Effects of insulin on cellular growth and proliferation

Life Sciences, Vol. 29, pp. 2131-2139 Printed in the U.S.A.

Pergamon Press

MINIREVIEW EFFECTS OF INSULIN ON CELLULAR GROWTH AND PROLIFERATION Daniel S. Straus Biomedical Sciences Division and Biology Department University of California, Riverside, California 92521

Summary Insulin stimulates the growth and proliferation of a variety of cells in culture. The growth-stimulatory effects of insulin are observed in Go/.G 1 arrested cells limited for serum growth factors or essential nutrients, ana m cells growing in hormone-supplemented serum-free media. Some, but not all, of the effects of insulin on growth require superphysiological concentrations of insulin. The action of insulin on growth is synergistic with the action of other hormones and growth factors, including FGF, PDGF, PGF_ and vasopressin. 2 This observation, as well as other observations regarding the temporal sequence of action of growth factors, suggests that different growth factors act on different intracellular biochemical events. Several hypotheses have been proposed to explain the effect of insulin on cellular proliferation, including regulation of essential metabolic processes and interaction of insulin with receptors for insulin-like growth factors. Evidence supporting these various hypotheses is reviewed. In addition to the growth-stimulatory effect of insulin observed in cell culture, a number of clinical examples suggest that insulin is an important growth-regulating hormone during fetal development. Insulin is an anabolic hormone with a wide range of effects on metabolism in vivo, including stimulation of the synthesis and inhibition of the breakdown of glycogen, proteins and lipids. In addition to its well-known effects on metabolism, insulin has long been known to stimulate the growth and proliferation of cells in vitro. The growthstimulatory effect of insulin in tissue culture was first observed by Gey and Thalhimer in 1924 (1). Subsequently, insulin has been found to stimulate the growth and proliferation of many cell types (Table 1), under a variety of experimental conditions. Insulin, or insulin in combination with other hormones, stimulates DNA synthesis and cell cycle progression of cells that have been arrested in G! by deprivation for serum (2-8, 12, 1#) or an essential nutrient such as phosphate (32). Iffsulin is also required by a variety of cells for optimal growth in hormone-supplemented serum-free media (for recent review, see reI. 20). The relationship between the growth promoting activity of insulin observed in cell culture and its other well known effects on metabolism has remained an interesting unsolved problem. This review summarizes some recent observations and hypotheses regarding the growth-stimulatory action of insulin. Hypotheses Regarding the Action of Insulin on Growth One possible explanation for the stimulatory effect of insulin on cellular growth and proliferation is that it stimulates anabolic processes in vitro similar to those which it stimulates in vivo, and this regulatory effect on metabolism has a positive effect on growth (metabolic regulation hypothesis). Almost all cells examined to date have been found to have high affinity insulin receptors, and insulin stimulates such processes as amino acid and hexose transport (3,33), glycogen synthesis (22,25,34,35), and f a t t y acid synthesis (22,25) in cells not usually thought of as typical targets for insulin action. One problem with the metabolic regulation hypothesis is that the effects of insulin on growt~ 0024-3205/81/212131-09502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

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TABLE l Cell types in which insulin has been found to have growth-stimulatory effects (partial list) References

Cell type Fibroblasts a (chick, human, mouse)

2-4

Swiss 3T3 cells a'b and BALB/c 3T3 cells a'b

5-9

SV40-transformed 3T3 cells b

10

Normal rat hepatocytes c and rat hepatoma cells a

11, 12

Normal m a m m a r y epithelial cells a and m a m m a r y carcinoma cells a'b

13-16

Kidney epitheJial cell lines b, and normal kidney epithelial cells b

17, lg

Aortic smooth muscle cells d

19

Aortic intimal cells b

20

Myoblasts b

21

Rat pituitary tumor cells b (GH3, GC) b Human cervical carcinoma cells (HeLa)

22 23

Human prostate carcinoma cells b (PC 3)

20

Neuroblastoma b and glioblastoma cells b

20, 24

Melanoma cells b

25

Fibroblast x melanoma hybrid cells a'b b Ovarian follicular ceils

4, 26 27

Sertoli cells b

20

Mouse t e r a t o c a r c i n o m a cells b

28-30

Human intestinal tumor cells a

31

astimulation resting bstimulation CStimulation serum. dstimulation

of DNA synthesis and/or cell growth in serum-deprived or serum=depleted cultures. of growth in hormone-supplemented serum-free medium. of growth in medium containing 1596 heat-inactivated dialyzed fetal bovine of growth in medium containing 196 fetal bovine serum.

are often observed to be optimal only at superphysioiogicai concentrations while its acute metabolic effects in the same cells are often optimal at low concentrations, (see, for example, ref. 26). This has raised the possibility that the growth effects of insulin might be caused by a contaminant (36), or that insulin might stimulate growth by interacting with receptors for some other hormone, such as an insulin-like growth factor. These two possibilities are discussed in greater detail below. The dose=response relationship observed for the growth effects of insulin, and the requirement for superphysiological doses observed in some experiments, are d i f f i c u l t to interpret directly in terms of receptor occupancy. In order to stimulate growth, insulin generally must be present for an extended time period. Many ceils actively degrade insulin (26), and insulin is unstable in some cell culture media even in the absence of cells (20,37). Therefore, insulin is generally not present throughout the course of growth experiments at the concentration i n i t a l l y added to the medium. In regard to the

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possibility that the effects of insulin on growth may be attributable to a contaminant, single component insulin has recently been subjected to analysis by HPLC on a highresolution reverse phase column (38). The single component insulin was found to contain at least nine minor contaminant peptides. However, the insulin peak recovered from reverse phase chromatography retained its ability to stimulate -H-thymidine incorporation in 3T3 ceils. Moreover, semisynthetic porcine insulin and synthetic human insulin were also active on t h e - H - t h y m i d i n e incorporation assay (38). Thus, this a c t i v i t y of insulin appears to be an intrinsic property of the hormone rather than the a c t i v i t y of a contaminant.

I n t e r p r e t a t i o n of the e f f e c t s of insulin on growth has been further c o m p l i c a t e d by the observation t h a t insulin binds weakly to receptors for the insulin-like growth factors IGF I and II (39) and MSA (40-42) on some cells such as chick and human fibroblasts. On the basis of this observation, and the apparent nonadditivity of MSA and insulin action in chick fibroblasts, Rechler, Podskalny and Nissley (40,41) proposed several years ago t h a t insulin might s t i m u l a t e growth by binding to r e c e p t o r s for insulin-like growth factors. This hypothesis has the advantage of explaining the apparent lack of correlation observed in some experiments between low doses required to elicit e f f e c t s on metabolism as compared with high doses required to elicit e f f e c t s on growth. Recently, King et al. Q43) have obtained direct evidence indicating that in human fibroblasts, stimulation of - H thymidine incorporation by insulin is not caused by the binding of insulin to its own high affinity receptor. These investigators observed t h a t blockade of the high affinity insulin r e c e p t o r with Fab fragments of a n t i - r e c e p t o r 3 a n t i b o d y blocked high affinity insulin binding but did not prevent the stimulation of H-thymidine incorporation. Moreover, i n t a c t anti-insulin-receptor IgG, which elicits a3number of acute (4#) and chronic (45) insulin-like metabolic e f f e c t s , did not s t i m u l a t e H-thymidine incorporation. King et al. i n t e r p r e t e d their d a t a as indicating that the growth e f f e c t s of insulin were m e d i a t e d by its binding to r e c e p t o r s for an insulin-like growth factor (43). It appears, however, that some effects of insulin on growth cannot be explained by the binding of insulin to IGF/MSA receptors, and are very likely caused by binding of insulin to its own high a f f i n i t y receptor. An early indication that this might be the case came from the observation that insulin did not bind, or bound extremely weakly, to MSA and IGF receptors on some ceils, such as normal rat liver cells (46), but insulin nevertheless stimulated the growth of rat liver ceils in culture (I I). Furthermore, the observation that very high concentrations of insulin are required to stimulate growth is not universal. Recently, insulin at physiological concentrations has been shown to stimulate DNA synthesis in serum-deprived resting rat hepatoma cells, with a pharmacological specificity suggestive of interaction of insulin with ~ts own high affinity r e c e p t o r (I2). Physiological concentrations of insulin also s t i m u l a t e H-thymidine incorporation in ZR-75-1 human m a m m a r y tumor ceils (14) and promote the growth of the ZR-75-1 ceils and another human m a m m a r y tumor cell line (MCF-7) in hormone-supplemented serumfree medium (15,16). A further indication that in some cells, insulin and insulin-like growth f a c t o r s s t i m u l a t e growth via different pathways comes from the observation that GH~ pituitary tumor ceils and TM-4 Sertoli ceils require both insulin and somatomedin C for ~optimal growth in s e r u m - f r e e medium (20,22). Thus, high concentrations of insulin do not c o m p l e t e l y replace somatomedin C and vice versa. Finally, F9 mouse embryonal c a r c i n o m a cells grow in hormone-supplemented s e r u m - f r e e medium containing either insulin or MSA (30). Since insulin does not c o m p e t e for MSA binding to these cells, its growth e f f e c t s are apparently not m e d i a t e d by its binding to MSA r e c e p t o r s (30). In summary, then, t h e r e is evidence supporting the idea t h a t the growth stimulation of some cells by insulin is caused by binding of insulin to MSA/IGF receptors, however, some e f f e c t s of insulin on growth are apparently caused by binding of insulin to its own receptors. Synergy Between Insulin and Other Hormones in Promoting Growth Although insulin itself stimulates DNA synthesis and growth in some cells under certain culture conditions (2,12,26,37) its Stimulatory effect on the growth is typically

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enhanced by, and in some cases requires, the presence of other hormonal growth factors. An early observation of synergy between insulin and other growth factors was made by Holley and Kiernan (5), who studied the stimulation of DNA synthesis in quiescent Swiss 3T3 cells by insulin, pituitary £ibroblast growth factor (FGF) and dexamethasone. In the presence of FGF, insulin at a c o n c e n t r a t i o n of 50 ng/ml stimulated DNA synthesis in these cells (5), while in the absence of FGF, insulin was active only at very high concentrations (47). Conversely, in the presence of insulin, FGF was as active at a concentration of 5 ng/ml as was 50 ng/rnl FGF in the absence of insulin. 3imenez de Asua et al. (6,7) have studied in detail the synergistic action of insulin and prostaglandin F~ ~ ( P G F g ~ ) in stimulating DNA synthesis in quiescent Swiss 3T3 cells. Plotting thrift data 15y the transition probability method of Smith and Martin (4g), Jimenez de Asua et al. found that addition of insulin at any time after the addition of PGF2g increased the rate of entry of cells into S period. The time of addition of insulin did not appear to influence the duration of the lag period observed before cells entered S; the lag period was rather "established" by addition of P G F ) ~ (7). Insulin also acts synergistically with vasopressin in stimulating DNA synthesis i~ Swiss 3T3 cells (49). In the presence of insulin, vasopressin exhibits maximal activity at a concentration of 2 ng/ml, while in the absence of insulin, vasopressin is virtually without effect, even at much higher concentrations. Recently, insulin has been shown to increase the number of vasopressin receptors on the surface of a cultured kidney epithelial cell line in serum-free medium (50). It is possible that this effect could explain the potentiation by insulin of vasopressin action on the Swiss 3T3 cells. Stiles, Pledger, Scher and coworkers have developed a scheme for classifying growth factors that act synergistically in promoting entry into the cell cycle of quiescent Go/G 1 BALB/c 3T3 cells (51). One class of factors, including platelet-derived growth factor (PDGF) and pituitary fibroblast growth factor (FGF), is required for the entry of growtharrested cells into early G 1. These factors are termed competence factors. The other class of factors, including insulin-like growth factors and insulin, is required for progression of "competent" cells through G. and into S. Under the conditions of Stiles et al. (52), insulin shows weak progression a c n v l t y (160 ng = one progression unit), while the insulin-like growth factor somatomedin C exhibits strong progression activity (2 ng = one progression unit). The progression factors must be present throughout G 1 in order to promote entry of cells into S period (51). The universality of the requirement for insulin or insulin-like growth factors for progression of cells through G. has been questioned recently by Heldin et al (53). They have found that m the nutr~Uonally balanced medium MCDB-105 normal human glial cells are able to undergo several rounds of division with PDGF alone, in the absence of progression factors. Others have also observed that the balance of low molecular nutrients in the medium is crucial in determining whether cells require insulin or other "progression factors" for growth (see below). •

.

.

J.

Biochemical Events Regulated by Insulin Holley (54) suggested several years ago that one of the functions of the growth factors present in serum is regulation of the intacellular concentration of low molecular weight nutrients. Insulin stimulates the uptake of a number of essential nutrients, including glucose (33), phosphate (55), and type A neutral amino acids (3), and it also stimulates release of membrane-bound magnesium (56). Insulin stimulates reentry into the cell cycle of cells made quiescent in media that have been manipulated to make one of these low molecular weight nutrients growth-limiting (32). That one function of insulin might be regulation of the intracellular balance of low molecular weight nutrients is further suggested by the observation of Wu and Sato (57) that HeLa-S3 cells are able to form clones in hormone-supplemented serum-free media minus FGF and insulin, if the basal medium is MCDB-105 (a medium in which nutrients have been carefully balanced for cloning of human fibroblasts). In contrast, insulin and FGF are required for formation of colonies in serum-free F12 medium. Along similar lines, Heldin et al. (53) have found that human glial cells proliferate in MCDB-105 supplemented with PDGF alone and have

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suggested that the growth requirement for insulin or other "progression" factors depends on the composition of the nutrient medium. The biochemical mechanism by which insulin stimulates intracellular events (enzyme activation and inactivation, protein synthesis, RNA synthesis and DNA synthesis) is a subject of great current interest. Considerable evidence has accumulated to indicate that insulin may exert many of its effects by regulating specific protein phosphorylation and dephosphorylation reactions (58-69). Recent evidence suggests that a peptide, released into the cytoplasm from the plasma membrane when insulin binds to its receptor, stimulates a phosphoprotein phosphatase that dephosphorylates (and hence activates) pyruvate dehydrogenase (61,6#,67-69). The possible involvement of this peptide as a secondary chemical mediator of the action of insulin on other intracellular events, including growth-related events, remains to be determined. Growth Requirement for Insulin and Malignant Transformation An early observation regarding the growth of mouse fibroblastic cell lines and their SV#0 viral transformants was that the SV#0 transformants had a lower quantitative requirement for serum (70), as well as an apparent qualitative change in the combination of serum factors that were required (7l). With the recent development of hormonesupplemented serum-free media for the culture of mammalian cells, there has been considerable interest in determining which growth factor requirements might be lost by transformed cells. The results of these studies have indicated that in general the growth requirement for insulin is not lost when cells become malignantly transformed. For example, Sato and coworkers have recently studied the growth factor requirements of BALB/c 3T3 cells and SV40-transformed BALB/c 3T3 ceils (9,10). They have found that viral transformation leads to a loss of the requirement for EGF, FGF and a partially purified factor from rat submaxillary gland (Gimmel), but not a loss of the requirement for insulin. Similar results have been obtained by Cherington et al. (72) with tumorigenic and nontumorigenic Chinese hamster embryo fibroblast cell lines. The tumorigenic clones had a lower requirement for EGF than the nontumorigenic clones but retained the insulin requirement. Cherington et al. showed further by flow cytometry that the tumorigenic clones could be arrested in G 1 by deprivation for insulin. The fact that malignant transformation is not generally accompanied by a loss of the growth requirement for insulin is further underscored by the observation that most tumor cell lines require insulin for growth in serum-free medium (20). Genetic Approaches The "pathway" by which insulin exerts its effects on growth has recently been studied by several laboratories using techniques of somatic cell genetics. The PG19 mouse melanoma cell line is unresponsive to the growth-stimulatory action of insulin, both in growth-arrested confluent monolayers in medium with low serum, and in hormonesupplemented serum-free medium (4,26). Insulin stimulates DNA synthesis in resting mouse embryo fibroblasts and fibroblast x melanoma hybrids, and it also stimulates the growth of the hybrids in hormone-supplemented serum-free medium (26). Thus ability to respond to the growth-stimulatory action of insulin behaves as a dominant characteristic in the hybrids, inherited from the fibroblast parent. The refractoriness of the mouse melanoma cells to growth stimulation by insulin does not appear to be attributable to a lack of insulin receptors (26). Shimizu and Shimizu (73) have recently selected for insulin-unresponsive variants of the BALB/c 3T3 cell line. The technique used involves selection against ceils that undergo mitosis following stimulation with the hormone. One insulin-insensitive variant has been studied in detail and appears to have an insulin receptor defect (73). Miskimins and Shimizu (74) have also isolated variants of the Swiss 3T3 cell line that are resistant to a cytotoxic insulin derivative comprised of insulin crosslinked to fragment A of diphtheria toxin. Two of the variants are deficient in insulin binding. Finally, Kahn et al. (75) have isolated variants of the Cloudman S91 melanoma cell line having an altered growth

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response to insulin. The parental S91 melanoma cell line has the unusual property of being inhibited by insulin in medium containing serum. (This property is not shared by three other melanoma cells lines (26,75).) Variants of the melanoma~e~lls resistant to growth inhibition by insulin have been isolated. One such variant (Ins ~ v ) was found to require insulin for growth. Insulin stimulated the^ growth of this variant at physiological concentrations (half maximal response at 10- - u M insulin). Possible Effects of insulin on fetal growth There are a number of clinical examples suggesting that insulin is an important growth-regulating hormone during human fetal development. Infants born to diabetic mothers with hyperglycemia late in pregnancy often have excessive body weight and size, and neonatal hypoglycemia (76). This has been a t t r i b u t e d to hyperinsulinemia in the fetus, in reaction to elevated maternal blood glucose, and subsequent action of insulin as a growth factor. Roth has suggested that the hypoglycemia and excess deposition of stored fat, glycogen and protein observed in such infants is a t t r i b u t a b l e to excess insulin i n t e r a c t i n g with insulin receptors, while the excess length and body size may be due to "spillover" action of insulin on IGF receptors (77). The reverse of this syndrome is observed in infants with insulin deficiency at birth. Islet cell agenesis is accompanied by very low birth weight, decreased fat deposition and deficient muscle development, and is fatal shortly a f t e r birth (78,79). Congenital permanent diabetes of the newborn is also characterized by small body size for gestational age (80). Fetal hypoinsulinemia may also be a contributory factor to low birth weight observed in other clinical situations (81). Finally, a class of patients with leprechaunism in which this syndrome is associated with insulin resistance has been described. In two such patients, the resistance to insulin appears to be a t t r i b u t a b l e to a postreceptor defect (82,83), while in two other patients, there is a primary defect in the insulin receptor (g4-g6). Whether the growth retardation observed in these patients arises directly from insulin resistance, a requirement of insulin receptors for the function of growth factor receptors (86), or whether it is an unrelated consequence of the syndrome remains to be demonstrated. Although the growth anomalies observed in the above examples have been considered in terms of direct effects of insulin on growth, i t should be added that some of the growth mediating effects of insulin in vivo may be indirect. An example of one such indirect effect has been reported by Baxter et al. (87), who have found that insulin regulates somatogenic receptors in the rat liver. These authors suggest that the low somatomedin levels and growth retardation observed in insulin-deficient diabetes may be attributable to deficiency in this action of insulin (87). Conclusions Insulin stimulates the growth and proliferation of a variety of cultured mammalian ceils. The results of King et al. (43) suggest that some effects of insulin on cellular proliferation are a t t r i b u t a b l e to interaction of insulin with IGF/MSA receptors, while other effects of insulin on growth appear to be caused by binding of insulin to its own high affinity receptor. The difference in the growth response to insulin observed in different ceils probably depends to a large e x t e n t on what processe(s) are growth-limiting in different cell types under different sets of experimental conditions. In experiments in which insulin alone, at physiological concentrations, stimulates DNA synthesis and/or growth, the target ceils are very likely already "competent" and limited principally for processes regulated by insulin. More typically, insulin acts synergistically with other growth factors to promote cell proliferation. The isolation by Kahn et al. (75) of an insulin-dependent variant cell line appears to represent a case in which cell cycle control has been altered in such a way as to make a process regulated by insulin growth-limiting. Very little is known at present about the nature of the biochemical mechanism by which insulin exerts its effects or growth, although there is some evidence to suggest that one function of insulin may be to regulate the intracellular balance of nutrients. The extent to which the growth effects of insulin observed in cell culture are related to those that occur in vivo during fetal development remains an interesting question.

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Acknowledgments I thank C.V. Byus, 3.E. Lever, 3.M. Podskalny, M.M. Rechler, and M.L. Taub for helpful comments and suggestions. This work was supported by NIH grant AM21993. References 1. 2. 3. 4. 5. 6. 7. 8.

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