Developmental Regulation of Insulin and Type I Insulin - Diabetes

Developmental Regulation ofInsulin and Type I Insulin-Like GrowthFactor Receptors and Absence of Type IIReceptors in Chicken Embryo TissuesLLUIS BASSAS, MAXINE A. LESNIAK, JOSE SERRANO, JESSE ROTH, AND FLORA DE PABLO

Chicken embryos are a suitable model for studying therole of insulin, insulin-like growth factors I and II (IGF-Iand IGF-II), and their receptors in embryogenesis. Weshow that plasma membranes from heart, liver, andlimb buds, as reported earlier for brain, each have adistinct developmental profile for insulin receptors andtype I IGF receptors. In heart and limb buds, IGFbinding is higher than insulin binding, but in liver,insulin receptors dominate. Expression of thesereceptors is, therefore, developmental^ regulated andtissue specific. The wide distribution of high-affinityreceptors capable of mediating insulin and IGF actionsin early organogenesis further supports the possibleimportance of this family of peptides for differentiationand growth in vertebrates. In all chicken embryotissues studied, both IGF-I and IGF-II appeared to bindto a type I IGF receptor. We have not detected areceptor with the peptide binding and structuralcharacteristics of the mammalian type II IGF receptor.The type II receptor was absent in embryos, liver fromnewly hatched chicks, and adipocytes from olderchicks, which suggests that the chicken may lack thissubtype of IGF receptor. Diabetes 37:637-44,1988

Insulin and insulin-like growth factors I and II (IGF-I andIGF-II) are structurally related polypeptides with over-lapping metabolic and growth effects on multiple celltypes (1). In addition, insulin and IGFs induce cellular

differentiation of many embryonic or fetal cell types (2-8).Because rapid growth and tissue differentiation are typicalof embryogenesis, insulin and IGFs may be important growthfactors in this period. Synthesis of IGFs has been demon-strated in many tissues, e.g., fetal tissue in rats and mice

From the Diabetes Branch, National Institute of Diabetes and Digestive andKidney Diseases, National Institutes of Health, Bethesda, Maryland.Address correspondence and reprint requests to Flora de Pablo, MD, PhD,Building 10, Room 8S-243, National Institutes of Health, Bethesda, MD 20892.Received for publication 1 December 1986 and accepted in revised form 1October 1987.

(9,10), chicken embryo liver (11) and cartilage (12), anddeveloping human brain (13). Insulin-related mRNA hasbeen detected in rat yolk sac (14); rat hypothalamus (15);rat, mouse, and hamster pituitary (16); and human placenta(17).

Receptors capable of mediating insulin and IGF actionsare widespread in fetal tissues and placenta during late de-velopment (18). Sara et al. (19) followed the ontogeny ofinsulin and IGF binding in human brain and liver after ~12wk of gestation, when human organogenesis is almost com-plete.

We describe insulin immunoactivity and bioactivity in2-day-old chicken embryos (postneurulation, with ~20somites), when pancreatic rudiment is not yet recognized(20). We detected in membranes from 2-day-old chickenembryos specific insulin receptors and IGF receptors (21).We found that IGF receptors dominated over insulin recep-tors in brain during organogenesis and that insulin receptorswere more abundant at the time of hatching. In this tissue,we suspected, on the basis of developmental binding pro-files and specificity studies, that both IGF-I and IGF-ll/mul-tiplication-stimulating activity (IGF-II/MSA) bound exclu-sively to type I IGF receptors.

We provide evidence for a distinct tissue-specific devel-opmental regulation of insulin receptors and IGF receptorsin selected chicken embryo tissues with well-defined pat-terns of organogenesis. We also confirm the absence of typeII IGF receptors in embryonic chicken tissues, suggestingthat these receptors do not mediate development, at leastin this avian species.

MATERIALS AND METHODSMaterials. Highly purified human IGF-I and partially purifiedIGF (lot 1932, a 10% pure preparation containing IGF-I andIGF-II in a 1:1 ratio) were a kind gift from R.E. Humbel (Bio-chemisches Institut der Universitat, Zurich, Switzerland).Purified IGF-I was labeled with 125I (New England Nuclear,Boston, MA) according to a modification of the Chloramine-T method (22) and was further purified by hydrophobic-

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INSULIN AND IGF RECEPTORS IN EMBRYOGENESIS

exclusion chromatography (23). Alternatively, unlabeled orlabeled recombinant human IGF-I was purchased fromAmersham (Arlington Heights, IL). [125l]monoiodinated por-cine insulin was purchased from New England Nuclear (Bos-ton, MA). 125I-IGF-II/MSA of rat origin (MSA peak III-2) wasgenerously provided by M.M. Rechler (National Institute ofDiabetes and Digestive and Kidney Diseases, NIH, Be-thesda, MD). Porcine insulin was purchased from Elanco(Indianapolis, IN), and rat IGF-II/MSA-CR was purchasedfrom Collaborative Research (Waltham, MA). The experi-ments in which full competition curves are presented wereperformed with highly purified or recombinant IGF-I both astracer and unlabeled peptide; however, to compete for bind-ing of highly purified IGF-II/MSA, IGF-II/MSA-CR (whichprimarily contains MSA peak II) was used. Acrylamide, bis-acrylamide, and sodium dodecyl sulfate (SDS) were pur-chased from Bio-Rad (Richmond, CA), and 1,4-dithiothreitoland dissuccinimidyl suberate were purchased from PierceChemical (Rockford, IL). All other products were of analyticalgrade.

Preparation of tissue membranes. Fertilized eggs of whiteLeghorn chickens were incubated at 38.5°C in 55-65% rel-ative humidity. Embryos at selected stages of developmentwere dissected on ice and pooled to obtain enough tissuefor the preparation of membranes (24). Tissues were ho-mogenized with a hand-held glass-glass homogenizer in 20vol of 1 mM NaHCO3 at 0°C, with 2 mM phenylmethylsulfonylfluoride and 1 jxg/ml leupeptin as protease inhibitors. Crudemembrane pellets were obtained by differential centrifuga-tion (25). The 20,000 x g pellet was washed and resus-pended in HEPES buffer (100 mM HEPES, 120 mM NaCI,15 mM Na acetate, 10 mM glucose, 2.5 mM KCI, 1.2 mMMgSO4l and 1 mM EDTA, pH 8.0). The protein concentrationwas determined by the fluorescamine method and adjustedto -1.5 mg/ml (26). Aliquots were stored at -70°C for upto 6 mo, a period in which binding activity remained un-changed, as demonstrated by testing several aliquots of thesame preparations.Binding studies. Binding of 125l-labeled peptides (total of0.3 ng, -10,000 counts/min) to membranes (75 jxg protein)was performed in 150 pj (total vol) HEPES buffer with 1%bovine serum albumin (BSA), pH 8.0, as described by Hav-rankova et al. (25) except that 0.5 mg/ml bacitracin wasincluded in all assay tubes. Binding was expressed as theratio between the radioactivity in the membrane pellet andthe total radioactivity added. Nonspecific binding, definedas the radioactivity bound in the presence of excess unla-beled hormone (i.e., 500 ng/ml IGF-I when the tracer was125l-labeled IGF-I, 5 ng/ml IGF-II/MSA-CR for 125I-IGF-II/MSAtracer, or 10 |xg/ml porcine insulin for 125l-insulin tracer), wassubtracted from total binding to give specific binding. Deg-radation was determined by measuring the difference in the5% trichloroacetic acid precipitability of the radioligands be-fore and after the incubation. Average precipitability of thetracers before binding assays was 95% for 125l-insulin, 85-90% for 125I-IGF-I, and 80-85% for 125I-IGF-II/MSA. The de-crease in trichloroacetic acid precipitability for each tracerwas <5% after binding studies were done at 15°C for 5.5 h,the standard conditions.

The 20,000 x g supematants of heart and liver membranepreparations from early and late development were testedfor 125I-IGF-I binding to determine if the method of membrane

preparation was equally efficient throughout embryogenesis;the binding assay was performed as described (21). Theresults showed a negligible proportion of IGF-I—specificbinding (<0.2% in liver and <0.6% in heart) in the super-natants of the preparations of membranes.

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FIG. 1. Binding patterns of 125l-labeled peptides to chicken embryotissue during ontogeny. Labeled insulin-like growth factor I(IGF-I) ( • ) , 125I-IGF-II/multiplication-stimulating activity peak III-2(IGF-II/MSA) (O), or 12sl-insulin (A) was incubated with membranesobtained from heart (top), liver {middle), and limb buds (bottom) ondesignated days of development with standard incubation conditions(see MATERIALS AND METHODS). Highly purified IGF-I and IGF-II/MSA weretracers. To calculate nonspecific binding of IGF-I and IGF-II/MSA, largeexcess of partially pure IGF I and II (2500 ng/ml) or IGF-II/MSA fromCollaborative Research (5000 ng/ml), respectively, was added.Nonspecific binding was subtracted from total binding to give resultsshown. Three or more different membrane preparations were used foreach age tested, except for 3-day-old heart assays, in which only 1preparation was used. Results are means ± SE of duplicatedeterminations in 4 studies done at each age of development.

638 DIABETES, VOL. 37, MAY 1988

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10 10 10 U>-10 10* 103 10*

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FIG. 2. Specificity of insulin-like growth factor (IGF) and insulin binding to receptors in 8- and 18-day-old membranes from chicken embryo hearts(top) and livers (bottom). Membranes were incubated with 125l-labeled IGF-I (left) or 125l-insulin {right) without or with a range of concentrations ofunlabeled IGF-I ( • ) , IGF-ll/multiplication-stimulating activity peak III-2 (IGF-II/MSA) (O), and insulin (A). Highly purified IGF I was used both astracer and competing peptide. To compete for binding of highly purified IGF-II/MSA, however, peptide used was IGF-II/MSA-CR (see Table 1legend). Binding is expressed as percentage of maximum binding obtained in absence of unlabeled peptides. Each point represents average oftriplicate samples in 1 study.


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TABLE 1Specificity of binding of insulin-like growth factor I, insulin-likegrowth factor ll/multiplication-stimulating activity, and insulin todifferent chicken embryo tissues

t25l-labeledpeptide

EDso

In heartIGF-IIGF-II/MSAInsulin

In liverIGF-I

IGF-II/MSA

Insulin

In brainIGF-I

IGF-II/MSA

Insulin

In adipose tissueIGF-IIGF-II/MSAInsulin

Age ofembryo(days)

8 and 188 and 188 and 18

8188

188

18

4 and 812 and 184 and 8

12 and 184

1218

*

Concentration of unlabeled peptide

IGF-I

157

250

6845

350100

2015656

1520

1015

150

(ng/ml)

IGF-II/MSA-CR

200100500

6015010075

350200

10010060

100755075

100150ND

Insulin

600500

5

200250200200

32

750100500100

545

150200

CM

The data correspond to 1 experiment for heart and adipose tissue,mean values of 2 experiments for liver, and 4 experiments for brain.Embryos of different ages were studied separately; where valueswere the same for both ages, only 1 value is given. In each exper-iment, binding was determined at least in duplicate tubes. IGF-I,insulin-like growth factor I; IGF-II/MSA, insulin-like growth factor l l /multiplication-stimulating activity peak III-2; IGF-II/MSA-CR, IGF-II/MSA from Collaborative Research (Lexington, MA) with activity peakII; ND, not determined.

*Adipose tissue from 3-wk-old chick was used because it is notreadily available in embryos. In all tissues, insulin (10 M-g/ml) com-peted for s 9 0 % of the 1Z5l-labeled IGF-II/MSA bound. With 2 se-lected membrane preparations from embryo liver and brain, we wereable to use highly purified IGF-II/MSA as the unlabeled competitorfor 125I-IGF-I binding (data not shown). IGF-II/MSA was 3- to 5-foldbetter as competitor than IGF-II/MSA-CR but still was only 5 0 - 6 0 %as potent as IGF-I. These data indicate that the affinity of IGF-II/MSA for the chicken embryo IGF receptor is greater (closer to theaffinity of IGF-I) than reflected in this table and in Fig. 2.

Affinity cross-linking of membrane receptors with la-beled peptides. Aliquots of membranes (150 |ig protein)were incubated with 0.3 ng of labeled peptides in HEPESbuffer plus 1% BSA and 0.5 mg/ml bacitracin under stan-dard conditions. Affinity labeling was performed in HEPESbuffer with 0.1 mM dissuccinimidyl suberate, pH 8.0, for 15min on ice according to a previously described procedure(27). After terminating the reaction with 100 mM Tris-HCIbuffer, samples were centrifuged and washed in HEPESbuffer, then formed into pellets and solubilized by boiling at100°C for 6 min in a solution of 125 mM Tris, 10% glycerol,0.002% bromphenol blue, and 2% SDS in the presence of100 mM dithiothreitol. Samples were electrophoresed in one-dimensional, discontinuous SDS-polyacrylamide gel slabsaccording to Laemmli (28). The dried gels were apposed toXAR-5 film (Eastman-Kodak, Rochester, NY) for 5-7 days togenerate autoradiograms.

RESULTSTissue-specific developmental regulation of insulin andIGF binding. We studied the specific binding of 125l-insulin,125I-IGF-I, and 125I-IGF-II/MSA to embryo heart, liver, and limbbuds from early organogenesis to late development (Fig. 1).In all tissues we studied, the developmental profile of insulinbinding was different from that of IGF binding. In contrast,125I-IGF-I and 125I-IGF-II/MSA binding were remarkably par-allel for a wide range of values. Binding of IGF-I was ap-proximately twofold greater than IGF-II/MSA binding in everytissue at every embryonic age. Similar results were evidentduring chick ontogeny in our initial analysis of brain receptors(21). The specificity studies confirmed that 125l-insulin boundto specific insulin receptors and that both 125I-IGF-I and125I-IGF-II/MSA bound to type I IGF receptors. In addition,all developmental changes in binding were mostly ac-counted for by changes in the number of receptors.

Interestingly, each tissue had characteristic develop-mental changes in the relationship of insulin and IGF binding(Fig. 1). In heart, binding of labeled IGFs to constant amountsof membrane protein was greatest at the earliest age studied(3 days), with a progressive decline to the lowest values atage 18 days, 3 days before hatching. In contrast, insulinbinding was low at age ^ 1 wk, increasing only slightly afterage 12 days. In growing limb buds, specific binding of la-beled IGFs was much higher than labeled insulin binding,but both peptides showed a moderate increase betweendays 4 and 8 of embryogenesis. In liver, in comparison with

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FIG. 3. Scatchard plots of t25l-labeled insulin-like growth factor I (IGF-I)and 125l-insulin binding (inset) to 8- ( • ) and 18-day-old ( • ) chickenembryo heart membranes. Each point represents mean of 3 replicatesamples in 1 experiment. Specific binding expressed as bound-to-freeratio (B/F) of labeled peptide is plotted as function of total hormonebound to receptor. Linear regression of IGF-I binding data wascalculated by least-squares. Scales for plots of both peptides wereidentical.


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Age ofembryo(days)

8188

18

IGF-I(x io-1 2mol /mg

membrane protein)

0.280.080.070.06

Insulin(xio~1 2 mol/mg

membrane protein)

0.020.040.100.05

TABLE 2Maximum binding capacity of chicken embryo tissues for insulin-like growth factor I and insulin

Tissue

Heart

Liver

*Only high-affinity sites were considered. The Scatchard plot wasanalyzed by a 2-site model.

heart and limb buds, specific binding of 125l-insulin washigher than labeled IGFs' binding at all stages of develop-ment; a slight decrease was observed with age with alltracers.Specificity of insulin and type I IGF receptors in earlyand late ontogeny. Traditional competition studies with 125I-insulin and 125I-IGF-I as tracers in a range of concentrationsof unlabeled insulin, IGF-I, and IGF-II/MSA were performed.Heart and liver membranes of representative stages of early(8-day) and late (18-day) development were chosen (Fig. 2).Binding of 125l-insulin to heart tissue was inhibited by unla-beled peptides with the rank of potencies characteristic ofan insulin receptor, i.e., insulin > IGF-I > IGF-II/MSA. Bind-ing of 125I-IGF-I to heart membrane was, on the other hand,competed for by IGF-I > IGF-II/MSA > insulin, as expectedfor a type I IGF receptor (Table 1). Further confirmation ofthe typical binding characteristics of the embryonic insulinand IGF receptors was obtained by Scatchard analysis ofthe competition curves (Fig. 3). Scatchard plots for IGF-Ibinding to heart membranes were typically linear, whereasthose for insulin binding were curvilinear. Similar specificityresults were obtained with liver membranes (Fig. 2). The totalnumber of high-affinity insulin receptors did not changemarkedly in heart or liver from early to late development. Thenumber of IGF receptors, in contrast, decreased markedlywith developmental age in heart (Fig. 3; Table 2).

In a limited series of competition experiments, we used125I-IGF-II/MSA as a tracer to study IGF receptors from heart

and liver. In all cases, unlabeled IGF-I was more potent thanIGF-II/MSA-CR in competing for labeled IGF-II/MSA bind-ing, and insulin had a significant affinity for such IGF recep-tors (Table 1). These results were consistent with data fromcompetition-binding studies in brain from our previous study,suggesting that both IGF-I and IGF-I I were binding to a type1 IGF receptor (21). They were also similar to the resultsobtained in chicken fibrobiasts (29). We think, however, thatthe low potency of IGF-II/MSA-CR competing for highly pur-ified IGF-II/MSA or IGF-I is in part due to the composition ofthis preparation (predominantly MSA-II). As we found in acontrol experiment on liver and brain membranes, MSA-III-2 may have only slightly lower affinity than IGF-I for the IGFreceptors of chicken embryo tissues. To pinpoint which typeof receptor was binding IGF-I I, a type I or type II receptor,we compared the structural characteristics of the IGF re-ceptors and the insulin receptors by cross-linking analysis.Structural analysis of IGF receptors. The two distinct fea-tures of a type II IGF receptor, as defined in mammals, arethe monomeric subunit structure {M, 250,000 under reducingconditions) and the lack of affinity for insulin. We could notfind a receptor with these characteristics with affinity cross-linking followed by gel electrophoresis in brain (Figs. 4 and5), heart, liver, and limb buds from embryos (Fig. 5) orin adipose tissue from adult chicken (Fig. 6). 125l-insulin,125I-IGF-I, and 125I-IGF-II/MSA were cross-linked to tissuemembranes. Despite the tissue and the radioligand used, aradioactive band appeared in the region expected for thebinding (a) subunit of either an insulin receptor or a type IIGF receptor (Mr -130,000). Other radioactive bands ofhigher molecular weight (-250,000 and >300,000) were ap-parent in all tissues with all ligands. Because all the molec-ular species in each gel lane (a-subunit and Iarger-Mr bands)were inhibited to the same extent by incubation of the mem-branes and tracers with the homologous unlabeled peptide,they probably represent the monomeric subunit and poly-meric aggregates of receptor subunits, respectively.

Brain membranes were cross-linked to 125I-IGF-II/MSAwithout or with a range of concentrations of unlabeled IGF-I or insulin. A band compatible with the a-subunit of the typeI IGF receptor was detected, and binding of labeled IGF-II/MSA was fully inhibited not only by IGF but also by unlabeledinsulin, albeit at a higher concentration (Fig. 4). This spec-

FIG. 4. Specificity of cross-linked insulin-likegrowth factor (IGF) and insulin receptors in6-day-old chicken embryo brain. Membranes (150jig protein) were incubated with 125l-labeled IGF-ll/multiplication-stimulating activity peak III-2 or12Sl-insulin (~0.3 ng) in presence of range ofconcentrations of unlabeled highly purified IGF-Iand insulin. Ligand-receptor complexes werecross-linked as described in MATERIALS ANDMETHODS. Samples were solubilized, reduced, andrun on discontinuous sodium dodecyl sulfate-polyacrylamide gel slab (running gel 5.5%).Left, autoradiograms generated by dried gel,with band {arrow) at position of monomerica-subunit of either type I IGF or insulin receptor.Each a-subunit band was cut from gels, andradioactivity was counted and represented (right)as percentage of maximum binding obtained inabsence of unlabeled hormones IGF-I ( • ) andinsulin (A). The radioactive bands of highmolecular weight in the autoradiograms showsame specificity for binding as correspondinga-subunits of receptors.

[125I] IGF-II [125I] Insulin

(ng/ml) 0 10 50 200 10 103 104 0 10 100

IGF-I Insulin

10 102 103

PEPTIDE CONCENTRATION (ng/ml)


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ificity corresponds again to a type I IGF receptor. When 1 2 5 I-insulin was cross-linked to brain membranes, the apparentreceptor affinity derived from the quantitation of the radio-activity corresponding to the a-subunit (Fig. 4) was consis-tent with what we had obtained by the traditional bindingstudies shown in Fig. 2.

All the tissues from young embryos studied (heart, limbbuds, and liver) repeated the electrophoretic pattern ob-

["•n INSULIN ["HIIGF-I [1MI]IGF-II

Mr x 10"3

B

205 —

a -»116 —

94 —

205 —

116 —

94 —

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— 205

— 116

205 —

116 —

205 —

a —116 —

94 —

INSULIN - + - - +IGF(I + II) - - - + - + -

FIG. 5. Affinity labeling of chicken embryo receptors from brain (4)and limb buds (B) (6-day-old) and heart (C) and liver (D) (8-day-old).125l-labeled insulin, 12Sl-insulin-like growth factor I (125I-IGF-I), and125l-IGF-ll/multiplication-stimulating activity peak III-2 (IGF-II/MSA) wereincubated with 150 fig of membrane protein without or with 10 jxg/mlunlabeled insulin or 5 fig/ml IGF (I and II). Samples were then cross-linked, solubilized, and electrophoresed on sodium dodecyl sulfate-polyacrylamide gel slab under reducing conditions. Autoradiogramsgenerated by dried gels are shown with positions of a-subunits ofinsulin and type I IGF receptors (arrows). Gel slabs (7.5%) were usedin every case except when 125I-IGF-II/MSA was cross-linked to liver; inthat study, we used 5.5% gel to enhance detail of radioactive bands ofhigh molecular weight, thereby enhancing detection of possible type IIIGF receptors.

[125I]IGF-I [125I]IGF-II [125I] Insulin

Mr x 10" 3

205 —

116 —

9 4 —

66

f

4 5 —

IGF (l + ll)Insulin

FIG. 6. Affinity labeling of insulin-like growth factor (IGF) and insulinreceptors on adult chicken adipose tissue. Membranes were incubatedwith 125Mabeled peptides. Unlabeled insulin (10 ng/ml) or partially pureIGF (I and II) (5 |ig/ml) was added to incubation mixture, and sampleswere further processed as described for Fig. 5.

tained with brain membranes* (Fig. 5); 125I-IGF-II/MSA wascross-linked to molecular species of the same electropho-retic mobility as IGF-I binding subunit and was fully inhibitedby IGF and insulin. The absence of type II IGF receptors inmultiple chicken embryo tissues, particularly liver, was in-triguing. Because adipocytes are rich in type II IGF receptorsin mammals (30), we chose to study membranes from adultchicken adipose tissue and again found results identical tothose of the embryonic tissues; 125I-IGF-I and 125I-IGF-II/MSAwere cross-linked to an ~130,000-/Wr band with the speci-ficity of the type I IGF receptor (Fig. 6).

DISCUSSIONSpecific insulin receptors and type I IGF receptors are pres-ent in several chicken embryo tissues from early stages oforganogenesis. The specificity studies done with traditionalbinding techniques as well as with quantitation of the cross-

*We have reported elsewhere (Endocrinology 121:1468, 1987) that themonomeric a-subunits of both insulin and IGF-I receptors of chick embryobrain, muscle, and liver differ slightly (<10%) from tissue to tissue in apparentmolecular weight and sensitivity to neuraminidase. In this discussion, we haveignored these differences.


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L. BASSAS AND ASSOCIATES

linked a-subunits show that insulin binds to typical insulinreceptors, whereas both IGF-I and IGF-II/MSA bind to typeI IGF receptors. Our data show that these two populationsof receptors are independently and distinctly regulatedthroughout embryonic development in each tissue. The typeI IGF receptors clearly dominated over insulin receptors dur-ing early organogenesis of heart and limb buds, whereasinsulin binding was higher in liver.

There seems to be some chronological correlation be-tween the overall rate of cell growth in developing heart andthe number of IGF receptors (31). In brain tissue and in theeye lens, such a correlation may also exist (21,32,33). Al-though no causal relationship has been established, wespeculate that the mitotic rate of developing tissues may inpart be regulated by changes in the number of receptorsfor IGFs, which modulate the peripheral response to localconcentrations of IGFs.

Many aspects of growth regulation probably depend onautocrine or paracrine mechanisms (34). Local concentra-tions of IGF-I are increased in cells from rat tissues undergo-ing active growth or regeneration (35,36). IGFs are synthe-sized in multiple fetal tissues (9,37), but detailed quantitationthroughout development has not been done. It is possiblethat the expression of IGFs and their receptors may be in-creased in parallel in tissues undergoing growth and differ-entiation. Because we know that insulin receptors are func-tional at postneurulation stages (between days 2 and 5 ofdevelopment) and growth is retarded when insulin action isblocked (38), it is possible that a complex regulation of bothinsulin and IGF actions is required for normal early embryo-genesis.

Several factors other than a change in the receptors couldartifactually influence quantitation of IGF binding to devel-oping tissues, and we have excluded a few of them. Bindingaffinities did not change during development in brain or heart(or eye lens; unpublished observations). The relative yieldin the preparation of membranes was not significantly af-fected throughout development. We did not detect carrier-binding proteins in the tissue homogenates of chicken em-bryos when we tested the supernatants of the membranepreparations either by binding analysis or polyacrylamidegel electrophoresis. In fact, IGF-carrier proteins have notbeen found in chicken serum (39). On the other hand, spe-cies differences between ligand and receptor may have af-fected the results, and developmental changes in membraneand tissue properties (i.e., the membrane preparations maynot be exactly comparable due to physical changes in theplasma membrane at different stages) could have biasedthe data. However, continuous comparison of IGF and insulinbinding minimizes the overall influence of these variables.

We have been unable to identify typical type II IGF re-ceptors in chicken embryo tissues early or late in develop-ment. These receptors probably would not appear duringpostnatal life, because they are absent from liver 3 days afterchicks hatch as well as from fat tissue in adult chickens. Inmammals, both liver- and fat-tissue cells have type II IGFreceptors (30). This absence of IGF-I I receptors could becharacteristic of the tissues from the chicken species, andthe absence of type II IGF receptors in chicken embryofibroblasts is consistent with this notion (40). More extensivestudies are necessary to determine whether type II IGF re-

ceptors are present in any avian or other nonmammalianspecies or whether they are exclusive to mammals. Althoughpeptides with remarkable resemblance to human IGF-I seemto be the largest component of the IGF produced by chickenembryo tissues (11,12), a peptide with the characteristics ofIGF-I I, distinct from IGF-I, has been detected by radiore-ceptor and protein binding assays in adult chicken serum(39). IGF-II has been a preferred candidate in fetal growthin mammals (41,42). The type of receptor that mediates theeffects of IGF-II is not clear even in mammals. Althoughseveral reports indicate that IGF-II mediates many of its ef-fects through the type I IGF receptors (43,44), recent studiesappear to indicate that there are effects unique to IGF-II,probably mediated through a type II IGF receptor (45,46).It is possible that the type II IGF receptor may be unique tomammals.

ACKNOWLEDGMENTSWe thank Drs. Rene E. Humbel (Biochemisches Institut derUniversitat, Zurich, Switzerland) and Matthew M. Rechler(NIH, Bethesda, MD) for their kind gifts of IGF-I and IGF-II/MSA, respectively. The expert secretarial assistance ofSheila Ogoh and Esther Bergman is gratefully acknowl-edged.

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human serum: chemical and biological characterization and aspects oftheir possible physiological role. Curr Top Cell Regul 19:257-309, 1981

2. Milstone LM, Piatigorsky J: S-Crystallin gene expression in embryonicchick lens epithelia cultured in the presence of insulin. Exp Cell Res105:9-14, 1977

3. Kato Y, Nasu N, Tasake T: A serum-free medium supplemented withmultiplication stimulating activity supports both proliferation and differ-entiation of chrondrocytes in primary culture. Exp Cell Res 125:167-74,1980

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5. Schmid CH, Steiner TH, Froesch ER: Preferential enhancement of myo-blast differentiation by IGF-I and II in primary cultures of chick embryoniccells. FEBSLett 116:117-21, 1983

6. Puro DG, Agardh E: Insulin-mediated regulation of neuronal maturation.Science 225:1170-72, 1984

7. Ewton DZ, Florini JR: Effects of the somatomedins and insulin on myoblastdifferentiation in vitro. Dev Biol 86:31-39, 1981

8. Chapman AB, Knight DM, Dieckmann BS, Ringold GM: Analysis of geneexpression during differentiation of adipogenic cells in culture and hor-mone control of the developmental program. J Biol Chem 259:15548-55, 1984

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Developmental Regulation of Insulin and Type I Insulin - Diabetes