Insulin and insulin-like growth factor I in early development: peptides, receptors and biological events.

0163-769X/90/1104-0558$02.00/0 Endocrine Reviews Copyright © 1990 by The Endocrine Society

Vol. 11, No. 4 Printed in U.S.A.

Insulin and Insulin-Like Growth Factor I in Early Development: Peptides, Receptors and Biological Events * FLORA DE PABLO, LAURIE A. SCOTT, AND JESSE ROTH Section on Receptors and Hormone Action, Diabetes Branch, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

T

HE goal of this paper is to sketch the present understanding of the role that insulin and insulin receptors, as well as insulin-like growth factor I (IGF-I) and its receptors, seem to play in early embryogenesis. This approach overturns ideas of previous decades which have suggested that hormones function relatively late in embryogenesis, i.e. only after the individual glands .„velop (1, 2); that insulin acts largely as a regulator of metabolic events in a narrow range of metabolically important target tissues {e.g. muscle, fat, and liver); and that IGF-I is mainly a postnatal growth stimulator (3). In contrast, it is now broadly accepted that 1) insulin is expressed before there is an embryonic pancreas, 2) IGFI is expressed before liver develops, and 3) both insulin and IGF-I influence metabolic, growth, and differentiation processes in a very wide range of cell types (4). We shall begin with an overview of the receptors, essential components of the hormone action cascade. Over the last decade it has been recognized that the receptors for insulin and IGF-I have similar biochemical activities, (5, 6) both in the basal and stimulated states, and both are very widely distributed throughout tissues at all ages and stages (7). The developmental models pertinent to studies in this area will be introduced next. The relevant data are fragmentary in all the models; we shall present the chick embryo first because it is the most studied (8), followed by an amphibian, the South African frog Xenopus laevis, and, an insect, the fruit fly Drosophila melanogaster. The studies in mammals, still small in number, will be discussed at the end of each section. The following aspects will then be discussed: 1) expression of receptors for insulin and IGF-I; 2) actions of Address requests for reprints to: F. de Pablo, M.D., Ph.D., Building 10, Room 8-S-243 National Institutes of Health, Bethesda, Maryland 20892. * Studies reviewed here were in part funded by the US-Spain Joint Committee for Scientific and Technological Cooperation.

insulin and IGF-I in early embryos; 3) probable sources of insulin and IGF-I in early ontogeny; 4) neutralization of biological effects by antibodies against insulin, IGF-I, and the receptors; and 5) conclusions.

I. Introduction Overview of receptors and hormone action It is now clear that hormones function to activate their receptors and that the activated receptor has the program that we ascribe to hormone action (5-7, 9, 10). In the case of insulin and IGF-I, each binds to its own receptor to activate an endogenous tyrosine-specific protein kinase (11-13). Autophosphorylation of the receptor, an early event, enhances further the kinase activity (14); this results in enhanced phosphorylation of cellular proteins which are presumed to be essential physiological intermediates in many or all of the actions of these hormones (15-18). The specificity of the two receptor kinases for artificial substrates is quite similar. Likewise, the endogenous substrates that can be phosphorylated by each of the receptors are similar (19, 20). It is a matter of intense study as to whether and how the insulin receptor and the IGF-I receptor differ in their intrinsic ability to mediate metabolic and growth responses (6, 21). There are still many missing links in understanding the chain of events by which insulin or IGF-I binding to their receptors leads to biologically relevant events in the target cells. It is still not at all clear how tyrosine phosphorylations lead to the production of soluble intracellular glycoinositides that have been suggested to mediate some of insulin actions (22-24), or to the serine and threonine phosphorylations and dephosphorylations linked to other events in insulin action (25-27). In addition to binding to its own receptor with high affinity, each of the ligands binds to the receptor of its homolog (Fig. 1) with a reduced affinity but full intrinsic activity

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INSULIN AND IGF-I IN EARLY DEVELOPMENT

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(6, 7, 28). The resultant cellular or biochemical response is that characteristic of the receptor, irrespective of which ligand is activating it. That both ligands can activate both receptors generates uncertainties in interpreting experimental data in vitro and especially in vivo. In our discussions we oversimplify the situation by speaking about cell surface receptors for insulin and for IGF-I. It is now clear that these two receptors, like many other cell surface receptors, continuously recycle between the surface and membranous sites within the cell (35). Further, binding of the hormone to the cell surface receptors leads to an acceleration of its internalization (36). The internalized receptor and especially the internalized hormone appear to be more susceptible to degradation; cells vary widely in how much of the internalized ligand and receptor return intact to the cell surface (37). While the internalization mechanisms appear to be important for regulation of degradation of both ligand and receptor, other possible roles for internalization in hormone action or translocation of hormonal receptors are as yet unclear. There are also provocative findings which suggest that insulin and IGF-I may have direct access to intracellular binding sites, and exist unbound in the nucleus (38-40).

Overview of developmental models

The developmental studies reviewed in this article have taken advantage of several well characterized ani-

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mal models (Table 1). In addition to mammals, we include nonmammalian vertebrates and invertebrates that are more accessible in early embryogenesis. In perusing this table, it is clear that the pancreas has an early appearance in organogenesis in a variety of vertebrates. In the present review we do not include studies done in late fetal stages and most cell lines since they have been comprehensively reviewed by others (50-55); rather, we focus on early development and the small but growing body of data available on oocytes and eggs.

II. Expression of Receptors for Insulin and IGF-I a. Chicken The chick embryo has undergone multiple cleavage divisions at the time the egg is laid. Gastrulation takes place post laying during day 1 of incubation and neurulation during day 2 (56). Overall the data at early stages are sparse, but it appears that both insulin receptors and IGF-I receptors are expressed at least as early as gastrulation, and that IGF-I receptors are dominant. At 28 h, before the first somite is formed in the chick embryo, specific binding to both receptors is widespread in the blastoderm (57) (Fig. 2). In addition to providing sensitivity, autoradiographic analysis enhanced by computerassisted densitometry showed localization of the receptors. During neurulation in the chick embryo, insulin receptors and especially IGF-I receptors are widely distributed throughout the nervous system. (The presence

Hormones:

Insulin IGF-I IGF-II

Metabolism Growth Differentiation

FIG. 1. Schematic representation of insulin and IGFs, their receptors, and biological actions. The insulin receptor and the IGF-I receptor have similar heterotetrameric structures. The insulin receptor binds insulin with high affinity and IGF-I and II with lower affinities while the IGF-I receptor binds IGF-I with high affinity and IGF-II and insulin with lower affinity. Each of these two receptors, activated by any of the three peptides, mediates a broad range of intracellular effects specific for that receptor in that particular cell type, and typically including stimulation (or inhibition) of metabolic events, growth and differentiation, as well as cell-specific processes (27). The function of the third receptor, now known as IGF-II/mannose-6-phosphate receptor, monomeric in structure, is much less well understood. Besides its ability to bind IGF-II and IGFI (but not insulin), it also binds, at another site, proteins that have mannose-6-phosphate (M6P) residues. Among these proteins are lysosomal enzymes that are secreted and then internalized via these receptors and thereby targeted to the lysosomes. The IGF-II/M6P receptor is more abundant intracellularly than in the cell-membrane. This will be our only discussion of IGF-II and the IGF-II/M6P receptor; we recognize their possible important role in early development, and refer you to recent articles (29-34).


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TABLE 1. Developmental models and some important events Human

Rat/mouse

Chicken

Xenopus

Sea urchin

294

22/20 Birth

21

3-4

3-4

1

Hatching

Swimming tadpole

Swimming tadpole

Hatching

3-8

2-3

b

3; 4

1-3 2;?

2 1

20 h 12 h

Duration (days)" Developmental landmark

Birth

Organogenesis postneurula Pancreas; j8-Cellsc Neurulation Gastrulation

28-70 40; 49 20-27 8-20

10-16 11; 12d 9-10 6-8

Drosophila

e

e

10 h 36 h

5h

° The time given for all events is approximate, since variations depending on season, temperature, and subspecies are common. The unit is days except when marked h (hours). [Adapted from Refs 40-48]. 6 The embryo (larva) undergoes a series of molts, and the imaginal discs of the larva give rise to adult organs. c The criteria for recognition of /3-cells are based on RIA of extracts, immunohistochemistry, and electronmicroscopy. Molecular biological approaches to detect insulin mRNA, while they are becoming standard, are not considered. d These are days of appearance of the pancreas and the /3-cells, respectively, in the rats; in mouse these occur about 1.5 days earlier. e Invertebrates do not develop a separate pancreas, but "pancreatic" hormones are found in the gut. (Adapted from Refs. 41-49).

Non-Specific Binding

18 h, 1 2 5 IIGFI

28 h, 125llnsulin

28 h, 125I-IGFI

52 h, 125llnsulin

52 h. 125IIGF-I

FIG. 2. Autoradiographic localization of receptors in early chick embryos. Whole-mounted embryos at 18 h, 28 h, and 52 h of development were incubated in situ with either [125I]insulin or [125I]IGF-I. In parallel experiments, an excess of unlabeled peptide was added to similar embryos together with the labeled ligand to determine nonspecific binding. Autoradiograms were obtained and the images were converted to color-coded (scale shown) computerized presentations. Red indicates highest binding, green and yellow intermediate and blue lowest. [Adapted with permission from M. Girbau et ai: Proc Natl Acad Sci USA 86:5868,1989 (57).]


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of IGF binding proteins was not studied directly but interference with receptor binding was excluded by competition studies). The binding of both labeled insulin and labeled IGF-I to receptors is high in similar regions of the embryo, namely the neural folds, neural tube, and optic vesicles. These structures are derived from the ectoderm. Mesoderm-derived tissues, like the heart, were relatively less rich in receptors for insulin and IGF-I. By day 2, when neurulation is complete and membrane preparations can be used in typical binding-competition assays, the specificity of insulin receptors and IGF-I receptors was confirmed (58, 59). Further enrichment for receptors by adsorption and elution from a wheat germ agglutinin column was necessary to study their tyrosine kinase activities. Wheat germ agglutinin eluates of extracts from whole day 2 embryos contained insulin and IGF-I stimulatable protein-tyrosine kinase activity. The /3-subunits of both receptors as well as an exogenous substrate, poly (Glu-Tyr), were phosphorylated in the presence of insulin and IGF-I (57). These kinase activities increased many fold between day 2 and day 4 in whole embryos, while binding of labeled insulin and labeled IGF-I increased only slightly; the biochemical basis for these differences in the coupling of the insulinbinding a-subunit and the kinase-containing /3-subunit of the receptor is unclear. The a-subunit of the receptors of the youngest embryos studied by affinity labeling, (i.e. day 2) had an electrophoretic mobility similar to brain receptors of older embryos (Fig. 3). Like the adult brain receptor, desialylation by treatment with neuraminidase changed neither the mobility of receptors from whole day 2 embryos or from developing brain; the enzyme treatment increased the mobility of the a subunit of embryonic liver and heart (59) which is similar to results with adult forms of these receptors. These data suggest that the glycosylation pattern of the receptors predominant in the earliest stages of chicken development is similar to the "neural" type. Also, the pattern found in an adult tissue is very similar to that found in that type of tissue in the early embryo. The physiological significance of the differences in posttranslational modifications of the receptors in the different tissues is not known. Initial studies of chick embryos during organogenesis postneurula (day 3 to day 7) identified insulin receptors in whole cells (60). We isolated organs for analysis of receptors using membrane preparations for competition binding assays with radiolabeled insulin and IGF-I. Both types of receptors were identified in all embryonic tissues studied, including brain, heart, muscle, liver, limb buds, and the lens of the eye (61, 62). Low levels of insulin and IGF-I receptors were also detected in extraembryonic membranes in early and midembryogenesis (63). In each organ, we found a distinctive pattern of developmental

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[125l]lnsulin Mrx10"3

Neuraminidase Whole Embryo Day 2

Brain Day 6

Chick Embryo Liver

Rat Liver

FIG. 3. Affinity-labeling of insulin receptors of chick embryo tissues and rat liver. [125I]insulin was cross-linked to the a-subunit of the receptors on membranes from whole chicken embryos or head, brain, and liver and from liver of rat; later some samples were also incubated with neuraminidase (+), while others were not (—). The samples were then solubilized in Triton, immunoprecipitated with antiinsulin antibody, solubilized in sodium dodecyl sulfate and electrophoresed. The position of the receptor a-subunit (arrow) and the mol wt markers are

indicated. [Adapted with permission from L. Bassas et al:. Endocrinology 121:1468, 1987 (57).®The Endocrine Society]

changes in the level of binding and the relationship of IGF-I to insulin binding. IGF-I binding is more prominent than that of insulin early in embryogenesis. The liver is an exception, however, with insulin binding dominating throughout development. The early and widespread expression of the two receptors in avian embryos as well as the complex tissuespecific patterns of change through embryogenesis suggest an important functional role of the receptors for normal embryonic development. Studies with antibodies against the receptors, as well as antibodies against insulin (described later), support this suggestion. b. Amphibians The use of amphibians is helpful to study development of receptors because eggs and embryos are available at each stage from early oogenesis through fertilization, up to the free swimming tadpole. Among amphibians, Xenopus laevis, the South African clawed toad or frog, is the current subject of intense studies related to growth factors in many laboratories (64). As yet the potential of


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(mRNA) transcripts corresponding to the tyrosine kinase domain of the IGF-I receptor and the insulin receptor homologs of Xenopus, in stage VI oocytes and unfertilized eggs. Ligand-binding studies with membrane preparations as well as intact oocytes suggest that full grown oocytes express many more IGF receptors than insulin receptors, and that unfertilized eggs express almost only IGF-I receptors at the protein level. We did find both insulin and IGF-I receptor-related mRNA transcripts in embryos, from blastula stage to the free swimming tadpole. At all of these stages, studies with [125I]IGF-I and [125I] insulin in binding-competition assays revealed clearly the presence of IGF-I receptors, while the presence of typical insulin receptors, measured by binding of labeled ligand, was equivocal until the tadpole stage. The transcriptional and posttranscriptional regulation of the two types of receptors in Xenopus appears to be highly regulated in development.

this amphibian to study the insulin-related response systems has not been fully exploited, e.g. studies with young embryos concerning insulin receptors and action are virtually absent. Several reports described receptors for insulin in full grown (stage VI) Xenopus oocytes. There, insulin and IGF-I are capable of stimulating a cascade of biological events associated with the reinitiation of meiosis, a process presumably mediated through receptors (65-68). It is still not totally clear which effects of insulin in this system are exerted through its receptor and which are mediated by IGF-I receptors. Mailer and Koontz (67) studied [125I]insulin binding to stage VI intact Xenopus oocytes and found numerous (40 million/cell) high affinity binding sites with an affinity typical of other insulin receptors [dissociation constant (Kd) = 1.4 nM]. The total specific binding was low, and the specificity of the receptors for insulin analogs was not studied. There were other low affinity binding sites, but they were not characterized, nor was [125I]IGFI binding measured. An antihuman insulin receptor antiserum blocked approximately 50% of the [125I]insulin binding, while it did not inhibit the effect of insulin on oocyte maturation. The authors of this pathfinding study concluded that this biological effect of insulin was probably mediated through a putative IGF receptor (see later section). The final interpretation, though, must await further studies to define the antibody's affinity for amphibian forms of receptors, the magnitude of its preference for insulin receptors vs. IGF-I receptors, and its insulinomimetic vs. insulin-blocking properties. In another study with stage VI oocytes, microinjection of monoclonal antibodies against the cytoplasmic portion of the insulin receptor blocked the effect of added insulin in reinitiation of meiosis (69). This elegant study is not definitive in showing that insulin is acting through the insulin receptor because these antibodies also recognize the cytoplasmic domain of the IGF-I receptor. Both tyrosine and serine protein kinase activities stimulated by insulin have been demonstrated in solubilized plasma membranes of stage VI Xenopus oocytes. The half-maximal stimulation of both activities was observed at approximately 100 nM insulin (70). More recently, Janicot and Lane (71) reported preliminary data on IGFI and insulin stimulation of receptor autophosphorylation as well as phosphorylation of tyrosines of an exogenous substrate. The concentration of peptide that yielded 50% of the maximal response for both processes was 3 nM for IGF-I and 200-300 nM for insulin, from which they concluded that IGF-I receptors mediated these events.

The structure and binding characteristics of the insulin receptor are highly conserved throughout the vertebrates (the IGF-I receptor has been studied much less extensively from an evolutionary point of view). Drosophila has been chosen as a valuable nonvertebrate in which to study the insulin receptor due to its well defined genetics and its availability during embryogenesis. Molecular cloning analysis together with the use of sitespecific antibodies have shown that the structural features and intrinsic functions of the Drosophila insulin receptor homolog have been highly conserved despite the evolutionary divergence of vertebrates and insects more than 600 million years ago (72). The Drosophila insulin receptor is similar to the human insulin receptor in deduced amino acid sequence, organization of the subunits, and ligand-stimulated protein kinase activity (73-77). Like the human insulin receptor, the mature Drosophila receptor is derived by processing from a proreceptor (Mr ~205,000) into a cellsurface glycoprotein composed of a-subunit with insulin binding domain (Mr ~115,000) and transmembrane /3subunit (Mr ~95,000) with an intrinsic insulin-stimulatable tyrosine kinase activity (77). In addition, a 170 kilodalton receptor component has been described, which may represent an incompletely processed proreceptor molecule. The receptor precursor has affinity for insulin binding that is markedly diminished, but it is phosphorylated on tyrosine residues in response to high doses of insulin; these observations are similar to those on the precursor of the human receptor.

In our own studies (Scavo L., A. R. Shuldiner, J. Serrano, R. Dashner, J. Roth, and F. De Pablo, manuscript in preparation) we have detected messenger RNA

The insulin receptor of Drosophila binds mammalian forms of [125I]insulin and unlabeled insulin with high affinity; overall it is very similar to the mammalian

c. Drosophila


November, 1990


receptors, except that the Drosphila receptor has a very much lower affinity for IGF-I and IGF-II (of mammalian origins) than do the insulin receptors of mammals. It is not yet known whether Drosophila has a separate receptor for IGF-I (77). It has recently been shown that a classical developmental hormone of insects, i.e., prothoracicotropic hormone from the silkworm, Bombyx mori, has a structure that is remarkably like that of insulin (78). This hormone appears not to interact with the insulin receptor of Drosophila, measured as either stimulation of tyrosine kinase activity or as competition with labeled insulin. Presumably it acts through its own receptor. However, when this material was interacted with the human insulin receptor, competition was observed, again suggesting that the mammalian receptor may have a somewhat broader specificity for insulin-related peptides than does the Drosophila receptor (77). The studies that led to the cloning of the Drosophila insulin receptor were done with embryos, and the stageand tissue-specific expression was highly suggestive of a role in embryo development. Insulin-stimulated tyrosine kinase activity, first detected in membranes of 6-12 h embryos, was high at 12-18 h of embryogenesis but was hardly detected in the adult flies (73). More recently, the developmental regulation and tissue distribution of the insulin receptor mRNA have been analyzed in greater detail. Two species of mRNA transcripts were detected, one in unfertilized eggs and 2-3 h embryos and another that reached an apparent peak at 8-12 h, which corresponds to the time of major growth of the embryonic nervous system (Fig. 4) (76). By in situ hybridization, the insulin receptor transcripts were ubiquitously distributed in the egg and early embryo. Interestingly, after 12 h, the highest levels were localized in the developing nervous system (Fig. 5) (79) which further suggests a possible role for the insulin response pathways in neural growth. In larvae the insulin receptor mRNA was most prominent in the imaginal disks, (which contain actively dividing cells) and, again, the nervous system. Cells of the larvae, which grow in size but do not divide, did not express insulin receptor mRNA. The authors also expressed surprise at finding very high levels in the adult ovaries. Hybridization appeared prominent in nurse cells which produce mRNAs subsequently stored in oocytes, as well as in mature oocytes, while it was absent in follicle cells. This pattern indicates that the insulin receptor mRNA is maternally transmitted, which is consistent with a role for this receptor in very early developmental events. d. Mammals Because of technical obstacles in obtaining tissue, studies of receptors for insulin and IGF-I in mammals

1 2 3 4 5 6 7 8 9

563

10 111213

11 k b -

FIG. 4. Developmental changes in insulin receptor mRNA from Drosophila. RNA blot of adult Drosophila and embryos at different stages hybridized to a probe that contains a region encoding a portion of the kinase domain. A large (~11 kb) mRNA is the most prominent hybridizing band; a smaller mRNA is apparent in lanes 2 and 3. Lane 1 is 20 ng poly(A) RNA from unfertilized egg; all other lanes are 5 ng poly(A)+ RNA. Lane 2, Unfertilized eggs; lane 3, embryos 0-4 h; lane 4, embryos 4-8 h; lane 5, embryos 8-12 h; lane 6, embryos 12-16 h; lane 7, embryos 16-20 h; lane 8, first instar; lane 9, second instar; lane 10, third instar; lane 11, pupae; lane 12, male adult; lane 13, female adult. [Reproduced with permission from L. Petruzzelli et al.: Proc Natl Acad Sci USA 83:4710,1986 (76).]

are sparse before implantation and soon after implantation. From very limited data, we have tentatively sketched the following conclusions. Granulosa cells in the ovary have receptors that specifically bind [125I] insulin and [125I] IGF-I, but the presence of these receptors on the oocyte has not yet been demonstrated (80). Likewise, in the mouse the receptors have not yet been demonstrated in two-cell embryos but are demonstrable by eight-cell and morula stages, with a clear increase in the blastocyst (81, 82) (Table 2). Electron immunocytochemical studies have confirmed the binding of goldlabeled insulin and its internalization by the cells of blastocyst stage embryos (83). By the use of the highly sensitive polymerase chain reaction, mRNA transcripts for insulin receptors and IGF-I receptors were detected at the eight-cell stage as well as in blastula (84) and postimplantation embryos (day 7.5) (85); the receptor kinase and other structural features of the receptor protein have not yet been characterized, due to the small amounts of tissue available. Studies in the rat are even more scant: insulin recep-



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a \ 1 FIG. 5. Autoradiographic localization of insulin receptor mRNA in 18 h Drosophila embryo. Bright field (a) and dark field (b) micrographs hybridized to an antisense RNA probe. The horizontal section shows hybridization in the cellular cortex in the brain {arrowheads). The neuropil (N) hybridized poorly. Insulin receptor transcripts were expressed in most cells of the embryo. [Reproduced with permission from R. S. Garofalo et al: Mol Cell Biol 8:1638, 1988 (79).]

TABLE 2. Insulin, IGF-I, and receptors in oocytes and preimplantation mouse embryos Oocyte 2-Cell 8-Cell Morula Blastocyst Receptors mRNA Insulin IGF-I Insulin binding IGF-I binding Ligands mRNA Insulin IGF-I

+ + ND

+ + + ND

+ + + +

0 0

0 0

0 0

0 0 0

0 0 0

o/+

ND

ND

0 0

0 0

ND, Not determined. [Adapted from Refs. 82 and 84.]

tors were found in membrane preparations of the neurula-stage embryo (days 10.4 and 11.6) by direct binding of labeled ligands as well as by photoaffinity cross-linking (Fig. 6) (86). Competition studies identified these as insulin receptors. The specific binding of labeled insulin was greater in yolk sac, an extraembryonic structure, than in the embryo itself. Human antiinsulin receptor antiserum inhibited insulin binding approximately 75% in embryos and 92% in extraembryonic membranes. Rat embryos before neurulation have not been examined, and studies of IGF-I receptors have not been reported. One study in pigs showed that specific binding of [125I] IGF-I (there referred to as somatomedin-C) was higher than [125I] insulin binding during late organogenesis in most tissues (87). In humans, binding studies have revealed receptors for

both insulin and IGF in membranes from brain, liver, kidney, lung, and adrenals of fetuses 10-17 weeks of gestation (88). The IGF receptor of brain had a specificity intermediate between the specificities typical of IGF-I and IGF-II receptors, but further studies with pure peptides are needed to corroborate this finding. Summary of receptors In early development, in general, based on all species studied, insulin receptors and IGF-I receptors are both very widely expressed, consistent with the observations in adults where these two receptors are very widely expressed in many tissues througout all species studied. That they are coordinately expressed under some conditions but differentially expressed under a wide range of conditions is again consistent with the observations in adults where the two receptor types may be coexpressed on the same cells, or differentially expressed. The very wide shifts in concentrations of both of these receptors during embryogenesis is but an exaggeration of the observations in adults where these receptors undergo fluctuations in concentrations of a substantial degree, often of physiological and pathological importance. In summary, while the role of the IGF-I and insulin receptors in early embryogenesis is just beginning to be understood, it is clear that these receptors undergo a whole panoply of systematic changes in many target cells


November, 1990

A


B C D E F G

565

scant, they can elucidate physiology more directly and more convincingly. a. Chicken

205-

116-

9766-

45-

FIG. 6. Photoaffinity cross-linking of receptors from day 11.6 rat embryo and yolk sac. Embryo homogenates (A and B), yolk sac homogenates (C and D), purified liver membranes (E and F), and liver homogenate (G) were incubated with [125I]insulin in the presence (+) or absence (—) of an excess of unlabeled insulin. The autoradiogram of a 1% polyacrylamide gel is shown. The mol wts of protein markers are indicated. [Reproduced with permission from T. Unterman et al.: Diabetes 35:1193, 1986 (86).]

including heavy representation in the developing nervous system, which suggests that they are playing an important role.

III. Actions of Insulin and IGF-I in Early Embryogenesis We shall discuss studies in each of the major developmental models examined, with occasional reference to lines of embryonic origin. Demonstrations of specific effects of added hormones provide pharmacological evidence for the presence of functional receptors and postreceptor pathways. These studies, like those that show the presence of the hormone-like agents, leave unanswered the questions of the physiological role of these hormones and their receptors. In a later section, we review studies with neutralizing antibodies; while still

In one study, low concentrations of insulin (0.1-1 ng/ ml) increased by 50% glucose consumption in the explanted chick embryo gastrula (day 1) (89). In our studies, the effects of insulin reported in the chick embryo in vivo at days 2 to 4 indicated two types of actions of insulin, stimulatory of growth at low doses and teratogenic at high doses. Insulin, at 10-100 ng/embryo, in a dose-dependent manner increased the whole embryo's content of protein, creatine kinase activity, creatine kinase MB-isozyme, triglycerides, cholesterol, phospholipids, RNA, and DNA (90). Human IGF-I was less potent in stimulating most of the parameters except the creatine kinase MB, the marker of muscle differentiation. Based on the potencies of insulin analogs (proinsulin and desoctapeptide insulin) the authors inferred that at low doses, insulin stimulates developmental processes mainly through the insulin receptor, with the possible exception of muscle cell differentiation. When insulin and IGF-I were applied together to embryos in early organogenesis, the effects were not additive; while it is possible that both were acting largely or exclusively through only one of the insulin-related receptors, we believe that each operates through its own receptor but that postbinding pathways for insulin and IGF-I converge to a common limiting step during embryogenesis (91). High doses of insulin and proinsulin (micrograms per embryo) cause death or abnormal growth, accompanied in survivors by a decrease in protein, RNA, and DNA content, measured at day 4 (92). These teratogenic effects were not mitigated by the simultaneous injection of glucose. In this study, the potency of high doses of proinsulin approached that of high doses of insulin, in contrast to the usual situation where proinsulin has only 1-10% the potency of insulin. Two possible explanations for this finding are 1) differences in peptide degradation rates, and 2) that at high concentrations, insulin and proinsulin interfered with embryonic development via an IGF receptor. These studies confirmed and extended the classic work of Landauer and Bliss (93) on the teratogenicity of insulin in chick embryos. In contrast to its teratogenic effects when used alone at high doses, insulin at intemediate doses has been found to "rescue" embryos from the teratogenic effects of phorbol ester (94). In addition to growth and metabolic effects, a number of studies have demonstrated in culture systems that insulin can stimulate growth and differentiation of several cell types and influence gene expression. Chicken



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0.10 r

2

o

Q. O O

Insulin/

c O 0.05

o (0

Hours

2

3 4 days in culture

5

10

FIG. 7. Effect of insulin on maturation of chick embryo neurons in culture. Retinas from day 7 chicken embryos were used to establish neuronal cultures in serum-free medium supplemented with 1(A), 10(0), 50(»), 100 (A), and 500 ng (•) of insulin per ml. Choline acetyltransferase activity was determined for each day of the culture, defined as picomoles of [12C]acetylcholine (ACh) formed per min/mg of protein. [Reproduced with permission from Kyriakis et al.: Proc Natl Acad Sci USA 84:7463, 1987 (103).]

embryonic muscle was one of the very first tissues in which insulin-induced differentiation was described (95). While in these studies the concentration of insulin used was high, more recent reports showed that insulin and IGF-I in nanomolar concentrations are capable of inducing myoblast differentiation (96-98). Increased amino acid uptake has been reported in chick embryo heart cells treated with high insulin concentrations (99). Curiously, insulin at nanomolar concentrations slowed the rate of spontaneous beats of chick embryo heart aggregates (100). At intermediate concentrations (100 ng/ml) insulin caused an increase in ornithine decarboxylase activity in day 11 chick embryo pelvic cartilage explants (101). Several effects in nervous tissue have been also reported. [3H]uridine incorporation was shown to increase in sensory ganglia of day 8 chick embryo after culture for 4-6 h with high levels of insulin (102). Insulin at very low concentrations (1 ng/ml) stimulated acetylcholine transferase activity in retinal neurons cultured from day 7 chick embryos (103) (Fig. 7); the authors suggest that insulin may play a role in mediating cholinergic differentiation of the embryonic retina. In a neuronal culture from chick brain that had been grown in a defined serumfree environment, insulin enhances the growth of neurons during the early proliferative stage. Nerve growth

FIG. 8. Effect of microinjected insulin on RNA synthesis in stage IV Xenopus oocytes. Cells were injected with 0.01% BSA, 3H-labeled GTP, and 0 (control) or approximately 18 fmol insulin. At various timepoints during a 3-h period, GTP incorporation was determined in groups of 4 to 12 oocytes. (*, P < 0.05, **, P < 0.01). [Reproduced with permission from D. S. Miller: Science 240:506, 1988 (116).]

factor, T3, and progesterone were inactive; IGFs were not tested (104). In similar neuronal cultures, a 2-fold increase in [3H]leucine incorporation was caused by picomolar concentrations of insulin (105). In an unusual system, the otic vesicle of day 3 chick embryos in culture, high insulin concentrations potentiated the effect of bombesin increasing cell proliferation and morphological differentiation (106). The chick embryo lens (107) has been a particularly useful system with which to analyze effects of insulin and IGF-I on cell differentiation and gene expression in early development. It had been learned some years ago that moderately high concentrations of insulin (0.1-1 ng/ ml), as well as chick embryo vitreous humor, promote the elongation of lens epithelial cells and their emergence as fully differentiated fiber cells, specialized in the synthesis of 5-crystallin (108). By using primary cultures of epithelial and fiber cells from day 6 chick embryo lens, Alemany et al. demonstrated that: 1) nanomolar concentrations of either insulin or IGF-I stimulate the accumulation of 5-crystallin mRNA; 2) this effect appears to be mediated by each hormone acting through its own receptor, i.e. insulin receptor antibodies block the effect of low concentrations of insulin; 3) both hormones increase transcription of the 5-crystallin gene and, in transient transfections, stimulate transcription of a reporter gene driven by the 5-crystallin promoter. The transcriptional component of the IGF-I effect can account for the observed stimulatory effect on 5-crystallin mRNA accumulation, while the transcriptional component of insulin's effect is too small to account for the increase in mRNA levels caused by insulin (109, 110). The embry-


November, 1990


onic lens is an ideal system to study the paracrine/ autocrine role of IGF-I on differentiation of an epithelial tissue, including possible effects of IGF-I on transcription factors (110). b. Amphibians As noted earlier, Xenopus laevis is used widely in studies of oocyte maturation and regulation of early development. Oocytes grow during several months of oogenesis (111). The full grown stage VI oocyte is arrested in prophase of the first meiotic division. Maturation involves breakdown of the germinal vesicle, chromosome condensation, and completion of meiosis I. Although progesterone is thought to be the major physiological hormone involved in triggering resumption of meiosis, there are a number of agents, including insulin, that are capable of inducing maturation (112,113). Rana pipiens, another amphibian, has also been used with similar results (114, 115). Before focusing on representative studies of this process in stage VI oocytes, we shall recount an interesting series of experiments with stage IV oocytes, which have accumulated some yolk but are still not able to fully mature (note that we find these studies difficult to interpret precisely because they are limited, have been reported by only a single laboratory, and are hard to fit into a broad biological context). Miller (116, 117) has reported that insulin applied directly to the cytoplasm of stage IV oocytes stimulated synthesis of total protein as well as total mRNA (Fig. 8). Interestingly, insulin in the external medium, interacting with intact oocytes, also stimulated an increase in RNA, protein, and glycogen synthesis. Further, insulin applied to isolated nuclei stimulated RNA synthesis. Surprisingly, with many of the effects observed, proinsulin and IGF-I were equally potent with insulin. Also the effects of cytoplasmic insulin and external insulin were additive. One possible interpretation is that insulin receptors as well as IGF-I receptors are present, and both are mediators of cellular effects. The author suggests that there are intracellular sites of insulin action. These experiments present new challenges to the field that invite further exploration. In stage VI oocytes, as was already mentioned, insulin induces meiotic maturation and cell division. However, the observation that a high concentration of insulin (50 nM) was required, while IGF-I was effective at low concentration (0.5 nM), suggested that the effect of insulin might be mediated via interaction with IGF-I receptors (67). This suggestion was supported by the observation that an antiinsulin receptor antibody which blocked insulin binding up to 50% had no effect on the doseresponse curve for induction of oocyte maturation by insulin.

567

Insulin and IGF-I do not induce oocyte maturation by a mechanism identical to that of progesterone, the natural inducer, but some signaling pathways are affected concordantly by the three hormones. One of the earliest events associated with triggering of the maturation response is a rapid decrease in basal levels of cAMP, due in part to an inhibition of the adenylate cyclase of the oocyte membrane. Insulin, IGF-I, and progesterone all inhibit oocyte adenylate cyclase (118,119). Further, cholera toxin, a potent irreversible activator of adenylate cyclase, inhibits reinitiation of meiosis induced by these hormones (120). By contrast, phosphodiesterase activity was stimulated by insulin and IGF-I but was unaffected by progesterone. The concentration of insulin and IGFI required for phosphodiesterase activation in oocytes was the same as the concentration required to inhibit adenylate cyclase in vitro, suggesting a concerted regulation of cAMP levels by insulin and IGF-I that affects both synthesis and degradation of cAMP. The concentration of insulin required for stimulation of phosphodiesterase activity and for inhibition of adenylate cyclase was 20 times lower than the concentration required for induction of oocyte maturation (121). One of the late events in the maturation response of oocytes is the appearance of a cytoplasmic maturation promoting factor (68,120). This factor has been detected in Xenopus oocytes after exposure to insulin or progesterone. Microinjection of the p21 ras protein also induces maturation via production of this factor; p21 and progesterone acted synergistically. Antibodies against p21 blocked both ras and insulin-induced maturation but did not inhibit oocyte maturation induced by progesterone (122-124). Unlike progesterone, but similar to insulin and IGF-I, ras protein stimulates phosphodiesterase activity (125). Therefore, it is possible that a ras protein may be required in the pathway of insulin-induced, but not progesterone-induced maturation. Two other intermediate biochemical events in the maturation process, an increase in intracellular pH (pHj) and phosphorylation of ribosomal protein S6, have been induced in stage VI oocytes by both insulin and progesterone to an equivalent extent and with the same time course (126, 127). That cholera toxin inhibits both of these effects of insulin but not of progesterone indicates that the regulation of these events is different for the two homones (126). The insulin-dependent increase in S6 phosphorylation is due, in part, to activation of a ribosomal S6 protein kinase (127, 128). In addition, in stage IV oocytes, which cannot mature in response to insulin or progesterone (but do mature in response to microinjection of maturation promoting factor), both pHj and S6 phosphorylation increased significantly after exposure to insulin; progesterone induced only a partial increase in pH; with no S6 phosphorylation. The stimu-



568

lation of S6 phosphorylation induced by insulin in stage IV oocytes was less than the stimulation by insulin in stage VI oocytes. These results suggest that, although these insulin-induced responses are present in stage IV oocytes, the magnitude may not be sufficient to induce maturation, or that a biochemical step besides S6 phosphorylation and production of maturation promoting factor is absent in stage IV oocytes. Another type of biological event has been recently reported to be sensitive to insulin and IGF-I but not to progesterone in the stage VI oocyte. Janicot and Lane (71) have shown a 3- to 4-fold increase in 2-deoxyglucose uptake when insulin at high nanomolar concentrations or IGF-I at low nanomolar concentrations was added to the culture (Fig. 9). They concluded that the biological effect was mediated via IGF-I receptors, a suggestion supported by their finding that microinjection of an antireceptor-kinase antibody blocked the effect. How-

i

ever, it should be noted that this antibody also reacts well with the kinase region of the insulin receptor. c. Sea urchins The sea urchin, an echinoderm used widely to study development from fertilization to organogenesis, has several well characterized lineage-specific, developmentally regulated genes (129), which can be used as biochemical markers of cell differentiation under hormonally stimulated conditions. In the sea urchin Strongylocentrotus purpuratus, addition of insulin modulated the levels of mRNA of different lineages, one ectoderm-specific, one mesoderm-specific, and one found in all cell types (130). Insulin, at 10-100 ng/ml added to the culture at the time of fertilization, increased several fold the levels of mRNA at blastula for a collagen gene, Spec-1, which is ectodermspecific, and the early gene for histones. Proinsulin was slightly less potent than insulin, and no effect was obtained with human IGF-I at 10 ng/ml. In another species of sea urchin, Paracentrotus lividus, addition of 100 nM insulin to unfertilized eggs elicited a 7-fold increase in the content of cAMP (131). These experiments do not clarify whether an endogenous insulin molecule has any role in sea urchin development but provide phamacological evidence that the machinery for insulin action is present in this species.

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d. Drosophila

a. **

Insulin-responsive cells also arise early in embryogenesis in Drosophila melanogaster. Cultured Drosophila cells from gastrula stage embryos require insulin for survival and differentiation into a variety of cell types (132). The pattern of expression of insulin receptor transcripts, very widespread through all embryonic stages, is also consistent with a general metabolic or multifunctional role for insulin during Drosophila development. However, except for the activation of the receptor kinase discussed earlier (73, 74), there have been no reports of direct experiments in embryos with insulin, IGF-I, or their antibodies. .0001 .001

.01

.1

1

HORMONE CONCENTRATION

10 (uri)

FlG. 9. Insulin and IGF-I effects on 2-deoxyglucose uptake and oocyte maturation in stage VI Xenopus oocytes. Groups of 10 to 35 oocytes were incubated in the presence of insulin (•) or IGF-I (O) at the concentrations indicated. A, Incubation was for 90 min at 18 C; then 2-deoxy-D[U-14C] glucose was added, and incubation was continued for 10 min before glucose uptake was determined. B, Incubation was overnight at 18 C, and then the oocytes were examined for the maturation response by the appearance of a white spot in the pigmented pole, characterizing germinal vesicle breakdown (GVBD); without added hormone, GVBD was less than 2%. [Reproduced with permission from M. Janicot and M. D. Lane: Proc Natl Acad Sci USA 86:2642, 1989 (71).]

e. Mammals Insulin at picomolar concentrations increased protein synthesis in morula and blastocysts of mouse (Fig. 10) (133). At a slightly higher concentration (4 ng/ml) insulin stimulated RNA and DNA synthesis in morula and, more markedly, in blastocyst stage mouse embryos (83). Insulin at very high concentrations (>20 /ug/ml) was embryotoxic and interfered with development of the preimplantation mouse embryo in vitro (134); 62% of the blastocysts were abnomal. Several groups have contributed to an elegant series


November, 1990

INSULIN AND IGF-I IN EARLY DEVELOPMENT Dose Response

01

I

10

I0 2

I05

I04

I05

O6

INSULIN CONCENTRATION (pM)

FIG. 10. Effect of insulin on protein synthesis in mouse blastocysts. Incorporation of [3H]leucine into protein by preimplantation mouse embryos cultured for 48 h with or without insulin, is expressed as counts per min/2 h per embryo. [Reproduced with permission from M. B. Harvey and P. L. Kaye: Endocrinology 122:1182 (133).® The Endocrine Society[

of experiments on rats during neurulation (135). Using a whole embryo culture system, they showed that conditions that mimic the intrauterine environment provided by a mother with hypoinsulinemic diabetes, including hyperglycemia and hyperketonemia, are deleterious to the embryo, causing malformations and death. They also found that large doses of insulin are not harmful (in terms of malformations) at this stage but that the addition of antiinsulin antibodies which presumably neutralize the small amounts of endogenous insulin are deleterious (136). Overall it would appear that in this experimental system distortions of fuel homeostasis have a dominant effect, but physiological concentrations of insulin are also contributing to the well being of the embryo at this stage. IV. Insulin and IGF-I: Sources in Early Ontogeny A. Insulin Traditionally, embryologists and endocrinologists searched for insulin only in the emerging pancreas or occasionally at other sites in the gastrointestinal tract using immunocytochemistry. Over the last decade, the recognition that insulin-related growth factors can be manufactured at multiple sites, the recognition that many peptide hormones are manufactured at extraglandular sites, and the detection of vertebrate peptide hormones in nonvertebrates that do not have the typical vertebrate endocrine glands (137, 138) has prompted a wider search for insulin, as well as the insulin-related peptides in embryos at early stages, and in eggs and oocytes. As can be seen in Table 1, insulin producing 0-

569

cells appear in the rudimentary embryonic pancreas relatively early in organogenesis. Because the data are sparse, we cannot present a full chronology of each species as we have done earlier with the receptor. Rather we shall present pancreatic insulin; nonpancreatic sources of insulin in vertebrates; insulin sources in nonvertebrates that never have a pancreas; and maternal sources of insulin supplied to the egg. Table 1 summarizes the developmental time-table of several species and, for each of the vertebrates, also notes the appearance of the rudiment of pancreas. Most of the studies have utilized immunohistochemistry with antimammalian insulin antibodies to localize cells that store insulin. Insulin storage is a highly specialized cellular process that develops later than secretion or synthesis. A more recent approach is to search in the pancreas, other organs, or in whole embryos for insulin mRNA, which appears contemporaneous with or earlier than insulin synthesis, secretion, or storage. a. Chicken. In chick embryos, pancreas begins to develop at days 3-4. Total RNA from the body of embryos at days 4 and 5 and from pancreas after day 10 showed a major mRNA transcript of approximately 0.6 kilobase (kb), detected by specific oligonucleotides representative of the chicken insulin gene (139). The abundance of this polyadenylated transcript increases remarkably during the last week of embryogenesis. In situ hybridization in the pancreas confirmed the developmental pattern of insulin gene expression and indicated that the increase in the level of expression was accounted for by an increase in the number of functional islets, as well as by an increase in the level of insulin mRNA per islet. An important postranscriptional control of insulin synthesis and insulin secretion is likely to occur in the chick embryo since plasma insulin levels, measurable at day 5 and later, are virtually unchanged during late embryonic development (140, 141). Embryonic extrapancreatic andprepancreatic. In chicken, the insulin mRNA transcript can be detected in liver during the second and third weeks of embryogenesis with an abundance 1 to 2 orders of magnitude lower than in the pancreas at the same stages (139). Immunoreactive insulin has also been detected in the duodenum of chick embryos. The insulin content in duodenum is comparable to that in pancreas at days 12 and 14 and decreases thereafter (142). Studies of insulin gene expression in prepancreatic embryos (days 2 and 3) revealed an insulin transcript of 0.6 kb, the same size detected later in pancreas, with RNA blots using large amounts of poly (A)+ RNA from whole embryos (Fig. 11). It was absent in poly (A)~ RNA indicating that it was a polyadenylated transcript, typical for a mature mRNA. Its abundance was 3 to 4 orders of


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Prepancreatic Chicken Insulin mRNA FIG. 11. RNA blot of insulin mRNA in prepancreatic embryos. Poly(A)+ RNA or poly(A)" RNA was extracted from whole chicken embryos at day 2 and day 3 of development and fractionated on a formaldehyde gel. Total RNA extracted from embryonic limbs and pancreas was included for comparison. The RNA was transferred to a nylon membrane and hybridized to a chicken insulin riboprobe. The autoradiogram shows an insulin transcript (arrow) in the pancreas, the E3 poly(A)+ and E2 poly(A)+ lanes but not in the limb lane. [ Reproduced with permission from J. Serrano et al.: Dev Biol 132:410, 1989 (139).]

magnitude less than the pancreatic insulin mRNA of late development (139). In previous studies we detected a material with the immunological and biological characteristics of chicken insulin, in extracts of whole embryos from day 2 and day 3 at concentrations that are about 4 orders of magnitude less than in mature pancreas (143). When the "head" region of day 3 embryos was studied separately from "body," about 25% of the total immunoactive insulin at that age was in the "head." Thus, insulin gene expression and insulin production appear to precede development of the pancreas. The cellular origin of the insulin in the early embryos remains to be determined. Maternal. In avian species, the egg provides a continuous source of nutrients. It may also contain hormones and growth factors required by the early embryo. Insulinrelated material was demonstrated in the white and yolk of unfertilized and fertilized eggs (143, 144). This material, distinct from IGF-I, has been partially purified by HPLC from the yolk of the unfertilized chicken egg (145). b. Amphibians. The pancreatic primordium in Rana pipiens at 2.5 days of development (stage 22 tadpole) shows immunostaining for insulin (146). The hepatopancreas primordium in Xenopus laeuis at 7 days of development (stage 46 tadpoles) has clusters of insulin-positive FITC immunostaining (our unpublished observations). As in the chicken, the insulin gene in Xenopus embryos is expressed before the development of the pancreas, e.g. embryos undergoing neurulation contain insulin mRNA. In addition, insulin mRNA and insulin are detected in the stage VI oocyte but only insulin in the unfertilized Xenopus egg (A.R. Shuldiner, F. de Pablo and J. Roth,

E2

E3 i POLY (A)

+

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i

( A ) " POLY ( A ) + ( A > ~

Kb -1.35 -0.24

manuscript in preparation). c. Sea urchin. Studies using antiporcine insulin antiserum in two species of sea urchins, Strongylocentrotus purpuratus and Lytechinus uariegatus, detected a diffuse pattern of immunostaining in eggs and blastula stage embryos (130, 147). In the free swimming larva, insulin immunostaining was well localized to cells of the gut epithelium (128) (Fig. 12). d. Drosophila. Many studies point to the presence of insulin-related material in both larval and adult forms of many species of insects (see Refs. 137 and 138). Adult Drosophila have been shown to have a material that resembles the insulin of vertebrates (148) but we are not aware of any studies of insulin or insulin mRNA in Drosophila embryos. e. Mammals. In contrast to nonmammals and adult foms of mammals where the evidence for extrapancreatic insulin comes from several sources (138), such evidence is lacking in the developing mammalian embryo. In rats, immunoactive insulin has been detected in pancreas at approximately day 11, when the pancreatic rudiment develops (149, 150). Proinsulin mRNA was undetected in some studies until day 16 (148) but was measurable in other studies at day 12 (151). In the mouse, insulin was detected by immunocytochemistry at day 11, while it was undetected earlier in the pancreatic rudiment (47). An mRNA transcript larger than the pancreatic insulin transcript was detected in fetal rat liver, but its translation into protein was not studied (152). In mouse embryos before implantation, insulin mRNA has not been detected using a very sensitive method,


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FIG. 12. Insulin immunocytochemistry with sea urchin embryos. Cells that contain insulin-related material are detected by indirect immunofluorescence with an antipork insulin antibody, a, Bright field view of a saggital section of a day 3 embryo to illustrate anatomy; b, indirect immunofluorescence with insulin antibody stains some individual endodermal cells in gut; c, with control serum no signal is detected. [Reproduced with permission from F. De Pablo et al.: Dev Biol 130:304,1988 (130).]

amplification with the polymerase chain reaction (84) (Table 2), while insulin has been detected immunocytochemically using electronmicroscopic techniques (83). One potential source of insulin in the mammalian embryo before implantation is the oviduct fluid; the authors suggested that the mouse blastocyst takes up insulin from the maternal oviduct by a receptor-mediated mechanism. Insulin mRNA also was not detected in postimplantation mouse embryos between 7.5 and 9.5 days (85). A mouse teratoma-derived cell line (1246-3A) is a unique example of secretion of an insulin-related factor and proliferative response to insulin by the same cell, i.e. perhaps a model for an autocrine action of insulin in embryonic cells (153). In humans, the earliest detection of immunoactive insulin in the pancreas is at approximately 7 weeks of gestation (154, 155). There are no reports of extrapancreatic insulin production in the embryo. B. IGF-I Although ample evidence exists of expression of IGFI in multiple tissues in late fetal development in mammals (156, 160), the expression of IGF-I in early embryonic stages has only begun to be studied. a. Chicken. Circulating IGF-I levels in chick embryos increase sharply between days 6 and 15 and decrease at later stages of embryogenesis (161). The sources of this serum IGF-I are probably multiple. Cartilage explants from day 9 embryos synthesized a peptide that crossreacted in a human IGF-I RIA (162). With very sensitive methods, IGF-I mRNA transcripts were detected in embryos at days 12-16 in multiple tissues, including pancreas, brain, stomach, heart, and limbs. In whole chick embryos, IGF-I was detected in blastula and gastrula embryos and increased markedly during the second week of embryogenesis (163) in rough parallelism with the serum IGF-I levels (our unpublished observations).

The vitreous humor of chick embryos also contains a material with the immunological and biological characteristics of IGF-I, (Ref. 164 and Caldes T., J. Alemany, M. L. Robins, and F. de Pablo, manuscript in preparation), termed lentropin by Beebe et al. (164). With HPLC we have recently partially purified the IGF-I from vitreous humor of embryos at days 6-15. IGF-I is present in the unfertilized chicken egg (thus, is maternal in origin) in concentrations at least as great as those of insulin (165). b. Other species. Genes for two very similar but clearly distinct IGF-I molecules have been cloned from Xenopus, but as yet there is no information on developmental expression (166). In Drosophila it is not known whether IGF-I related genes are present. In the mouse embryo preimplantation, IGF-I mRNA was not detected (84). However, early postimplantation, between days 7.5 and 9.5, the mouse embryo has IGF-I mRNA transcripts (85). Whole mouse embryos at days 9-12 contain immunoreactive IGF-I (154); explants of liver, and limb buds of day 11 mouse embryos produced IGF-I after 3 days in culture. Other tissues, including intestine, heart, and brain, from older embryos also synthesized IGF-I, measured in that pioneer study as immunoassayable "somatomedin-C" (167). In the rat, very small quantities of IGF-I mRNA were detected by in situ hybridization in embryos after day 16 in one study (166). With a sensitive RNase protection assay, IGF-I mRNA was found in rat embryo liver of day 11 but it was only a few percent of the content in adult liver. A major induction (8.6-fold) occurred between days 11 and 13 (169). In humans, early studies detected IGF-related materials during midembryogenesis (170). More recently, D'Ercole et al. (171) measured IGF-I in serum and tissues of human fetuses from 9-19 weeks of gestation. The serum level was approximately 27% of nomal adult val-



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ues. In tissues, IGF-I was highest in the lung and intestine and, surprisingly, lowest in the liver. The presence of IGF-I mRNA in the human fetus has been confirmed by in situ hybridization (172).

WEIGHT 100 r

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C. Summary of sources Until recently, insulin and IGF-I had been looked for only in relatively late development with an emphasis on the pancreas as the sole source of insulin and the liver and some other tissues as the source of IGF-I. The data from individual species are very sparse; overall it appears that the oocyte and egg of nonmammals may be a source of insulin and IGF-I during oocyte maturation, fertilization, and early stages of embryonic development. The expression of insulin and IGF-I in embryos of nonmammals clearly occurs before the pancreas and the liver differentiate. More complete studies are required to evaluate in a definitive manner the maternal vs. embryonic contribution of these peptides in the early mammalian embryo. We should be alert in any embryo species to the possibility of mRNA and peptide production at multiple sites.

V. Effect of Insulin Antibodies and Insulin Receptor Antibodies a. Chicken Glucose metabolism is apparently regulated by insulin physiologically in chick embryos after organogenesis. Leibson et al. (140) injected an insulin antiserum iv in chick embryos between days 9 and 16 and observed that the antiserum had caused a "diabetic state" 24 h later, i.e. an elevation of blood glucose and reduction in liver glycogen. We have analyzed in the chick embryo the developmental perturbances caused by both insulin antibodies and insulin receptor antibodies (173, 174) (Fig. 13). Treatment of the embryo at the beginning of organogenesis (day 2) with antiinsulin antibodies produced developmental retardation or arrest over the next 2 days, presumably due to neutralization of the embryo's endogenous insulin. A partially purified polyclonal antiinsulin antibody killed approximately 20% of the embryos. Many of the embryos that survived were morphologically and biochemically retarded, typically manifested as a reduction in weight, total DNA, RNA, protein, triglycerides, and creatine kinase. The MB isozyme of the creatine kinase, a marker for muscle differentiation, was also decreased. Antibodies against other factors including epidemal growth factor, nerve growth factor, and somatostatin were without effect on these developmental indices; antibodies against IGF-I were not tested. While neutralization of endogenous insulin clearly interferes

TOTAL CK

r-i

f t f

f rT Ab-I

i

Ab-R

DNA

RNA

CK-MB

100

i

75

Ab-I

Ab-R

Ab-I

Ab-R

Ab-I

Ab-R

ANTIBODY DOSE l^g/embryo)

FIG. 13. Effects of antiinsulin and antiinsulin receptor antibodies on growth and biochemical development of early chick embryos. Antiinsulin antibody (Ab-I) or antiinsulin receptor antibody (Ab-R) (200 or 400 /ig total Immunoglobulin G) were applied to embryos at day 2. Another group of embryos received normal immunoglobulin G (designated "0"). Two days later the dead embryos were enumerated and discarded. Live embryos were weighed and homogenized and the content of protein, DNA, RNA, total creatine kinase (CK), or its isozyme MB (CK-MB) was measured. (*, P < 0.05, **, P < 0.005). [Reproduced with permission from M. Girbau et al: Biochem Biophys Res Commun 153:142,1988 (174).]

with embryo development, it is unclear which effects are direct on growth and differentiation or indirect via metabolic disturbances, or both. Based on the specificity of the antibodies for insulin, the presence of insulin, insulin-related receptors, and insulin-responsive pathways, and the symmetry of results obtained by adding vs. substracting insulin (i. e. acceleration of development with added insulin), we have concluded that insulin in chick embryos at prepancreatic stages, acting through insulin-related receptors, is playing a physiological role. This conclusion is further supported by the finding that the adverse effects of antiinsulin antibodies are mimicked closely by antibodies to the insulin receptor (174). The antireceptor antibody applied at day 2 also increased the death rate of embryos, and the survivors showed a reduction in weight, total protein, DNA, RNA, triglycerides, and creatine kinase. The simplest interpretation is that at this early age endogenous insulin is acting via insulin receptors, and interference by specific antibodies with either the hormone or the receptor has serious developmental consequences. However, the antireceptor antibodies may cross-react to a variable degree


November, 1990


with the receptor for IGF-I as well. Thus the effect of antireceptor antibody may be due to interference with both insulin-mediated and IGF-I mediated events (IGFI, like insulin, is present in early embryos), while antiinsulin antibody interfered with only insulin's effects. There is one striking difference in biological effects between the antibodies: the MB-isozyme of creatine kinase (a useful differentiation marker) was retarded with antiinsulin but unaffected by anti-receptor antibodies. Both insulin and, even more, IGF-I added simulataneously with the antireceptor antibody, were capable of increasing the embryo's content of creatine kinase-MB isozyme (91). This suggests that an IGF-I receptor may be primarily involved in the regulation of this enzyme. The interpretation is less precise yet because our antireceptor antibody, in addition to its blocking effect, has some hormone-mimicking properties, which can vary with time (i.e. hormone-mimicking effect is often early), as well as among species and individual tissues. In summary, we again conclude that insulin is acting as a physiological agent via insulin receptors to achieve normal development in pre-pancreatic chick embryos, but as yet unwarranted are more precise interpretations of which receptors or of the directness of the effects. In a sense, the in vivo model is more satisfying because of its closeness to real life, but less satisfying in that multiple interpretations are possible. b. Other species.

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which demonstrate that 1) The two peptides are present in eggs. 2.) Both peptides are synthesized very early by embryonic tissues. 3) The insulin receptors and the IGFI receptors are widespread in young embryos. 4) Insulin and IGF-I, acting through specific receptors, stimulate growth and differentiation of embryonic cells. 5) Antibodies to insulin or its receptor have deleterious effects on the embryo. Possibly IGF-II, IGFs binding proteins, and many other hormone-like agents (as well as intercellular matrix molecules) may also play a role in the development of the early embryo. In a recent essay we put these ideas in a more general context (4). Other reviews have highlighted findings in Drosophila (25) and mouse (84) consistent with the concept that the family of insulin-related hormones and receptors, as well as their role in development, is very highly conserved in evolution. As we have indicated (4), it should be emphasized that proof of function for each factor will come via an updated version of a classic endocrinological approach: 1) demonstration of a developmental malfunction by withdrawal of the endogenous product {e.g. the peptide or its receptor by an antibody or specific antagonist) or inactivation of its corresponding mRNA by an antisense sequence or the gene by a specific mutation; followed whenever possible by 2) reestablishment of the normal developmental function by an appropriate readdition. Acknowledgments

Some studies using antireceptor antibodies in Xenopus oocytes have already been discussed. We are not aware of reports in the literature of early mammalian embryos treated with antibodies against insulin or its receptor, except as described above; neutralization/depletion of insulin by antibody in the culture medium of mammalian embryos (day 9.5 rat embryos) caused retardation of growth and development, which was restored to normal by the readdition of insulin. These studies indicated that the early mammalian embryo has an absolute requirement for maternally derived insulin even in the presence ofIGFs(138). Antiserum to human IGF-I has been shown to inhibit the growth of rat embryos at day 10, when they had been transplanted under the kidney capsule of a syngeneic host. After 6 days of IGF-I antibody infusion, the treated embryos were only approximately 20% growth retarded, while 9 days of IGF-I antibody treatment caused 40-45% growth retardation (175).

Conclusions We conclude that insulin and IGF-I play an important role in the early embryo, based on work in many systems but especially in chicken and to a lesser extent in frog,

This review is dedicated to the memory of Ora Mendelsohn Rosen whose vivacity, intelligence, and creativity inspired us. We thank R. S. Garofalo, J. A. D'Ercole, P. Rotwein, D. LeRoith, and M. A. Lesniak for critical reading and suggestions on the manuscript, and Ms. Esther Bergman for careful preparation of the manuscript.

References 1. Ham RG, Veomett MJ 1980 Hormonal mechanisms in cellular differentiation. In: Mechanisms of Development. The CV Mosby Co., St. Louis, p 429 2. Tata JR 1984 The action of growth and developmental hormones: evolutionary aspects. In: Goldberger RP, Yamamoto KR (eds) Biological Regulation and Development. Plenum Publishing Co, New York, vol 3B:1 3. Williams RH, Wilson JD, Foster DW 1985 Williams Textbook of Endocrinology 1985, ed 7. Saunders, Philadelphia 4. De Pablo F, Roth J, 1990 Endocrinization of the early embryo: an emerging role for homones and homone-like factors. Trends Biochem Sci 15:339 5. Czech MP 1989 Signal transmission by the insulin-like growth factors. Cell 59:235 6. Ullrich A, Schlessinger J 1990 Signal transduction by receptors with tyrosine kinase activity. Cell 61:203 7. Gammeltoft S 1989 Insulin-like growth factors and insulin: gene expression, receptors and biological actions. In: Peptide Homones as Prohomones. Ellis Horwood Limited, p 176 8. De Pablo F 1990 Insulin and insulin-like growth factors (IGFs) in avian development. In: Rosenblum IY, Heyner S, (eds) Growth Factors in Mammalian Development. CRC Press, Boca Raton, FL, p 71


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Keystone Symposia on Molecular & Cellular Biology January 28, 1991 through April 7, 1991 For program and application information on 1991 conferences write to: Keystone Symposia, 2032 Armacost Ave., Los Angeles, CA 90025. Telephone: (213) 207-5042. Telefax: (213) 207-2397. Telex: 495012 attn. Keystone Symposia.


Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells.

Insulin receptors and insulin-like growth factor I receptors are functional during organogenesis of the lens.

Insulin-like growth factor-I receptors in nonfunctioning thyroid nodules.

Insulin-like growth factor-I receptors in human glial tumors.

Effect of puberty on insulinlike growth factor I and HbA1 in type I diabetes.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

Functional roles of insulin and insulinlike growth factors in preimplantation mouse embryo development.

Insulin and insulin-like-growth-factor-I (IGF-I) receptors in Xenopus laevis oocytes. Comparison with insulin receptors from liver and muscle.

Effect of tamoxifen on serum insulinlike growth factor I levels in stage I breast cancer patients.

Expression of insulin-like growth factor-I during chicken development.

Presence of insulin-like growth factor-I receptors and absence of growth hormone receptors in the antler tip.

insulin-like growth factor I hybrid receptors.

insulinlike growth factor II receptors.

Autoradiographic localization of insulin-like growth factor I receptors in rat brain and chick embryo.

Truncated and native insulinlike growth factor I enhance mucosal adaptation after jejunoileal resection.

Genes encoding receptors for insulin and insulin-like growth factor I are expressed in Xenopus oocytes and embryos.

Dephosphorylation of human insulin-like growth factor I (IGF-I) receptors by membrane-associated tyrosine phosphatases.

Insulin-like growth factor-I (IGF-I) attenuation of growth hormone is enhanced by overexpression of pituitary IGF-I receptors.

Insulin-like growth factor-I and insulin as growth and differentiation factors in chicken embryogenesis.

Progestin regulation of insulin and insulin-like growth factor I receptors in cultured human breast cancer cells.

Insulin-like growth factor I stimulates oligodendrocyte development and myelination in rat brain aggregate cultures.

Immunolocalization of insulin-like growth factor-I (IGF-I) and its receptors (IGF-IR) in the equine epididymis.

Characterization of insulin-like growth factor I receptors and growth effects in human lung cancer cell lines.

[Growth Hormone-Insulin Growth Factor I (GH-IGF-I) axis and growth].