6376081

Growth-Stimulatory Actions of Insulin in Vitro and in Vivo*

DANIEL S. STRAUS

Division of Biomedical Sciences and Department of Biology, University of California, Riverside, California 92521

Abstract

I NSULIN stimulates the growth and proliferation of a variety of somatic cells in culture, and evidence sug- gests that insulin is also an important regulator of growth in vivo. In cell culture, insulin interacts synergistically with other hormones and growth factors such as platelet- derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), tumor-promoting phorbol esters, and thrombin, to stimulate progression through the cell cycle of cells that have been arrested in G1 by deprivation for serum. In addition, insulin is re- quired by most cells for optimal long term growth in hormone-supplemented serum-free media. In some cells, such as human skin fibroblasts, the growth-promoting effects of insulin appear to be mediated primarily by its low affinity interaction with receptors for insulin-like growth factor I (IGF-I). In other cells, such as hepato- cytes, hepatoma cells, adrenocortical tumor cells, mam- mary carcinoma cells, and F9 embryonal carcinoma cells, insulin appears to stimulate growth by binding to high affinity insulin receptors. The insulin and IGF-I receptor proteins, like the receptor proteins for other growth- promoting hormones such as EGF and PDGF, are closely associated with tyrosine-specific protein kinase activi- ties. The mechanism by which the binding of insulin to its receptor and activation of the receptor-associated tyrosine protein kinase activity control intracellular pro- tein phosphorylation and dephosphorylation reactions, such as the phosphorylation of ribosomal protein S6, is a subject of considerable current interest. The phospho- rylation of ribosomal protein S6 may be related mecha- nistically to the activation by insulin of protein synthesis, and hence the passage of cells through the G1 phase of the cell cycle. Malignant transformation does not gen- erally result in a total loss of the growth requirement of cells for insulin or insulin-like growth factors, although transformation is accompanied in some cases by a qual-

itative reduction in the insulin/IGF requirement. Abnor- malities in insulin production or sensitivity in vivo are accompanied by abnormalities in growth; thus, insulin appears to be an important regulator of growth in vivo. Some of the growth-promoting effects of insulin in vivo may be attributable to direct action of insulin, while other effects may be caused by the regulatory effect of insulin on somatomedin production, and possibly on somatomedin action.

Introduction

Insulin regulates a wide spectrum of metabolic proc- esses in vivo, via mechanisms that are not fully under- stood. In addition to its well-known effects on metabo- lism, insulin stimulates the growth and proliferation of a variety of cells in culture, and evidence suggests that insulin may also be an important regulator of growth in vivo. The first observation of the growth-stimulatory effect of insulin in tissue culture was made by Gey and Thalhimer in 1924 (1). Subsequently, insulin has been shown to stimulate the proliferation of many cell types (2), under a variety of experimental conditions. Insulin acts synergistically with other hormones and growth factors to stimulate progression through the cell cycle of cells that have been arrested by G1 by deprivation of serum (3-11) or an essential nutrient such as phosphate (12). Insulin is also required by most cell types for optimal growth in hormone-supplemented serum-free media (2, 13, 14). The relationship between the effects of insulin on metabolism and on cell proliferation has remained an interesting unsolved problem. This review summarizes current evidence and hypotheses concerning the growth-stimulatory effects of insulin.

Interpretation of insulin dose-response curves, and action of insulin via insulin or IGF-I receptors

An early observation regarding the growth-stimulatory effect of insulin on some types of cultured cells, especially fibroblasts and fibroblastic cell lines, was that super-

* This work was supported by National Institutes of Health Grant AM21993.

physiological concentrations of insulin, typically on the order of 1 ug/ml or higher, were required to elicit a maximal response (3-6). There are a number of possible explanations for this observation including the following: 1) growth stimulation by insulin might not be caused by insulin itself but rather by a minor contaminant; 2) insulin might be degraded during the course of the growth assay, thus, the amount of insulin added at the beginning of an experiment may be much greater than the average concentration of insulin present during the course of the experiment; and 3) insulin might stimulate growth by binding with a low affinity to receptors for another hormone such as a somatomedin. Each of these possibil- ities is considered in detail below.

In regard to the possibility that the effects of insulin on growth might be attributable to a contaminant (15), “single component” insulin has recently been subjected to analysis by reverse-phase high-performance liquid chromatography (16). Using this high-resolution tech- nique, the single component insulin was found to contain at least nine minor contaminant peptides (16). However, the insulin peak recovered from the reverse phase column retained its ability to stimulate [H]thymidine incorpo- ration in 3T3 cells (16). Moreover, semisynthetic porcine insulin (prepared by reoxidizing purified A- and B- chains obtained from insulin) and synthetic human in- sulin were also active in the [H]thymidine incorporation assay (16). Fully synthetic human insulin is also equi- potent with porcine insulin in stimulating [3H]thymidine incorporation in human fibroblasts (17). Crystalline in- sulin is generally prepared as the zinc complex; thus, in experiments in which high concentrations of insulin are used, high concentrations of zinc are also present. How- ever, the effects of insulin on growth are not caused by zinc, since zinc-free insulin preparations also stimulate cell growth (16, 18) and are actually more active on some cells than zinc-insulin due to an inhibitory effect of high zinc concentrations (18). Therefore, the growth-stimu- latory activity of insulin appears to be an intrinsic prop- erty of the hormone rather than the activity of a contaminant.

Because of the very long time-course and high temper- ature (37 C) used in cell growth experiments, degradation of insulin during cell growth assays presents a major problem for relating dose-response data for a biological response to equilibrium binding constants for receptor binding. Insulin is degraded by cultured cells via recep- tor-mediated endocytosis (19-23) and in some cases by action of extracellular proteases (8). In addition, insulin is unstable in some cell culture media even in the absence of cells (9, 13). Thus, the actual concentration of insulin may change through the course of DNA synthesis and growth assays, with the average concentration of biolog- ically active insulin being considerably lower than that

initially added to the medium. Because of this, caution must be exercised in any attempt to relate dose-response data obtained from the bioassays to binding affinity of insulin to a specific class of receptors.

Rechler and coworkers proposed some time ago that stimulation of the growth of fibroblasts by insulin was mediated by its weak binding to receptors for insulin- like growth factors (IGFs) (24, 25). Initially, this hypoth- esis was based on equilibrium binding data indicating that insulin competed for IGF binding to chick embryo fibroblasts, and the observation that effects of insulin and MSA (rat IGF-II) on the growth of chick embryo fibroblasts were nonadditive (24, 25). Further support for this idea was obtained when highly purified prepa- rations of IGF-I and -II became available (26-30), and it was observed that much higher concentrations of insulin than IGF were required to stimulate the growth of fibro- blasts (31). This, together with the observation that insulin competed weakly for the binding of the purified IGF peptides to receptors on fibroblasts (31) suggested that the growth-promoting effects of insulin on fibro- blasts were mediated by the binding of insulin to an IGF receptor.

The nature of the interaction of insulin with IGF receptors has been greatly clarified by recent advances on the biochemistry and hormone binding properties of IGF receptors (32-39). Affinity-crosslinking studies have revealed the existence of two classes of IGF receptors (32-38). IGF-I receptors (also designated somatomedin C (37, 39) or “type I” receptors (38) have a higher affinity for IGF-I than IGF-II and have a low affinity for insulin (37, 38). IGF-I receptors have a very similar structure and subunit composition to insulin receptors. Both re- ceptors consist of four disulfide-linked subunits including two nonidentical subunits, a (M, ~ 135,000) and 6 (M, ~ 90,000), which are linked by disulfide bonding (37-39). One minor difference in the subunit structure of the two receptors is that the apparent molecular weight of the ß subunit of the IGF-I receptor is slightly larger than that of the @ subunit of the insulin receptor (39, 40). The IGF-I and insulin receptors appear to have at least some similar antigenic determinants (41); however, mono- clonal antibodies have been obtained that distinguish between the two receptors (39). Both insulin (42-45) and IGF-I (46, 47) receptors are associated with a tyrosine- specific protein kinase activity. Both receptors are syn- thesized as a single precursor polypeptide (M, ~ 180,000), which is processed to give one a and one 3 subunit (48, 49). IGF-II receptors (also designated “type II” receptors) bind IGF-II with a higher affinity than IGF-I and do not appear to have a significant affinity for insulin. IGF-II receptors consist of a single polypeptide chain, M, ~ 260,000 (reduced), or 220,000 (nonreduced) (32-36, 38). Since insulin binds to IGF-I receptors but not to IGF-II

receptors, the competition by insulin for IGF binding observed earlier in equilibrium-binding studies appears to represent competition for binding to IGF-I receptors. The close structural similarity between the insulin recep- tor and IGF-I receptor suggests that the genes for the two receptors may have evolved through a duplication event. Interestingly, a human insulin-resistant diabetic patient who exhibits a decreased number of insulin re- ceptors has also been found to have low levels of IGF-I receptors (50). This raises the interesting possibility that the two receptors may be co-regulated.

The strongest evidence for an effect of insulin on growth being mediated by its interaction with IGF recep- tors has been obtained with cultured human skin fibro- blasts by King et al. (51). These investigators observed that blockade of the high-affinity insulin receptor with Fab fragments of anti-receptor antibody blocked high- affinity insulin binding but did not prevent the stimu- lation by insulin by [H]thymidine incorporation. More- over, intact anti-insulin-receptor IgG, which elicits a number of acute (52) and chronic (53) insulin-like meta- bolic effects, did not stimulate [H]thymidine incorpo- ration. King et al. (51) interpreted their data as indicating that the growth effects of insulin in human fibroblasts were mediated by its binding to IGF receptors. Massague and Czech (38) have shown by affinity cross-linking experiments that human skin fibroblasts have IGF-I receptors, which presumably mediate the growth-stimu- latory effect of insulin in these cells. Less direct evidence suggests that some, or all, of the growth-stimulatory effects of insulin in chick embryo fibroblasts (3, 24, 25, 31), rat myoblasts (54), BALB/c 3T3 cells (55), and CCI39 Chinese hamster lung fibroblasts (56) are me- diated by interaction of insulin with IGF receptors.

In contrast to the above results, the growth-stimula- tory effect of insulin in other cells appears to be mediated by binding of insulin to high-affinity insulin receptors. An early indication that this might be the case came from the observation that insulin did not bind, or bound extremely weakly to IGF receptors on some cells such as rat liver cells (57), but insulin nevertheless stimulated the growth of rat liver cells in culture (58). [Insulin has also been implicated as an important regulatory hormone for liver regeneration in vivo (59).] The lack of competi- tion by insulin for IGF- or MSA-binding to liver cells observed in equilibrium binding studies has more re- cently been explained by affinity cross-linking experi- ments which have shown that liver cells have large numbers of IGF-II (type II) binding sites which do not bind insulin, and no detectable IGF-I (type I) binding sites (38). Thus the growth stimulatory effects of insulin in liver cells appear to be mediated by binding of insulin to the insulin receptor. This conclusion is strongly sup- ported by recent results on the effects of insulin on the

growth of cultured H35 rat hepatoma cells. Insulin strongly stimulates DNA synthesis and cell cycle pro- gression of G1-arrested H35 hepatoma cells (10). The low dose of insulin required to elicit this response (EC50 = 30-70 pmolar), as well as the relative potencies of insulin and proinsulin, strongly suggest that insulin is acting through the high-affinity insulin receptor (10). This con- clusion is further supported by the observation that the H35 hepatoma cells, like the normal liver cells from which they are derived, have IGF-II (type II) but not IGF-I binding sites, which indicates that insulin action in these cells is mediated by binding of insulin to insulin receptors (11).

The effects of insulin on the growth of a number of other cell types also appears to be caused by binding of insulin to high-affinity insulin receptors rather than IGF receptors. Physiological concentrations of insulin stim- ulate [3H]thymidine incorporation in human mammary tumor cells (8) and promote the growth of human mam- mary tumor cells in hormone-supplemented serum-free media (60, 61). Insulin at physiological concentrations also stimulates the growth of adrenocortical tumor cells in hormone-supplemented serum-free media (62). The EC50 for this effect is 0.08 nM and the EC100 is 2 nM, which strongly suggests interaction of insulin with high- affinity insulin binding sites (62). Other cells, such as GH3 rat pituitary tumor cells and TM4 rat Sertoli cells, require both insulin and an IGF for optimal growth in serum-free media (13, 63). The fact that neither hormone completely replaces the requirement for the other hor- mone suggests action of the two hormones through dif- ferent receptors. Finally, the growth of F9 embryonal carcinoma cells is stimulated by low concentrations of insulin (64) or rat IGF-II (65). Insulin appears to exert its effect in these cells by binding to insulin receptors since the effects of insulin are mimicked by anti-insulin receptor antibody (64), and the F9 cells appear to have primarily IGF-II rather than IGF-I receptors (64, 65).

From the above observations, it is apparent that “spill- over” action of insulin through IGF receptors occurs primarily in cells such as fibroblasts that have large numbers of IGF-I receptors, which bind insulin. In a number of epithelial cell types that have large numbers of IGF-II receptors, which do not bind insulin, and few if any IGF-I receptors, insulin appears to exert its actions exclusively through the insulin receptor.

Synergistic interactions between insulin and other growth-promoting hormones

Although insulin alone can stimulate progression through the cell cycle of some cells under certain culture conditions (9), growth stimulation by insulin has more commonly been observed to be potentiated by, and in

some cases to require, the presence of other hormones. One cell line in which the synergistic interaction between insulin and other growth-promoting hormones has been studied extensively is the Swiss mouse 3T3 cell line (66). These cells can be arrested in G1 by limitation for serum- derived growth factors, and then restimulated to reenter the cell cycle by readdition of serum or combinations of growth factors. Insulin has been found to act synergisti- cally with FGF (4, 67), EGF (68), PDGF (69), PGF20 (70, 71), vasopressin (72), TPA (69), and cyclic AMP analogs (73), to stimulate entry into S phase of resting cultures of the Swiss 3T3 cells. In each case, action of insulin in combination with the other hormones is synergistic in that: 1) the effects observed for insulin plus one of the other factors are more than additive, and 2) for a given response, lower concentrations of insulin are required when one of the other factors is present, and vice versa (66). Jimenez de Asua and coworkers have performed detailed studies of the kinetics of entry into S phase of the Swiss 3T3 cells in response to PGF2% (70, 71) and FGF (67) in combination with insulin. Plotting their data by the transition probability model of Smith and Martin (74), these investigators have measured the du- ration of the lag phase that occurs between stimulation with hormones and entry of the first cells into S phase, and also the rate of entry of cells into S phase after the lag phase. In each case, the principal effect of insulin is to increase the rate of entry into S phase after the lag phase, with little or no effect observed on the duration of the lag phase. Insulin can be added at any time during the lag phase and still stimulate the rate of entry into S phase. In contrast, the initiation of the lag phase is established by the time of additions of PGF2% or FGF, and not by the time of addition of insulin. Using a more advanced basal growth medium (MCDB 402) and an assay in which cell division as well as DNA synthesis is monitored as an endpoint, Shipley and Ham (75, 76) have reevaluated the effects of FGF, PDGF, EGF, dexa- methasone and insulin on growth of the Swiss 3T3 cells. Under the assay conditions employed by these investi- gators, insulin is not required for the initiation of DNA synthesis in response to FGF but is required for contin- uing multiplication over a period of several days (75, 76).

Similar studies performed with G1-arrested cultures of BALB/c 3T3 cells have also demonstrated synergistic action of insulin in combination with other growth fac- tors such as FGF (5), PDGF (55) and the phorbol ester tumor promoter TPA (77). Based on the temporal se- quence of action of different hormones observed with BALB/c 3T3 cells, Pledger, Stiles and coworkers have developed a scheme in which growth-promoting hor- mones are classified as “competence” or “progression” factors (55). Competence factors such as PDGF and the tumor promotor TPA are proposed to cause a stable

biochemical change early in G1 which, once established, is no longer dependent on the continued presence of that factor in the medium. Once cells become competent in this manner, “progression” factors such as insulin or IGF-I can stimulate progression through G1 and into S phase (55). Further studies of the growth factor require- ments of the BALB/c 3T3 cells in hormone-supple- mented serum-free medium have been performed re- cently by McClure (78), who has found that these cells grow well under serum-free conditions in medium sup- plemented with transferrin, hydrocortisone, insulin, and FGF or PDGF.

Two areas of controversy currently surround the com- petence/progression factor model. The first relates to the question of whether the competence state induced by factors such as PDGF and TPA persists after removal of these factors from the medium (69, 79, 80). The compe- tence factors, as a class, share the property of adsorbing nonspecifically to surfaces and are therefore difficult to completely remove by washing (69, 79, 80). To circum- vent this problem, Singh et al. (80) have used anti-PDGF antibodies to inactivate PDGF at various times after addition of PDGF to cells. These experiments support the idea that PDGF must be present for only a relatively short period of time to promote a mitogenic response. A second area of controversy relates to the universality of the requirement for progression factors such as insulin and insulin-like growth factors. Heldin and coworkers (81) have shown that in the nutritionally balanced me- dium MCDB-105, PDGF alone promotes the growth of human glial cells, in the absence of progression factors. These investigators concluded that a dependency on progression factors is not a general feature of PDGF- responsive cells (81). However, the possibility was not ruled out that the human glial cells produced an insulin- like growth factor that stimulated growth of the same cells (i.e. autocrine growth stimulation). In regard to this possibility, it should be noted that a growth requirement of human diploid fibroblasts for insulin-like growth fac- tors has also not been observed in all experiments (re- viewed in Ref. 82). However, in this case the difficulty in demonstrating a requirement for insulin or IGFs is very likely attributable to production by the cells of IGFs (82, 83). In experiments in which diploid human fibro- blasts are grown at clonal densities in hormone-supple- mented serum-free media, insulin is clearly required for optimal growth (84).

Biochemical mechanisms for growth stimulation by insulin and insulin-like growth factors

Insulin is an anabolic hormone with a wide range of effects on metabolism, including stimulation of the syn- thesis and inhibition of the degradation of glycogen,

protein, and lipids (85). Insulin stimulates the uptake of glucose (86), phosphate (87), monovalent cations (87), and type A neutral amino acids (88), and it modulates fluxes of divalent cations such as Mg++ and Ca++ (87). Evidence suggests that the positive effects of insulin on growth are closely related to its stimulation of anabolic processes. Some recent advances on the mechanism of insulin action and their relation to the effects of insulin on cell proliferation are discussed below.

Many of the early metabolic effects of insulin in re- sponsive cells are mediated by protein phosphorylation and dephosphorylation reactions (89-91). Recently, the insulin receptor has been shown to be a tyrosine-specific protein kinase, the activity of which is stimulated by the binding of insulin (42-45). Phosphotyrosine is an unu- sual amino acid that accounts for only 0.03% of the total phosphoamino acid content of normal cells (92), and a number of lines of evidence currently suggest that phos- phorylation of tyrosine on some key protein(s) may be an important signal regulating cellular proliferation. Phosphorylation of tyrosine has been linked to the mech- anism of cellular transformation by a number of onco- genic viruses, the transforming genes of which have been shown to code for tyrosine-specific protein kinases (93- 95). In addition, receptors for a number of growth-pro- moting hormones including EGF (96), PDGF (97), IGF- I/SmC (46, 47), and insulin (42-45) have recently been shown to be closely associated with tyrosine-specific protein kinases, and activation of the kinase activity by the binding of ligand very likely represents a major mechanism for transmembrane signalling by this class of hormones.

The molecular mechanism by which activation of the insulin receptor kinase is coupled to later events (e.g. activation and inactivation of intracellular enzymes, in- duction of protein synthesis, etc.) is the key unsolved problem in insulin action. Evidence suggests that induc- tion of protein synthesis during the G1 phase of the cell cycle is required for the initiation of DNA synthesis after the stimulation of growth-arrested cells by serum or insulin (98-104). A number of lines of evidence indicate that the enhanced synthesis and stabilization of a labile protein before the initiation of DNA synthesis is a key regulatory event leading to the initiation of DNA syn- thesis (101-104). One well-known anabolic effect of in- sulin in cultured cells is stimulation of protein synthesis (100) and inhibition of protein degradation (105). It is thus possible that insulin and other growth factors may influence the rate of entry into S phase by stimulating protein synthesis and inhibiting protein degradation.

There are a number of possible levels for control of protein synthesis including regulation of rates of tran- scription and translation. Recently, genes specifying mRNA species that are induced during growth stimula-

tion by serum (106) or PDGF (107) have been cloned. A similar approach might be feasible for identification of mRNAs induced by insulin and insulin-like growth fac- tors. Insulin has recently been shown to have positive (108, 109) and negative (110, 111) effects on levels of some specific mRNAs.

In relation to possible mechanisms of translational control, considerable interest has focused recently on the phosphorylation of ribosomal protein S6. This protein is a basic protein of mol wt ~ 31,000, and is a component of the 40S subunit (112). Ribosomal protein S6 becomes highly phosphorylated when Go/G1-arrested cells are stimulated with serum (113-115), EGF (114, 116, 117), PDGF (118, 119), insulin (113, 114, 116, 117, 120) and insulin-like growth factors (113). Since the amino acid that is phosphorylated on the S6 protein is serine rather than tyrosine (121), phosphorylation of S6 is not caused by direct action of the receptor-associated tyrosine-spe- cific protein kinase. There are two distinct pathways for phosphorylation of ribosomal protein S6. The first is activated by hormones that elevate cAMP and involves action of cAMP-dependent protein kinase (116, 121- 124). Purified cAMP-dependent protein kinases promote the incorporation of up to two molecules of phosphate per molecule of S6 (123, 124). A second pathway, acti- vated by insulin and growth factors, leads to the incor- poration of up to five molecules of phosphate per mole- cule of S6 (113-120). Phosphopeptide mapping of the S6 protein phosphorylated by the cAMP-dependent and insulin-dependent pathways has revealed differences in the sites that are phosphorylated (121, 125, 126). The amino acid sequence of one of the peptides that is phos- phorylated by the cAMP-dependent protein kinase has been reported recently (124). Considerable interest is currently centered on the identity of the cAMP-inde- pendent protein kinase that mediates the insulin and growth factor-stimulated phosphorylation of the S6 pro- tein. Perisic and Traugh (125, 126) have recently pre- sented evidence suggesting that protease-activated ki- nase II (PAK II kinase) (127) is the insulin-stimulated kinase that phosphorylates S6. The purified PAK II kinase utilizes ribosomal protein S6 as a preferred sub- strate (127), and phosphopeptide maps of the S6 protein phosphorylated by purified PAK II kinase in vitro are identical to those obtained following stimulation of 3T3- L1 cells with insulin (125, 126). Furthermore, stimulation of the 3T3-L1 cells with insulin leads to activation of the PAK-II kinase enzyme (125). In contrast to these results, Cobb and Rosen have described an insulin-sensitive pro- tein kinase associated with the particulate fraction from 3T3-L1 cells with properties similar to casein kinase I (128). The suggestion of these investigators that casein kinase I might be the insulin-regulated S6 kinase is in apparent conflict with the results of Perisic and Traugh,

who reported that casein kinase I does not phosphorylate ribosomal protein S6 (125). Recently, Le Peuch et al. (129) have shown that highly purified preparations of the Ca+2, phospholipid-dependent protein kinase C phos- phorylate the S6 protein in vitro. Recent evidence sug- gests that protein kinase C is the receptor for tumor- promoting phorbol esters (130, 131). Thus, it seems likely that this enzyme may mediate the effects of tumor pro- moters on S6 phosphorylation observed in intact cells (132). Protein kinase C shares some properties with the purified PAK-II kinase, but there are also distinct dif- ferences between the two enzymes (127).

There is evidence to suggest that phosphorylation of ribosomal protein S6 may be linked to the increased rates of initiation of protein synthesis observed when cells are stimulated with insulin and growth factors. The S6 protein has been localized in the head region of the 40S ribosomal subunit, near the site of binding of the ternary initiation complex (133). Terao and Ogata have obtained evidence suggesting that the S6 protein is in- volved in interaction of poly(U) with the 40S subunit (134, 135). Gressner and Van deLeur have demonstrated decreased rates of dissociation of poly(U) from highly phosphorylated 40S subunits (136). Duncan and Mc- Conkey (137, 138) demonstrated that 40S ribosomal sub- units containing newly phosphorylated S6 were prefer- entially incorporated into polysomes, and inferred from this that S6 phosphorylation enhanced the rates of ini- tiation by the 40S ribosomes. Recently, direct evidence linking phosphorylation of S6 to increased rates of ini- tiation has been obtained by Burkhard and Traugh (139), who have shown that phosphorylation of 40S ribosomal subunits in vitro with protease-activated kinase-II in- creases the binding of AUG and poly (A,U,G) and stim- ulates poly (A,U,G)-directed translation. In contrast, phosphorylation of 40S subunits with cAMP-dependent protein kinase inhibits the binding of poly (A,U,G) and inhibits poly (A,U,G)-directed translation.

Several proteins other than ribosomal protein S6 are also phosphorylated on serine residues in response to insulin. One such protein is the insulin receptor. When intact cells are treated with insulin, the ß subunit of the receptor protein is phosphorylated on serine and threo- nine residues as well as tyrosine residues (140). Since the purified receptor protein has only tyrosine kinase activity (43-45), a second insulin-sensitive protein kinase en- zyme apparently catalyzes the serine phosphorylation of the insulin receptor. The relationship between this pro- tein kinase activity and that which phosphorylates ri- bosomal protein S6 is an interesting question. Recently, Jacobs and coworkers have shown that the insulin and IGF-I receptor proteins are both phosphorylated when cells are treated with the phorbol ester TPA (141). As noted above, it has recently been proposed that TPA

action is mediated by its binding to protein kinase C (130, 131). Thus it is possible that protein kinase C is responsible for the phorbol-ester-stimulated phosphoryl- ation of the insulin receptor. Another protein that is phosphorylated in response to insulin is the lipogenic enzyme ATP-citrate lyase (142, 143). Like ribosomal protein S6, ATP-citrate lyase can be phosphorylated by an insulin-sensitive protein kinase and cAMP-dependent protein kinase; however, in contrast to results obtained with the S6 protein, insulin and cAMP-stimulated phos- phorylation of ATP-citrate lyase appears to occur on a single peptide (142, 143). A physiological role for the phosphorylation of ATP-citrate lyase has not yet been established.

A number of physiological effects of insulin are caused by its stimulation of protein dephosphorylation. Some progress has been made toward identification of an in- tracellular mediator of effects of insulin on protein de- phosphorylation. A low molecular weight factor from skeletal muscle of insulin-treated rats that stimulates glycogen synthetase by activating glycogen synthetase phosphoprotein phosphatase has been identified by Lar- ner and coworkers (144). A similar insulin-sensitive fac- tor that stimulates pyruvate dehydrogenase (PDH) in cellfree mitochondrial preparations has been observed in muscle extracts (145), adipocyte plasma membrane prep- arations (146), intact adipocytes (147), liver particulate preparations (148, 149), and rat hepatoma cells (150). The insulin-sensitive stimulator of PDH appears to act by stimulating the activity of a phosphoprotein phospha- tase rather than by inhibiting a protein kinase (151). This factor has a mol wt of approximately 1000-2000 (152, 153) and an isoelectric point of 4.5 (153). Partially purified preparations of the PDH-stimulatory factor have also been observed to activate the low Km cyclic AMP phosphodiesterase (150, 154) and (Ca2+-Mg2+)- ATPase (155). Crude preparations of the insulin-sensi- tive activators of pyruvate dehydrogenase and glycogen synthetase phosphoprotein phosphatase contain inhibi- tors of the same two enzymes; thus, these preparations typically exhibit biphasic dose-response curves (144, 148). The inhibitory molecules appear to be biochemi- cally distinct from the stimulatory molecules (149, 156). The biological role of the inhibitors is at present unclear; some evidence suggests that they may in fact be another class of mediators of insulin action (149, 156).

The molecular nature of the putative mediators of effects of insulin on protein dephosphorylation reactions is under investigation in several laboratories. Seals and Czech (157) reported data suggesting that the mediator that activates PDH is a peptide that is released by cleavage of a larger membrane protein. Seals (158) has reported at 5000- to 10,000-fold purification of the PDH activator, using procedures that again suggest that this

factor is a peptide. A further characterization of the purified mediator will be required before definite conclu- sions can be drawn regarding the role of this agent in the sequence of insulin action.

The relationship between the signal or signals that mediate effects of insulin on protein phosphorylation reactions and dephosphorylation reactions is a question of considerable interest. Although there is currently no direct evidence bearing on this question, it is of interest to note that two protein kinases, protease-activated ki- nase II and protein kinase C, which are possible candi- dates for the kinase that phosphorylates ribosomal pro- tein S6 in vivo, are both activated by limited proteolytic cleavage (127, 159). It is tempting to speculate that an early event in insulin action could be activation of a protease that activates one or both of the kinase enzymes and also cleaves the peptide mediator from a precursor in the plasma membrane.

Growth requirement for insulin and malignant transformation

An early observation regarding the oncogenic trans- formation of normal fibroblasts (160, 161) and fibro- blastic cell lines (161-163) by tumor viruses was that malignantly transformed cells required lower concentra- tions of serum for growth than their untransformed counterparts. This suggested that the process of trans- formation was accompanied by a loss of requirement for growth factors present in serum. With the recent intro- duction of hormone-supplemented serum-free media for the culture of mammalian cells (13, 14), considerable attention has been directed toward defining the specific hormone and growth factor requirements of malignant as compared with nonmalignant cells. The consensus of a number of studies (described below) is that complete loss of the growth requirement for insulin is not a general characteristic of oncogenic transformation, although a qualitative reduction in the insulin requirement has been observed in some cases.

Using an experimental protocol in which cells were first plated in medium with serum and then transferred to a serum-free medium, Cherington et al. (164) com- pared the growth requirements of BHK-21 hamster cells with transformed BHK-21 cells, and the growth require- ments of a nontumorigenic clone (CHEF/18) and a tu- morigenic clone (CHEF/16) of Chinese hamster embryo fibroblasts. In this study, the most consistent correlate for transformation was loss of the requirement for EGF. Although transformation was accompanied by loss of insulin requirement in one case (polyoma virus transfor- mation of the BHK-21 cells), several other transformed cell lines retained a requirement for insulin for optimal growth. Cherington et al. (164) demonstrated further,

using the technique of flow cytometry, that the tumori- genic CHEF/16 cell line, which did not require EGF for growth, had a clear requirement for insulin and could be arrested in the G1 phase of the cell cycle when deprived of insulin. McClure (78) and Rockwell et al. (165) co- workers have performed a detailed study of the growth factor requirements of BALB/c 3T3 cells and SV40 transformed 3T3 cells under completely serum-free con- ditions. In monolayer cultures grown on a fibronectin substratum, the BALB/c 3T3 cells and SV40-trans- formed BALB/3T3 clearly differ, in that the BALB/c 3T3 cells have a strict requirement for PDGF or FGF for optimal growth, while the transformed BALB/c 3T3 cells do not. Transformation of BALB/c 3T3 cells with SV40 does not result in a loss of the growth requirement for insulin (165). Powers et al. (166) have studied hormonal requirements of Swiss 3T3 cells and virus-transformed Swiss 3T3 cells, using an experimental protocol in which cells were plated in medium containing serum, or on serum-coated dishes before growth in serum-free me- dium. These investigators observed a dramatic decrease, although not a complete elimination, of the insulin re- quirement of the virus transformed cells (166). Similarly, by using [3H]thymidine incorporation as a short term assay for growth, Van Obberghen-Schilling et al. (167) found that tumorigenic variants of the CCI39 Chinese hamster lung fibroblast cell line had a reduced require- ment for the thrombin-potentiating action of insulin. Finally, the hormonal requirements of two rat embryo fibroblast cell lines, REF52 (168) and Rat-1 (169), and virus-transformed derivatives of the two cell lines, have been studied in serum-free medium. Both the REF-52 and Rat-1 cells require high density lipoprotein (HDL) for growth in serum-free medium. Both cell lines exhibit a small but significant growth response to insulin when grown in mass culture. The most consistent change in growth requirements accompanying SV40 transforma- tion of the REF-52 cells is a loss of the requirement for EGF and vasopressin. Transformation of the Rat-1 cells with Rous sarcoma virus leads to a decreased growth response to EGF and FGF and possibly to insulin.

The overall suggestion from the above experiments, performed with fibroblastic cell lines and their tumori- genic derivatives, is that the most consistent correlate of transformation of fibroblasts is loss of PDGF, FGF, or EGF requirement, and that the insulin/IGF requirement, although in some cases reduced, is generally not com- pletely lost. A number of cell lines derived from epithelial tumors also have been cultured in hormone-supple- mented serum-free media (13, 14). Most of these cell lines require insulin for optimal growth in the serum- free media, again suggesting that complete loss of the insulin requirement is not a general feature of malignant transformation.

Genetic approaches

A number of laboratories have applied techniques of somatic cell genetics to a study of the pathway of insulin action. One such study has involved an analysis of insulin action in the mouse melanoma cell line PG19, and so- matic cell hybrids formed by crossing this cell line with mouse embryo fibroblasts (120, 170-172). The PG19 melanoma cell line does not respond to the mitogenic effects of insulin either under conditions of serum limi- tation (170) or in hormone-supplemented serum-free me- dium (171), while the melanoma X mouse embryo fibro- blast hybrids exhibit a strong growth response to insulin under both conditions. The lack of a growth response to insulin of the melanoma cells is not attributable to a lack of insulin receptors (171) or receptors for insulin-like growth factors (172). Insulin stimulates protein synthesis and inhibits protein degradation in the fibroblast X melanoma hybrids, but not in the parental melanoma cells (172). Thus, in relation to the temporal sequence of events elicited by insulin in responsive cells, the defect in the response to insulin in the melanoma cells appears to lie at a step subsequent to the binding of hormone to its receptor but before the stimulation of protein synthe- sis. Recently, the phosphorylation of ribosomal protein S6 in response to insulin has been studied in the mela- noma and hybrid cells (120). In the hybrid cells, under conditions of growth arrest in medium with low serum, ribosomal protein S6 is rapidly phosphorylated in re- sponse to insulin. The phosphorylation of the S6 protein increases over a wide range of insulin concentrations, suggesting that insulin stimulates the phosphorylation by interacting with both high- and low-affinity receptors. In contrast, in growth-arrested cultures of the melanoma cells, an intermediate level of S6 phosphorylation is observed, and insulin causes only a marginal increase in the phosphorylation of S6 (120). A possible interpreta- tion of these results is that some process or processes normally controlled by insulin are constitutive in the melanoma cells, and that these cells are resistant to further stimulation by insulin. Hybridization of the mel- anoma cells to fibroblasts apparently restores the insu- lin-sensitive processes to hormonal control.

Shimizu and coworkers have isolated a number of insulin-unresponsive variants of the BALB/c 3T3 cell line (173). One of the variant cell lines, IN-2, appears to have a deficiency of high-affinity insulin binding sites. The same investigators have isolated variants of Swiss 3T3 cells (174) and 3T3-L1 cells (175) resistant to an insulin-diphtheria toxin conjugate. One of the conjugate- resistant Swiss 3T3 variants has severely diminished insulin binding (174), while other variants obtained in both selections have moderately decreased insulin bind- ing (174, 175).

Pawelek and coworkers have performed a detailed study of the growth response to insulin of Cloudman S91 melanoma cells (176, 177). These cells have the unusual property of being inhibited rather than stimulated by insulin (176). The growth inhibition by insulin is ob- served only in medium containing serum; the same cells are stimulated by insulin in hormone-supplemented serum-free media (177). Thus, growth inhibition of the S91 cells by insulin appears to depend on the presence of a factor or factors present in serum. A number of variants of the S91 cells have been isolated by selecting for resistance to insulin in medium containing serum (176). One of these insulin-resistant variants actually requires insulin for growth in medium with serum. The insulin-dependent variant cell line has an increased num- ber of insulin receptors in comparison with the parental S91 melanoma cells, and exhibits a growth response to insulin in a dose range consistent with interaction of insulin with high affinity insulin receptors (176).

Evidence for action of insulin as a growth-promoting hormone in vivo

Numerous lines of evidence implicate insulin as an important growth-regulatory hormone during human fe- tal development. Infants born to poorly controlled dia- betic mothers have a syndrome that is characterized by hyperinsulinemia, excessive size and weight for gesta- tional age, excessive body fat, organomegaly involving heart, liver, and spleen, and hypertrophy of the umbilical cord (178). The excessive size and weight of these infants has been attributed to transplacental passage of glucose which stimulates fetal insulin production, and subse- quent action of insulin as a growth factor (179). Roth and coworkers have suggested that some features ob- served in this syndrome, such as elevated deposition of fat, glycogen, and protein may be due to interaction of insulin with insulin receptors, while the excessive length and organ size may be due to interaction of high concen- trations of insulin with IGF receptors (179). An addi- tional possible mode of insulin action, discussed below, is that excess insulin may act indirectly by stimulating somatomedin production and potentiating somatomedin action. In contrast to the syndrome observed in the infant of a diabetic mother, neonatal diabetes is charac- terized by retarded fetal growth, poor deposition of adi- pose tissue, and small muscle mass (180). An extreme version of this is seen in the rare syndromes of pancreatic agenesis (180) and congenital absence of the islets of Langerhans (181), which are characterized by extreme growth retardation, absence of adipose tissue, and failure of muscle mass. Another rare congenital syndrome in which growth retardation is correlated with a deficiency in insulin action is leprechaunism. In this disorder, the

problem is not a deficiency in insulin production, but rather extreme resistance to insulin action at the tissue level. This syndrome is characterized by low birth weight, absence of subcutaneous fat, decreased muscle mass, characteristic facial features, hyperinsulinemia, insulin resistance, and early death (182-190). Four infants with leprechaunism, two males and two females, have been described in detail recently (182-190). In two of the patients, there is a clear decrease in insulin binding to cultured fibroblasts (182-185). With one patient, Scat- chard plots of insulin binding to cultured fibroblasts and B-lymphocytes transformed with Epstein-Barr virus both revealed a decrease in insulin binding, although the results differed in that the experiments performed with the fibroblasts suggested a lowered receptor affinity, while the experiments with the transformed lymphocytes suggested a decrease in receptor numbers (183-185). A third patient with leprechaunism has been reported to have normal numbers of insulin receptors (186), but these receptors have several qualitative abnormalities including an abnormal sensitivity to temperature and pH (187, 188). In a fourth patient, no abnormality in insulin binding has been detected (189), but fibroblasts cultured from the patient exhibit a resistance to insulin, SmC, EGF, and serum (190). Thus, the mechanism of insulin resistance in leprechaunism may be heteroge- neous. Another rare syndrome in which a growth anom- aly is found in combination with insulin resistance is lipoatrophic diabetes, which is characterized by either complete or partial lack of adipose tissue, abnormalities of carbohydrate and lipid metabolism, and severe resist- ance to endogenous and exogenous insulin. A recent detailed study of eleven patients with lipoatrophic dia- betes suggests that the mechanism of insulin resistance in this disorder is heterogenous and may involve both receptor and postreceptor anomalies (191).

Interactions between insulin and the growth hormone/ IGF system

Although some effects of insulin on growth in vivo are very likely caused by direct action of insulin, there is a growing body of evidence suggesting that insulin may also enhance growth indirectly by increasing hepatic production of somatomedins, and possibly by potentiat- ing the action of somatomedins in target tissues. By using the technique of liver perfusion, insulin has been found to stimulate somatomedin release by rat liver (192- 194). Hepatic production of IGF and its carrier protein is decreased in streptozotocin-induced diabetic rats, and this decrease is partially reversed by insulin (195). Serum somatomedin levels are also low in human type I dia- betics who are in poor metabolic control (196). Injection of fetal rabbits with insulin causes an increase in bioac-

tive somatomedin as well as an increase in sulfate and thymidine incorporation in costal cartilage (197). More recently, high concentrations of insulin have been re- ported to increase somatomedin A production in mono- layer cultures of rat hepatocytes (198). A possible mech- anism by which insulin might increase somatomedin output by the liver has been suggested by Baxter et al. (199), who have reported that insulin regulates the num- ber of somatogenic receptors in the rat liver. These authors proposed that the low somatomedin levels and growth retardation observed in insulin-deficient diabetes may be attributable to a deficiency in this action of insulin (199). In a more recent study, Maes et al. (200) also found a normalization of somatomedin C (SmC) levels in streptozotocin-induced diabetic rats after treat- ment with insulin. However, these investigators found only a partial correlation between SmC levels and GH receptor number in liver, and concluded that the decrease in SmC production in diabetes is primarily attributable to a postreceptor problem in GH action, and that the stimulation by insulin of SmC production may be due to a general improvement in the function of intracellular mechanisms involved in SmC synthesis (200).

In addition to modulating the number of GH-binding sites in liver, insulin has also been found to increase the binding of IGF-II to rat adipocytes in vitro (197, 198) and to cultured rat hepatoma cells (11). Scatchard analy- sis of the binding of IGF-II to intact adipocytes suggested that this increase in binding was due to an increase in receptor affinity with no change in the total number of binding sites (201, 202). However, Scatchard analysis of IGF-II binding to isolated plasma membranes suggested that insulin caused an increase in numbers of binding sites (201). To further investigate this phenomenon, Oka et al. (203) recently have examined directly the effect of insulin on numbers of cell surface receptors for IGF-II using antibodies prepared against the IGF-II receptor. The results of these experiments indicate that insulin increases the number of IGF-II receptors displayed on the cell surface (203). The regulation by insulin of IGF- II receptors raises the interesting possibility that, in addition to controlling the output of somatomedins by liver, insulin might also potentiate the action of IGF-II in some somatic target cells, although a direct demon- stration of this has not yet been reported.

Conclusions

Considerable progress has been made recently in iden- tifying cell surface receptors that mediate the effects of insulin on cellular growth and proliferation. In some cells, insulin appears to stimulate growth by binding to high-affinity insulin receptors, while in other cells, the growth-promoting effects of insulin appear to be me-

diated primarily by the low affinity interaction of insulin with IGF-I receptors. The major unsolved problem relat- ing to insulin action remains the elucidation of intracel- lular mechanisms that control the effects of insulin on metabolism and on growth. There are a number of strik- ing similarities between the action of insulin and the insulin-like growth factors, and that of other growth factors such as EGF and PDGF. Each of these hormones has a specific receptor associated with a tyrosine-specific protein kinase activity, and treatment of cells with each of the hormones yields a rapid stimulation of the phos- phorylation of at least one common protein: ribosomal protein S6. However, the synergistic effects of insulin and insulin-like growth factors in combination with PDGF, as well as the possible differences in their tem- poral sequence of action, suggests that there must be some differences in the pathway of action of the different hormones. It is possible that this difference may be explained ultimately by a difference in substrate speci- ficity of the different receptor kinases. Alternatively, it is possible that the different hormones act via branched pathways, with some branch or branches being shared by all of the hormones. One common branch in the pathways of action of insulin and the other growth factors appears to involve activation of a protein kinase that phosphorylates serine residues on ribosomal protein S6. Finally, the extent to which the growth-promoting effects of insulin in vivo are caused by direct action of the hormone, or by a regulatory effect on somatomedin release and somatomedin action, remains to be deter- mined.

Acknowledgments

I thank Drs. M. P. Czech, S. Jacobs, W. H. Daughaday and J. R. Gavin III for helpful comments on this manuscript.

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