oyama & Eagle, 1956). Glucose was measured by a glucose oxidase reagent (Clinton Laboratories) and lactic acid by the enzymic method of Schon (1965). Amino acids were measured on a Technicon Amino Acid Autoanalyser. Isotope labelling procedures The following radiochemicals, obtained from the Radiochemical Centre, Amersham, were used: (1) D-glucose-C 14 (U) (3-1 mci/mm), i-6/ici/culture; (2) L-leucine-C 14 (U) (10 mci/mm) and L-arginine-C 14 (U) (9-6 mci/mm) as precursors for protein synthesis, 1 //Ci/culture; (3) thymidine-6-t(n) (2 Ci/mM) as a,precursor of DNA synthesis, 10/tCi/culture; and (4) uridine-5-t (5 Ci/mM) as a precursor of RNA synthesis, 10 //.Ci/culture. Cell cultures were pulsed for 30 min in a medium deficient in either glucose, leucine or arginine at 37 C. Except in the measurement of glucose uptake, the pulse was poured off, the cell sheet washed once with phosphate buffer and the complete medium was added for 2 h. Cells were then harvested after 3 washes in buffer with a mixture of 0-25 % Difco trypsin and 01% versene, solubilized in 0-2N NaOH and added to the scintillation mixture (POPOP, 0-2 g; PPO, 6 g; naphthalene, 50 g and Dioxan to 1 1.). The cpm of the samples were determined in the Philips Liquid Scintillation Analyser.

3 Effect of insulin on WI-38 cells 577 RESULTS The effect of insulin on cell growth and metabolism in pre- and post-confluent cultures The effect of insulin on growth, protein and DNA synthesis was investigated during the culture cycle of WI-j8 cells in MEM(E). The rates of protein synthesis in the control and insulin treated cultures were similar until 24 h before the cultures became confluent, at which stage insulin prolonged the increasing rate of protein Fig. 1. The effect of insulin on protein and DNA synthesis during the culture cycle of WI-38 cells in MEM(E). O O, control;, insulin; 9, insulin added at 96 h; vertical dotted line represents the stage at which the cultures became confluent. Medium changed at 72, 120 and 168 h. synthesis for an extra 24 h (Fig. 1). After confluency, protein synthesis was slightly higher in the presence of insulin but the rates at which protein synthesis decreased were identical in both cultures. DNA synthesis was depressed in the presence of insulin by a consistent amount until the culture became confluent (Fig. 1), then insulin had the effect of stabilizing the rate of synthesis. In the control the rate of DNA synthesis declined at a constant rate from 24 h before confluency until the end of the

4 578 J. B. Griffiths culture. When insulin was added to a culture after 96 h of growth it immediately depressed the rate of DNA synthesis to the level in the culture with insulin initially present (Fig. 1). The effect on protein synthesis, however, was slower to manifest itself as there was no change for 72 h and the rate of protein synthesis did not become identical to that of the culture with insulin initially present until 96 h after the insulin was added. The cell yield was identical in both the control and insulin cultures, reaching a level of about 2 x io 6 /culture. This result was at variance with previous work which had shown that insulin stimulated an extra 30% growth during a 96-h period immediately after the culture had become confluent (Robinson, 1969). The effect of medium composition on the action of insulin on cell growth In the previous experiment insulin did not stimulate any additional growth, but earlier work had clearly shown that insulin could significantly increase growth. As the 2 experiments had been carried out in different media (MEM and MEM(E)), the Table 1. Effect of insulin on cell growth: the influence of medium composition (Cell yields* are given as io 6 cells/culture (inoculum 6 x io 6 ) after growth in 4-oz medical flats containing 10 ml medium with a medium change after 96 h.) Yield 96 h A % increase Yield 192 h % increass BME BME + insulin MEM MEM + insulin MEM(E) MEM(E) +insulin % 2' % 3' % * Mean count of 4 cultures. 16-5% 27-S % -5-5% effect of insulin on cell growth in various media was determined to find out if its action was dependent upon medium composition. The results are summarized in Table 1 and show that insulin is most effective in MEM, and not effective at all in MEM(E). Insulin has a limited effect in BME, but as this medium supports less growth than MEM (Griffiths, 1970), growth-limitation probably occurred before insulin could exert its full effect. These results explain the disparity in growth responses to insulin, and also show that additional glucose and glutamine increase cell growth in MEM by the same amount as does insulin. Additional glucose, glutamine and insulin did not have an additive effect on cell growth. The effect of insulin on the protein composition of cells was investigated because insulin has been shown to stimulate protein synthesis (Schwartz & Amos, 1968; Fig. 1) and also to switch on premature cell division which was presumed to result in smaller cells (Robinson, 1969). The results presented in Fig. 2 showed that cells grown in insulin have a significantly higher protein content, which becomes greater as the

5 Effect of insulin on WI-38 cells 579 culture cycle progresses (after 240 h the cells grown in insulin have 30% more protein than in the control). This demonstrated that protein synthesis is not affected to such a degree by cell contact in the presence of insulin, but that cell division is still inhibited. MEM MEM(E) Hours Fig. 2. The effect of insulin and medium composition on the growth and protein content of WI-38 cells. The media were changed at 96 and 192 h. Control cultures: A A, cell protein content; O O, cell protein per culture; D, cell numbers. Insulin cultures: A A, cell protein content; O #, cell protein per culture; I, cell numbers. Vertical dotted lines represent the stage at which the cultures became confluent. The effect of insulin on cell metabolism in MEM and MEM(E) It is now established that insulin has an effect on cell metabolism and growth and that the magnitude of its action is affected by the medium composition. The results in Fig. 1 may have been influenced by the nutritionally rich environment obtained with media changes every 48 h throughout the experiment. The influence of insulin on cell growth, protein, RNA and DNA synthesis was measured over a period of 5 days following a medium change, with no subsequent medium changes. The results, presented in Fig. 3, show that protein synthesis is stimulated by insulin in MEM, but not to any significant extent in MEM(E). DNA synthesis is lower in both media with insulin but the difference is very small. Insulin has very little effect on RNA synthesis as the rates with and without insulin, in both MEM and MEM(E) after confluency, are very similar. The only significant effect insulin has on cell metabolism, therefore, is to increase protein synthesis in MEM to the rate it is in MEM(E). This action accounts for the higher protein content of the cells grown in MEM and insulin (Fig. 2). 37 C EL 7

6 580 J. B. Griffiths MEM MEM(E) 1500 Jo 1000 " i 500» I i l 300 «= Hours Fig. 3. The effect of insulin on protein, DNA and RNA synthesis during the culture cycle of WI-38 cells in MEM and MEM(E). The media were changed at 96 h only. O O, control cultures in MEM;, insulin cultures in MEM; A A, control cultures in MEM(E); A> insulin cultures in MEM(E). The vertical dotted lines represent the stage at which the cultures became confluent. The effect of insulin on nutrient utilization The uptake of amino acids and glucose was measured to determine whether insulin altered the pattern or magnitude of nutrient utilization (Table 2). The uptake of amino acids was consistently lower in insulin, despite the fact that more cell protein was produced in these cultures (Fig. 2). Some of the metabolites that are produced in the cells by amino acid metabolism were measured (alanine, glycine and ammonia), and showed a higher production in insulin (Table 2). Glucose utilization was always greater in the presence of insulin, but not by a significant amount. The actual uptake values may be misleading as glucose became exhausted 24 h before the end of the experiment in both insulin cultures and in the MEM control. Since insulin has been reported to increase the rate of glycolysis, lactic acid production by WI-38 cells was measured (Table 2). Insulin had the effect of doubling the quantity of lactic acid produced. The percentage of glucose converted to lactic acid

7 Effect of insulin on WI-38 cells 581 was calculated (Table 2) and showed that aerobic glycolysis proceeded at a greater rate in MEM than MEM(E), and was doubled by insulin. The effect of insulin on glucose uptake and incorporation The main function of insulin is to facilitate the entry of glucose into the cell. This function of insulin was tested on WI-38 cells using [ 14 C]glucose. Uptake of glucose into the intracellular pool was measured by harvesting the cells rapidly after the Table 2. The effect of insulin on the metabolism of nutrients in MEM and MEM(E) by WI-38 cells during ig2 h growth Arginine Cystine Histidine Isoleucine Leucine Methionine Phenylalanine Tyrosine Valine Total Glucose Ammonia Alanine Glycine Lactic acid Glucose converted to lactic acid (%) In Control MEM, utilized In MEM, /tg produced 1 Control A Insulin Insulin In MEM(E), /tg utilized Control In /tg Control Insulin MEM(E), produced A Insulin !5i5 79 pulse; incorporation of glucose was measured by chasing the pulse for 2 h in a complete medium before the cells were harvested. These measurements were made on cells growing normally in a culture and also on cells which had been glucose-starved for 2 h prior to pulsing. These latter measurements were carried out in order to measure the ability, rather than the need, of a cell to take up glucose. The release of labelled metabolites into a medium during the 2-h chase period was also measured. The results of these investigations are summarized in Fig. 4. The uptake of [ 14 C]glucose was marginally greater before confluency and considerably greater after confluency in insulin. Glucose incorporation was higher in 37-2

8 582 J. B. Griffiths insulin, the difference increasing as the culture progressed. Insulin caused a consistently higher uptake of glucose into starved cells. Incorporation of glucose was also greater, but only until confluency at which stage it became identical to the control. The release of label during the chase period was greater in insulin in the normal cultures and identical in the starved cultures. The results, therefore, confirmed the ability of insulin to increase the glucose uptake, and this was especially noticeable in post-confluent cultures. A. Glucose uptake B. Glucose incorporation c. Glucose release Hours Fig. 4. The effect of insulin on the uptake, incorporation and release of [ 14 C]glucose by WI-j8 cells. O O, control cultures;, insulin cultures. The vertical dotted lines represent the stage at which the cultures became confluent. DISCUSSION The action of insulin on WI-38 cells was shown to be influenced by the medium composition as additional glucose and glutamine increased cell growth by the same amount as insulin. Serum has also been shown to have an effect on the action of insulin (Sidman, 1956) and Paul & Pearson (i960) suggested that insulin increases the synthesis of cell components only when the supply of energy-rich compounds is limited. Glucose and glutamine are both energy-rich compounds, readily available for cellular metabolism, and this would account for the lack of response to insulin in the cultures supplemented with these 2 nutrients. Glucose uptake and incorporation were significantly greater in the presence of insulin, especially after the culture became confluent (Fig. 4). The data on uptake of [ 14 C]glucose show that insulin was more effective than the utilization figures (Table 2) at first suggested, but this was due to the considerable quantity of labelled metabolites

9 Effect of insulin on WI-38 cells 583 that were exuded from the cell (Fig. 4, c). This label may not necessarily be glucose, as insulin greatly enhances the rate of aerobic glycolysis, doubling the amount of lactic acid formed. Paul, Broadfoot & Walker (1966) compared the rates of lactic acid formation in untransformed and polyoma-transformed BHK cells and confirmed the belief that malignant cells have a higher glycolytic rate than normal cells. These authors suggested that this was due to glucose being able to enter a malignant cell more readily than a normal cell. The stimulation of glucose uptake and of the glycolytic rate in normal cells by insulin, thereby enabling them to grow in a manner more characteristic of altered cells, has interesting implications in the study of contact inhibition. The effect insulin has on amino acid uptake is not so clear, as amino acid utilization is always lower in insulin cultures despite a higher rate of protein synthesis and a higher cell protein content. This difference is exaggerated by the fact that the products of nitrogen metabolism which were measured (alanine, glycine and ammonia) were produced in greater quantities in the insulin cultures. These metabolites are formed mainly from glutamine, especially ammonia, and indicate that insulin enhances glutamine utilization by acting on glutaminase and facilitating glutamic acid formation. It has been shown that amino acids are often used in far greater quantities than can be accounted for by the increase in cell protein, and that culture conditions can be altered to bring about a more economical utilization of amino acids for cell growth (Griffiths & Pirt, 1967; Griffiths, 1970). Insulin, apparently, has the effect of producing a more efficient utilization of amino acids for cell growth, and must control their metabolism in some fashion, either by regulating their uptake into the intracellular pool or by regulating their availability to the enzyme systems involved in their metabolism. Wool & Krahl (1959) also found insulin stimulated amino acid incorporation and Krahl (1962) suggested that this was due to a decompartmentation which rendered the protein synthesis mechanisms more accessible to each other or to their substrates. The results suggest that amino acid uptake is not stimulated by insulin but they do indicate a better utilization of the amino acids. When diploid cells become crowded together in a confluent culture their surface area becomes greatly reduced (Castor, 1969) and consequently they are likely to become starved due to the inability of the cell to take up sufficient nutrients (Griffiths, 1970). Carter (1968) has proposed that cell growth can occur only if the cell membrane is not stable, and that cells packed together do not divide because their membranes have attained a stable equilibrium with each other. The observation by Paul & Pearson (i960) that insulin induces pinocytosis means that insulin prevents, or at least slows down, stabilization of the cell membrane. The fact that protein synthesis and glucose uptake are not significantly stimulated by insulin until after confluency (Figs. 1 and 4) becomes very relevant as this is the period when normally cells have become stabilized and nutrient uptake has been reduced to levels insufficient for growth. The suggestion that insulin is effective only when energy-rich compounds are limited (Paul & Pearson, i960) is also relevant as limitation of these compounds would occur after confluency when the cell is unable to assimilate them in sufficient quantity without insulin. However, it is doubtful whether amino acid uptake is increased, as the evidence, discussed

10 584 J. B. Griffiths earlier, suggests that stimulation of protein synthesis by insulin is due to a reduced requirement for amino acid transport. Contact inhibition involves retardation of protein and nucleic acid synthesis (Levine, Becker, Boone & Eagle, 1965) and insulin is effective in removing the restriction from protein synthesis only. Insulin is reported to act on a process between RNA and protein synthesis (Wool et al. 1968) and as insulin has no effect on DNA metabolism it must be concluded that contact inhibition primarily affects DNA synthesis and that RNA and protein synthesis are inhibited as a result of DNA inhibition. This means that cells in a confluent culture are restricted to the pre-dna synthesis phase (G 1 phase) but protein synthesis, being a continuous metabolic process (as opposed to a gene-controlled or cyclic process), is able to continue, given suitable nutritional conditions. I wish to thank Mrs I. K. Strutt, Mrs J. Gray and Miss H. Jack for their technical assistance and Mr J. Slade for the amino acid analyses. REFERENCES CARTER, S. B. (1968). Tissue homeostasis and the biological basis of cancer. Nature, Lond. 220, CASTOR, L. N. (1969). Contact regulation of cell division in an epithelial-like cell line. J. cell. Physiol. 72, EBOUE-BONIS, D., CHAMBAUT, A. M., VOLFIN, P. & CLAUSER, H. (1963). Action of insulin on the isolated rat diaphragm in the presence of actinomycin D and puromycin. Nature, Lond. 199, GRIFFITHS, J. B. (1970). The quantitative utilization of amino acids and glucose and contact inhibition of growth in cultures of the human diploid cell, WI-38. J. Cell Sci. 6, GRIFFITHS, J. B. & PIRT, S. J. (1967). The uptake of amino acids by mouse cells (strain LS) during growth in batch culture and chemostat culture: the influence of cell growth rate. Proc. R. Soc. B 168, HAYFLICK, L. & MOORHEAD, P. S. (1961). The serial cultivation of human diploid cell strains. Expl Cell. Res. 25, HOLLEY, R. W. & KIERNAN, J. A. (1968). Contact inhibition of cell division in 3T3 cells. Proc. natn. Acad. Sci. U.S.A. 60, KRAHL, M. E. (1962). In The Action of Insulin. New York: Grune & Stratton. LESLIE, I. & DAVIDSON, J. N. (1951). The effect of insulin on cellular composition and growth of chick heart explants. Biochem. J. 49, xli. LESLIE, I. & PAUL, J. (1954). The action of insulin on the composition of cells and medium during culture of chick heart explants. J. Endocr. 11, LESLIE, I., FULTON, W. C. & SINCLAIR, R. (1957). The metabolism of human embryonic and malignant cells and their response to insulin. Biochim. biopliys. Ada 24, LEVINE, E. M., BECKER, Y., BOONE, C. & EAGLE, H. (1965). Contact inhibition, macromolecular synthesis, and polyribosomes in cultured human diploid fibroblasts. Proc. natn. Acad. Sci. U.S.A. S3, LIEBERMAN, I. & OVE, P. (1959). Growth factors for mammalian cells in culture. J. biol. Chem. 234, LOCKWOOD, D. H., VOYTOVICH, A. E., STOCKDALE, F. E. & TOPPER, Y. J. (1967). Insulindependent DNA polymerase and DNA synthesis in mammary epithelial cells in vitro. Proc. natn. Acad. Sci. U.S.A. 58, OYAMA, V. I. & EAGLE, H. (1956). Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteau). Proc. Soc. exp. Biol. Med. 91, PAUL, J. & PEARSON, E. S. (i960). The action of insulin on the metabolism of cell cultures. J. Endocr. 21,

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