Differentiation of 3T3-L1 fibroblasts to adipocytes

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Experimental Ceil Research 120 (1979) 395403

DIFFERENTIATION

OF 3T3-Ll

FIBROBLASTS

TO ADIPOCYTES

Loss of Stimulation of Uridine and 2-Deoxy-D-glucose Transport by PGFza in the Course of Differentiation of the 3T3-LI Cell Line TONG-HSUAN Depurtment

CHANG, IRENE H. WILLIAMS

and S. EFTHIMIOS

of Biological Chemistq. The University of Maryland Medicine, Baltimore, MD 21201, USA

POLAKIS

School of

SUMMARY Insulin and prostaglandin F2 (PGF,) stimulate uridine and 2-deoxy-o-glucose transport by 3T3-Ll and 3T3-C2 cells. Maximal stimulation of both transport systems is achieved with I .5 PM PGF,. Maximal stimulation of uridine transport is achieved with 34 nM (200 nglml) insulin. The basal (control) level of uridine uptake is lower in 3T3-Ll than in 3T3-C2 cells, while the basal level of 2-deoxy-o-glucose uptake is slightly higher in 3T3-LI cells. The stimulatory effect of PGF, on both transport systems is abolished in the course of differentiation of 3T3-LI cells to adipocytes. Removal of the inducers of differentiation (insulin plus indomethacin) from the medium of differentiated cells does not restore responsiveness of either transport system to PGFaa. In contrast the stimulatory effect of insulin on either transport system is not abolished during differentiation. Treatment of the non-differentiating 3T3-C2 cell line with inducers of differentiation reduces the basal level of 2-deoxy-u-glucose transport by 70% and only slightly decreases the basal level of uridine transport without affecting the stimulatory effect of PGF, on either transport system.

The differentiation of 3T3-Ll Iibroblasts into adipocytes is affected by prostaglandins [ 1, 21. The effects of prostaglandins on the process of differentiation are complicated and dependent on the stage of differentiation and on concentration [ 11.In addition, of all the hormones which interact with both cell types, i.e. adipocytes isolated from rats or mice and 3T3 mouse-embryo fibroblasts in culture, prostaglandins have the unique distinction of lowering CAMP levels in adipocytes and raising CAMP levels in 3T3 fibroblasts. This behavior suggested to us that the process of differentiation might be accompanied or even be dependent on changes in the nature of prostaglandin receptors. Consequently we have studied the effect of prostaglandins on the

transport processes of the two cell lines and prostaglandin binding to the receptors of cells during the process of differentiation. In this report we describe the effect of prostaglandins on the transport of deoxyglucase, and uridine in the differentiating 3T3Ll and non-differentiating 3T3-C2 cell lines. MATERIALS

AND METHODS

Materials Insulin was purchased from Elanco, indomethacin and bovine serum albumin from Sigma, [2-3H]deoxy-oglucose and E3H]uridine from New England Nuclear. PGF, was a gift from Dr John Pike of Upjohn Pharmaceutical Company.

Growth of cells Cells were grown as described before [3].

396

Chang, Williams and Polakis 3T

12

r-

Measurement of 2-deoxy-D-glucose transport

A 0

IO B

6 4 2

0

E

x

1234567

1234561

I. Abscissa: time (mitt); ordinate: [3H]uridine upin 10W3xcpm/35 mm plate at 37°C. X, Control; 0, insulin 3.5~10-~ M (200 rig/ml); 0, PGFa,, 1.5X 10m6M (531 rig/ml). For details see Materials and Methods.

Fig. take

Monolayers of confluent cells in 35 mm dishes were treated as described for measurement of uridine transport through the second 60 min incubation. Then the medium was again aspirated, the plates were washed three times with Dulbecco’s phosphate-buffered saline and the cell monolayer was covered with 1 ml of prewarmed Dulbecco’s phosphate-buffered saline with calcium, magnesium, albumin (I mglml), indomethacin (6 PM), [2-3H]deoxy-o-glucose (0.25 &i/plate; spec. act. 8.26 Ci/mmol) and with or without hormone. The plates were incubated in a water bath at 37°C for 5 min. At the end of the incubation the cell monolayers were washed six times with ice-cold Dulbecco’s phosphatebuffered saline, the cells were scraped off the plates and counted. Each assay was carried out in triplicate and the values presented are mean values I!Zone standard deviation.

RESULTS Measurement of uridine transport Confluent monolayers of cells in 35 mm dishes were washed three times with ice-cold Dulbecco’s phosphate-buffered saline. One ml of Dulbecco’s modified Eagle’s medium with 5 % charcoal-treated serum, and 6 PM indomethacin was added per plate and the cells were preincubated in a CO, incubator at 37°C for 60 min. At the end of this period the medium was aspirated and new medium of the same composition with or without hormone was added and the cells were further preincubated in the CO, incubator for 60 min. Subsequently, the plates were seeded with 50 nCi of [3H]uridine (spec. act. 9.0 Ci/mmole) and incubated for 5 min at 37°C in a water bath. At the end of the incubation period the plates were rinsed six times with ice-cold Dulbecco’s phosphate-buffered saline and the acid-soluble intracellular pool was extracted with 1 ml of 5% ice-cold trichloroacetic acid (TCA) for 20 min at 4°C. A fraction of the TCA extract (usually 0.5 ml) was counted to determine intracellular acid-soluble counts. The monolayer was washed three times with TCA followed by three washes with phosphate-buffered saline, the cells were scraped off the plates and counted. Each assay was carried out in triplicate and the values presented are mean values f one standard deviation. To avoid or severely reduce production of prostaglandins by the cell monolayers indomethacin at a concentration of 6 FM was included in the incubation medium. lndomethacin at concentrations higher than 30 PM significantly inhibits the transport of uridine. For example, indomethacin at 300 PM inhibits uridine transport 35%, and although the addition of a given prostaglandin concentration in the presence of 300 PM indomethacin results in a relative stimulation equal to the relative stimulation achieved at the same concentration of prostaglandin and 6 FM indomethacin, maximal rates of transport cannot be obtained at 300 FM indomethacin even at higher prostaglandin concentrations. Exp Cell Res 120 (1979)

Linearity of radioisotope uptake with time The uptake of uridine and 2-deoxy-D-glucase by 3T3-C2 cells is shown in fig. 1. The uptake is linear for the duration of the experiment, i.e. 7 min either in the presence

3T3-LI

C

12

12 e

0

4 p:l”k 00 0.3* 3. 34 340 3wo

1.5 I5 15015001E.000

2. Abscissa: (A, B) insulin (nM); (C, D) PGF, (nM); ordinate: [SH]uridine uptake in 10-3xcpm/35 mm plate/5 min incubation at 37°C. (A, B) Uridine uptake by 3T3-Ll and 3T3-C2 cells at various insulin concentrations and in the 0: absence or 0, presence of 30 nM PGF,. (C, D) Undine uptake by 3T3-Ll and 3T3-C2 cells at various prostaglandin concentrations and in the 0, absence, or n , presence of 1.31 nM insulin.

Fig.

Hormonal

stimulation

of transport and differentiation

of 3T3-Ll

cells

397

Again in absolute quantities per mg of cell protein PGFza stimulated uridine transport by 13 160 cpm in 3T3-C2 cells and by 11050 cpm in 3T3-Ll cells. In the time period the assay was conducted about 5 % of the uridine taken up was found incorporated into acid-insoluble material. Even at optimal iI:: 0 0.34 34 34 340 3400 0 15 15 150 1500 15000 concentrations of PGFzu (> 1.5 PM) the addition of insulin (1.31 x 1OWM) further inFig. 3. Abscissn: (left) insulin (nM); (right) PGFaa (nM); ordinate: r3H12-deoxy-o-glucose uptake for 5 creases the rate of uptake of uridine into min at 37°C expressed as % of the control.3T3-Ll and 3T3-C2 cells (fig. 2C, D). At Uptake was measured as described under Materials optimal concentrations of insulin (>34 nM) and Methods in the presence of various concentrations of insulin or PGF, . 0, 3T3-Ll cells; 0, 3T3-C2 cells. the addition of PGF, does not stimulate the Control values: (lefr) 6 109 cpm for 3T3-Ll cells and 6746 cpm for 3T3-C2 cells; (right) 4356 cpm for 3T3uptake of uridine into 3T3-Ll cells (fig. Ll cells and 4558 cpm for 3T3-C2 cells. 2A). It is probable that only a fraction of the transport system in 3T3-Ll cells is saturated with the receptor-prostaglandin or absence of insulin or PGF*. Similar re- complex at optimal prostaglandin concensults were obtained with 3T3-Ll cells with trations. The remainder free fraction of the the only difference that the extent of hor- transport system can interact with and be monal stimulation of uridine and 2-deoxy- stimulated by the receptor-insulin complex D-&COSe was in general more pronounced. formed upon addition of insulin. In contrast at optimal insulin concentrations all moleDose response of uridine uptake and cules of the uridine transport system of 3T32-deoxy-D-glucose uptake by Ll cells have interacted with the receptor PGF,, insulin complex and the addition of PGF, Maximum stimulation of uridine uptake by cannot stimulate the system any further. both cell lines is achieved with 34 nM (200 The uridine transport system of the 3T3rig/ml) of insulin (figs 2A, B) and 1.5 PM C2 cells is not maximally stimulated at the (53 1 nglml) of PGFza (figs 2C, 0). In the optimal concentrations of either insulin or presence of either hormone maximal rates PGF, , consequently the addition of PGF, of uridine transport per mg of protein are in the presence of optimal insulin concencomparable in both cell lines. The larger trations and vice versa results in further stimulation observed with 3T3-Ll cells is stimulation of uridine transport (fig. 2B, D). due to the lower rate of basal (control) uri- Presumably at optimal insulin or PGF, condine uptake in this line. Insulin stimulated centrations only a fraction of the transport uridine uptake about 1.7-fold by 3T3-C2 system of the 3T3-C2 cells has interacted cells and 2.4-fold by 3T3-Ll cells, however, with the receptor-hormone complex. quantitatively insulin increased uridine Maximal stimulation of 2-deoxy-D-glutransport per mg of protein in the 5 min in- case transport in the two cell lines is cubation period by 13840 cpm in 3T3-C2 achieved with 1.5 PM PGF, (fig. 3). In concells and by 13960 cpm in the 3T3-Ll cells. trast to the uridine transport system that PGFza stimulated uridine uptake 1.7-fold by becomes saturated with 34 nM insulin the 3T3-C2 cells and 2-fold by 3T3-Ll cells. glucose transport system is not saturated 26-791807

ExaCellRes

IZOf19791

390

Chang, Williams and Polakis

Table 1. Stimulation of uridine transport (expressed as cpm per 5 min of incubation at 37°C) by prostaglandins in the non-differentiating 3T3-C2 and differentiating 3T3-Ll cell lines Transport was measured in cultures treated with indomethacin (1.25x 10m4M) plus insulin (1.76~ 10m6M) and in control cultures two weeks after confluence. In this period more than 95% of the 3T3-Ll cells treated with indomethacin plus insulin had differentiated into adipocytes. The effect of prostaglandins at a concentration of 1.5 PM on the process of uridine uptake was measured as described in Experimental procedures. The specific activity of uridine employed was 9.0 Cilmmol Cell line...

3T3-LI

Treatment... Period of treatment..

None Two weeks

Addition to the assay mixture

cpmldish

cpm/mg of protein

% of control

cpm/dish

cpmlmg of protein

% of control

None PGF, PGF,a PGE, PGE,

1 674+ 66 5 291+352 4 966f363 3 778+289 2 937f246

10 810 34 179 32 080 24 405 18 972

100 310 297 226 175

3 028f217 3 291f237 3 697f 39 3 413+198 3 432+329

4 976 5 409 6 076 5 610 5 641

loo 109 122 113 113

Insulin+indomethacin Two weeks

with insulin concentrations as high as 3.4 PM (fig. 3). Since this insulin concentration is two orders of magnitude higher than the dissociation constant of the low affinity insulin receptor of the cells [3] it is probable that at this high (3.4 PM) concentration insulin cross-reacts with receptors of other peptide hormones which in turn interact with the glucose transport system of the 5x10 4

4. Abscissa: days after confluence: ordinate: cell no./35 mm plate. 0, No addition; A, insulin 1.76~ 1Om6M (10 pg/ml); X, indomethacin (1.25~10~~ M); 0, insulin (1.74x10-@ M) plus indomethacin (1.25~ lo-“ M).

Fig.

Exp Cd Res I20 (1979)

cells. The basal level of uridine transport per mg of protein is always substantially higher in 3T3-C2 than in 3T3-Ll cells, while the basal level of 2-deoxy-D-glucose transport per mg of protein is slightly higher in 3T3-Ll cells (13-20%). PGF2, (1.5 PM) stimulates 2-deoxy-D-glucose uptake by about 1.8-fold in 3T3-Ll cells and 2-fold in 3T3-C2 cells. In both cases PGFza increases sugar transport in the 5 min incubation period by about 6000 cpm. Insulin stimulates 2-deoxy-D-glucose transport by about 2-fold in both lines. Effect of various prostaglandins on the uptake of uridine in 3T3-LI and 3 T3-C2 cells Two weeks after confluence the transport of uridine by 3T3-Ll and 3T3-C2 cells was stimulated by all prostaglandins tested (table 1). At a concentration of 1.5 ,uM, PGF, was most potent followed by PGFI, , PGE, and PGE, in that order. The stimulation achieved with 3T3-Ll cells was more pronounced than the stimulation observed

Hormonal stimulation of transport and differentiation of 3T3-LI cells

399

3T3-C2 Insulin+indomethacin Two weeks

None Two weeks

:pm/dish

cpmlmg of protein

% of control

cpmidish

cpmlmg of protein

5%of control

5 730+504 I I 043+252 IO 9585340 9 856+345 8 730+ 298

19 549 37 676 37 386 33 626 29 785

100 193 191 172 152

4 181?218 7 675+348 7 54621 376 6 7785299 5 765+ 187

21 39 39 35 29

loo 184 180 162 138

with 3T3-C2 cells. On a protein basis the rate of uptake of uridine is comparable in the two cell lines in the presence of prostaglandins; however, in the absence of any prostaglandins 3T3-C2 cells appear to transport uridine almost twice as efficiently as 3T3-Ll cells. Thus the lower on a percentage basis stimulation of uridine transport by prostaglandins in 3T3-C2 cells is rather due to the higher control values obtained with these cells. 3T3-C2 cells treated for 2 weeks with insulin (1.76~ lop6 M) plus indomethatin (1.25~ lop1 M) exhibit only a slight and most probably insignificant reduction in the stimulation of uridine uptake by prostaglandins. However, 3T3-Ll cells treated for 2 weeks with insulin (1.76~ lop6 M) plus indomethacin (1.25~ lop4 M), differentiated into adipocytes with a frequency of almost 95 % and at the same time the stimulation of uridine uptake by prostaglandins was greatly reduced or abolished. The slight stimulation still observable may in fact be due to the presence of some undifferentiated fibroblasts (-5 %). We have separated the dif-

674 787 118 137 885

ferentiated adipocytes from undifferentiated fibroblasts on Percoll gradients following detachment of cells with collagenase treatment. However, since uridine uptake and the effect of prostaglandins can only be demonstrated in monolayers but not in suspension and not after attachment of separated cells on nitrocellulose Millipore filters, we have been unable to confirm whether the slight stimulation of uridine uptake by PGF,, with 3T3-Ll cells differentiated into adipocytes following treatment for two weeks with insulin plus indomethacin, is due to the small number of undifferentiated fibroblasts present in the plate. As indicated in fig. 4 cell numbers continue to increase in confluent 35 mm plates of 3T3-Ll cells (i.e. plates fully covered by cells). The final number of cells per plate differs after various treatments in the following order: number of cells in plates treated with indomethacin plus insulin > number of cells in plates treated with insulin > number of cells in control plates (no Exp Cell Res I20 (1979)

400

Chang, Williams and Polakis

Table 2. Effect of prostaglandin F,, and insulin on the uridine transport in differentiated and non-differentiated 3T3-Ll cells Cells were tested 20 days after confluence. Differentiation was induced by treating the cells with insulin (1.74X 10e6M) plus indomethacin (1.24~ lO-4 M) starting from the day of confluence. Transport assays were carried out in triplicate. The values presented are mean values f 1 S.D.

Cells Non-differentiated 3T3-Ll Differentiated 3T3-Ll

Transport assayed in the presence of

Uridine cpm/S min incubation/35 mm plate

No additions Insulin, 3.4~ IOV M PGF, 1.5 PM No additions Insulin, 3.4~ IO-* M PGF, I.5 PM

1 24Ok357 3 388rf:I75 29932 20 1 979*130 3 331+175 I98lt116

treatment) > number of cells in plates treated with indomethacin. The effect of indomethacin on cell proliferation may be attributable to the inhibition of prostaglandin synthesis. It is our experience that PGF, is strongly mitogenic with confluent cultures of 3T3-Ll cells but only mildly mitogenic with 3T3-C2 cells. As seen in fig. 5, 3T3-Ll cells induced to differentiate to adipocytes by indomethacin (fig. .5B), insulin (fig. SC) or a combination of insulin plus indomethacin (fig. 5D) lose their responsiveness to PGFza concomitant with their conversion to adipocytes. The higher the percentage of cells converted to adipocytes the lower the stimulation of uridine uptake by PGF,. In the case of cells treated with insulin plus indomethacin (fig. 5D), 96 % of the cells were converted to adipocytes and the stimulation of uridine uptake by PGF, was completely abolished. Control cultures, retained for the same time in the absence of any inducers of differentiation did not exhibit any adipocytic conversion and as indicated in fig. 5A the responsiveness of cells to PGF, increased after a lag period of about 7 days. Although it might have been coincidental, the lag period preceding any significant adipocytic ExpCellRes

120(/979J

% of control loo 273 241 100

168 100

conversion in the plates treated with indomethacin or insulin was also 7 days. We have already demonstrated that the uridine transport system of 3T3-Ll cells is also responsive to insulin (fig. 2), consequently, we investigated whether this transport sys-

2::ow50 5

IO

15

20

Fig. 5. Abscissa: days after confluence; ordinate: (left)

0, stimulation of uridine uptake by PGF, (1.5 PM) expressed as % of the control; (right) 0, % conversion of 3T3-Ll fibroblasts to adipocytes. Uridine uptake was measured in 35 mm plates for 5 min at 37°C as described under Materials and Methods. (A) Control; (B) indomethacin; (C) insulin; (D) insulin and indomethacin.

Hormonal

stimulation

Table 3. Effect of prostaglandin

of transport and differentiation

Fza on the by 3T3-Ll

transport of 2-deoxy-D-glucose and 3T3-C2 cells

Transport was assayed 20 days after confluence with and without PGF, (1.5 PM). A number of 3T3-Ll and 3T3-C2 plates were treated continuously from the dav of confluence with insulin (1.74x 10m6M) plus mdomethacin (1.25~ IO-* M). This treatment induced differentiation of 3T3-Ll cells at a frequency higher than 95 %. Transport assays were carried out in triplicates, and the average values were used in the calculations

Cell line 3T3-Ll

3T3-C2

Culture conditions No treatment Treatment with insulin+ indomethacin No treatment Treatment with insulin+ indomethacin

2-Deoxy-Dglucose transport % of control 223 104 231 243

tern also lost responsiveness to insulin in the course of differentiation. The data of table 2 indicate that the stimulation of uridine transport by insulin is not abolished in the differentiated 3T3-Ll cells although the extent ,of stimulation is reduced from 2.7fold in the undifferentiated to 1.7-fold in the differentiated cells. Notice again that stimulation of the transport system by PGFza is completely abolished. Since PGF, also stimulates 2-deoxy-D-glucose uptake by 3T3-Ll and 3T3-C2 cells we examined the effect of treatment of both cell lines with insulin plus indomethacin on the hormonal responsiveness of the 2-deoxy-D-glucose transport system. Treatment with insulin plus indomethacin abolishes the PGF, effect on the uptake of 2-deoxy-D-glucose by the differentiated 3T3-Ll but not by the non-differentiating 3T3-C2 cells (table 3). Although the extent of PGF2, stimulation of sugar uptake was not affected by the treat-

of 3T3-Ll

cells

401

ment of 3T3-C2 cells the basal level of transport in treated cells is reduced to about 40% of the rate observed with the control culture, so that the rate of transport achieved by the treated cells in the presence of PGFza is equal to the basal transport rate of the untreated 3T3-C2 cells. In contrast to 3T3-C2 cells, treatment of 3T3-Ll cells with insulin and indomethacin leads to differentiation and greatly increases the basal levels of 2-deoxy-D-glucose transport but it abolishes the effect of PGF, . DISCUSSION The data presented in this paper demonstrate for the first time the stimulation of uridine transport by PGF, in both 3T3-Ll and 3T3-C2 cells. The stimulatory effect is lost in the course of the differentiation of 3T3-Ll cells to adipocytes. The loss of responsiveness of the uridine and glucose transport systems to PGF, is either a prerequisite or the result of differentiation since treatment of the non-differentiating cell line (3T3-C2) with inducers of differentiation (insulin plus indomethacin) does not abolish the stimulatory effect of PGF, on the transport of either uridine or 2deoxy-D-glucose . Furthermore, 3T3-L 1 cells that have differentiated to adipocytes under the influence of insulin plus indomethacin do not recover responsiveness to PGF, after the removal of the inducers of differentiation. Since insulin still interacts with the transport systems of the differentiated 3T3-Ll cells, the lack of responsiveness of these systems to PGFza is suggestive of the following possibilities: (a) The differentiated cells do not possess a PGF, receptor; (b) the PGF, receptor of the fibroblasts is lost during differentiation and is replaced by a new receptor that does not interact with the uridine and 2-deoxy-D-glucose

402

Chang, Williams and Polakis

transport systems; (c) the PGFza receptor of the fibroblasts is modified during differentiation so that it does not interact with the transport systems; (d) the transport systems are either modified or replaced so that they no longer interact with the PGF, receptor but they still interact with the insulin receptor. Growth of cells requires among other things an adequate supply of precursors of DNA, RNA, protein and lipids. The movement of quiescent 3T3 cells to a growing state by the addition of fresh serum is accompanied by large increases in the rates of transport of inorganic phosphate, nucleosides and glucose [4-U]. Limitations of individual amino acids or phosphate in the medium of sparse 3T3 cells also lead to arrest of growth [ 161. However, as recent studies indicate [ 17-201these transport processes are not primary regulators of cell growth. In our laboratory we have observed that in confluent cultures insulin is mildly mitogenic for both cell lines while PGF*, is mildly mitogenic for 3T3-C2 cells but strongly mitogenic for 3T3-Ll cells. Transport processes associated with growth, for example uridine uptake per lo6 cells or per mg of protein are increased by PGF, in both lines but the increase is much higher in 3T3-Ll than in 3T3-C2 cells. The rate of uridine uptake at optimal PGF, concentrations is the same for 3T3-Ll and 3T3C2 cells, and the higher degree of stimulation of 3T3-Ll cells by PGF, is due to the lower basal transport levels of uridine in 3T3-Ll cells (table 1). In contrast to the basal level of transport of growth promoting substances, the basal level of 2-deoxy-Dglucose transport 3T3-Ll cells is slightly higher than in 3T3-C2 cells. Consequently, restriction of PGF% supply results in a more severe curtailment of growth in 3T3-Ll than in 3T3-C2 cells. Addition of indoExp CellRes 120(1979)

methacin (1.25~ 10m4M) into the medium of confluent 3T3-Ll cells at concentrations that completely inhibit the production of prostaglandins, endoperoxides, thromboxanes and prostacyclin, completely abolishes the stimulation of growth by serum. It is conceivable that in this case the transport of growth supporting substances is much more severely curtailed than the transport of the energy source, the cells become flooded with glucose and unable to grow, they differentiate to adipocytes and store the extra energy in the form of triglycerides. Complete inhibition of prostaglandin biosynthesis results in differentiation of 3T3-Ll cells even in the absence of added insulin [3]. Furthermore differentiation induced by 1.74~ lO-‘j M insulin can be prevented by the continuous presence of prostaglandins (5x 10-“-4~10-~ M) in the medium [l]. It follows, that the regulation of arachidonate metabolism in 3T3-Ll cells may play an important role in the process of differentiation of these cells. We wish to thank Dr R. Howard Green for providing the 3T3-Ll and 3T3-C2 cell lines. This work was supported by Grant GM 23057 (to S. E. P.) from the NIH.

REFERENCES 1. Williams, I H & Polakis, S E, Biochem biophys res commun 77 (1977) 175. 2. Russell, T R & Ho, R J, Proc natl acad sci US 73 (1976) 4516. 3. Chang, T H & Polakis, S E, J biol them 253 (1978) 4693. 4. Sefton, B M & Rubin, H. Proc natl acad xi US 68 (1971) 3154. 5. Vaheri, A, Ruoslahti, E & Nordling, S, Nature new biol 238 (1972) 211. 6. Jimenez de Asua, L, Rozengurt, E & Dulbecco, R. Proc natl acad sci US 71 (1974) 96. 7. Rozengurt, E, Stein, W D & Wigglesworth, N, Nature 267 (1977)442. 8. Goldenberg, G J & Stein, W D, Nature 274 (1978) 475. 9. Cunningham, D D & Pardee, A B, Proc natl acad sci US 64 (1969) 1049. 10. Grimes, W J & Schroeder, J L, J cell bio156 (1973) 487.

Hormonal

stimulation

of transport and differentiation

I I. Kram, R, Mamont, P & Tomkins, G M, Proc natl acad sci US 70 (1973) 1432. 12. Bradley, W E C & Gulp, L A, Exp cell res 84 (1974) 335. 13. Jimenez de Asua, L & Rozengurt, E, Nature 251 ( 1974)624. 14. Foster, D 0 & Pardee, A B, J biol them 244 (1969) 2675. 15. Oxender, D L, Lee, M & Cecchini, G, J biol them 252 (1977) 2680. 16. Holley, RW & Kiernan, J A, Proc natl acad sci US 71 (1974) 2042.

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17. Thrash, C A & Cunningham, D D, Nature 252 (1974) 45. 18. Barsch. G S. Greenberg. DB & Cunninaham, D D, J cell physio192 (1977)115. 19. Greenberg, DB, Barsch, G S, Ho, T S &Cunningham, D D, J cell physiol90 (1977) 193. 20. Naiditch, W P & Cunningham, D D, J cell physiol 92 (1977) 319. Received October IO, 1978 Revised version received December 7, 1978 Accepted December 8, 1978

Exp CdRes

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