insulin in insects and annelids

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Insulin in Insects and AnnelidsDEREK LEROITH, MAXINE A. LESNIAK, AND JESSE ROTH

SUMMARYThe fruitfly, Drosophila melanogaster and the earth-worm, Annelida oligocheta were extracted with acid-ethanol by a classic method for recovering insulinfrom the pancreas. When each extract was filtered ona Sephadex G-50 column, a distinct peak of insulin im-munoreactivity (equivalent to 0.1 to 2 ng of insulin/gwet weight) was recovered in the region typical of in-sulin. The material in this peak had reactivity in the in-sulin bioassay, measuring stimulation of glucose oxi-dation or lipogenesis by isolated rat adipocytes. Thebioactivity was partially or largely neutralized by anti-insulin antibodies. In concordance with previous workshowing the presence of material very similar to insu-lin in the blowfly and molluscs, we have confirmed thepresence of insulin in insects and extended the obser-vation to earthworms. These findings suggest that in-sulin is more widespread in invertebrates than waspreviously thought. In a companion study (Proc. Natl.Acad. Sci. USA 77:6184-88,1980), we have demon-strated material similar to insulin in unicellular or-ganisms. DIABETES 30:70-76, January 1981.

T

hough insulin is clas sical ly sy nthesized in the /3-cells of the pancreas, recent reports have de-scribed material that is very similar to insulin ingut-derived cells of primitive vertebrates and com -

plex invertebrates, and in neural elements of mammals andinsects. 1 10

In the present study, we confirm the presence of materialthat is very similar to insulin in drosophila heads. In addi -tion, we find similar m aterial in extracts of drosophila bodiesand in extracts of both the skin and internal structures of theearthworm. These findings suggest that insulin is much

Diabetes Branch, NIAMDD, National Institutes of Health, Bethesda, Maryland20205.Address reprint requests to Derek LeRoith, Diabetes Branch, NIAMDD, Build-ing 10, Room 8S-243, National Institutes of Health, Bethesda, Maryland20205.Received for publication 21 August 1980.

more widespread among the tissues of invertebrates thanwas previously thought, and not only in gut- or nervous sys-tem-derived ce lls. In addition, we have also shown, in a sep-arate study, the presence of similar material in unicellularorganisms, the ciliated protozoan Tetrahymena pyriformisand the unicellular fungus Neurospora crassa.

MATERIALS AND METHODSLarvae of the fruitfly, Drosophila m elanogaster, were main-tained at room temperature on medium 4-24 (Carolina Bio-logical Supply Company), which contained yeast, flour, su-crose, m inerals, and vitamins. Adult flies were anesthetizedwith ether, the heads separated from the bodies, and storedat -70C until extraction. The interior organs ( inside ) ofadult earthworms, Annelida oligocheta (Carolina BiologicalSupply Co.), were dissected out from the skin ( outside )and both were stored at - 70C.Extraction of insulin. A standard extraction p rocedure wasused 12 as previously described, 9-13 except that no albuminwas added at any stage. The organisms were weighed andhomogenized with a Brinkman po lytron in 10 vol of ice-c oldacid-ethanol (0.2 N H CI, 75% ethanol). The suspension wasmixed o vernight at 4C. After centrifu gation at 1500 x g for20 min at 4C, the precipitate was disca rded , and the super-natant was concentrated by air evaporation at room tem-perature, resuspended in 5 vol of 0.05 M (NH4)2CO 3, andneutralized with concentrated NH

4OH. After centrifugation

at 1500 x g for 20 min at 4C, the precipitate was dis-carded, and the supernatant was lyophilized and reconsti-tuted with distilled water.

The reconstituted extract was applied to a column of Se-phadex G-50 (medium) and eluted with 0.05 M (NH4)2CO 3.Each effluent fraction was lyo philized and reconstituted to 1ml with distilled water. The insulin content of each fractionwas determined by radioimmunoassay at 1:10 (final di lu-tion) of the sample. The fractions corres ponding to the peakof insulin immunoactivity were pooled, lyophilized, and re-constituted with distilled water. The reconstituted pool wasthen tested for bioactivity and reassayed for immunoreac-tivity.

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DEREK LEROITH, MAXINE A. LESNIAK, AND JESSE ROTH

Insulin radioimmunoassay. Porcine insulin was pur-chased from Eli Lilly; rat insulin was a gift of Dr. R. E.Chance of Lilly Research Laboratories. Guinea pig anti-por-cine insulin sera, designated 68 (gift of Dr. A. Kagan) and619 (purchased from the Department of Pharmacology, Indi-ana University, Indianapolis), were both used in the radioim-munoassy, whereas only 619 was used to neutralize the in-sulin effect in the bioassays. The insulin radioimmunoassaywas performed by standard m ethods using 12 5l-labeled por-cine insulin as the tracer and rabbit anti-guinea pig serum asthe second antibody. 14-15 Both antisera, 619 and 68, gaveidentical results in the radioimmunoassays when tested withthe various extracts.Controls for RIA. To exclude the possibility that the ap-parent reactivity of the gel-filtered extracts in the immunoas-say could be due to interference in the assay (e.g., degrada-tion of 12 5l-insulin or inhibition of the immunoprecipitation),two types of experiments were performed. In the first, at theend of 4 days of incuba tion the precipitates were separatedfrom the supematants, and the supernatants were added toTCA (5% final concentration); at least 92% of the radioactiv-

ity was precipitated in both the extracts and in the control(extract-free) samples. In the second , duplicate samples ofextract (or of control) were incubated in the radioimmuno-assay in the usual way. After 72 h, an excess (1:1000) ofanti-insulin antibody was added to one sample of eachextract and each control. Twelve hours later, the goat anti-guinea pig antibody was added to both samples to precipi-tate the antibody-bound insulin in the usual way. With ex-cess antibody, more than 90% of the labeled insulin wasprecipitated in both the extract and control samples.Insulin bioassays. Biologic activities of extracts were mea-sured either as glucose oxidation, i.e., conversion of [U-14C]glucose to 14 CO 2l16 or lipogenesis, i.e., incorporation of[3 -3H] glucose into toluene-extractable lipids17 by isolatedfat cells prepared from epididymal fat pads of youngSprague-Dawley rats. To show that the bioactive moleculeswere reactive with anti-insulin antibody, duplicate aliquotswere mixed with a 1:100 dilution of guinea pig anti-porcineinsulin antibody 619 or normal guinea pig serum beforetheir addition to the bioassay. Normal guinea pig serum atthis concentration had no effect on the bioassay.

RESULTSDrosophila. When acid-ethanol extracts of drosophila weregel filtered on Sephadex G-50, insulin immunoreactivitywas recovered in a discrete peak in the region typical of thatfor mammalian insulins (Figure 1A, Table 1) and was equiv-

alent to a few ng of insulin/g wet weight. The effluent frac-tions that corresponded to the insulin peak, when dilute d inthe radioimmunoassay, gave results very similar to themammalian insulins (Figure 1B). Results obtained with iso-lated heads were about twice those obtained with the restof the drosophila (labeled bod y in Table 1). Aliquots of thegel-filtered material, when tested with isolated rat adipo-cytes, stimulated glucose oxidation as well as lipogenesis,two classical responses to insulin (Figure 1C, Table 1). Themagnitude of the biologic effect of the extract was equiva-lent to its content of immunoreactive insulin, and the bio-logic effect was largely neutralized by the addition of anti-insulin antibody to the bioassay system.Earthworm. When acid-ethanol extracts of earthworm were

TABLE 1Insulin extracts from

Batch1. Whole2. Whole3. Whole4. Head

Body

Wetweight

(g)4.02.72.00.030.15

Drosophila melanogaster

Insulin (ng)from G-50

Columnfrac-tions

(RIA)10.513.2


3/7

20 25 30 35 40 45

FRACTION NUMBER

B30

25

20

15

10

Drosophila

Rat Insulin

Pork Insulin

i i 1

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8INSULIN (ng/ml)

x DROSOPHILA-HEAD (MD100 200

50 100 150 200DROSOPHILA-BODY M>


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DEREK L E R O I T H MAXINE A. LESNIAK, AND JESSE ROTH

1.50

1.25

1.00

0 75

0.50

0.25

80

-, 0.31

0.38

0.50

0.57

0.68

0.8540 60 80 100 120 140

FRACTION NUMBER

160 180

100

75

50

25

o 100

No Insulin

50

25 -

0 L

- No Insulin

0.1 0.2 0.5 1.0 2.0-^PORK INSULIN STANDARD (ng/ml)

0.5 1.0 2.0EARTHWORM EXTRACTng/ml RIA INSULIN (Pork Standard)

A OUTSIDE INSIDE

fot l

o

oCO

zQZCO

z_ J

05

r

5

60

40

20

0

_ \

\

\ \\xV\ \\ \

PORK INSULIN

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7INSULIN (ng/ml)

X EARTHWORM EXTRACT100 200

FIGURE 2. Earthworm extracts.

A. Gel filtration. Batch 3 in Table 2 was filtered on a Sephadex G -50column (5 x 90 cm; 10 ml per fraction). Each fraction was lyophlllzedand reconstituted In 1 ml of distilled water, and the Immunoreactlveinsulin was measured at a 1:10 final dilution as described In M E T H O D Sand Figure 1 legend.

B. Immunoreactlvity. Batch 4 In Table 2 was gel filtered; the peakfractions were pooled and assayed as described In the legend toFigure 1B. By adjusting the horizontal axis, the earthworm extractscan be shown to fit the pork Insulin curve about as well as thedrosophlla extracts (Figure 1B).

C. Bioactivlty. Extracts of inside ( ) and outside (A A)were compared with pork Insulin ( ) In the bloassay. As in Figure1C the Insulin concentration of the extract represents theImmunoreactlvity of the reconstituted pool measured in the RIAagainst the pork Insulin standard. In addition, the bioactivlty of threesamples were also determined In the presence of anti-Insulinantibody, and the degree of neutralization is indicated by thearrowheads.

magnitude of the effect was appropriate for the amount ofimmunoreactivity in the preparations (Figure 2C). Anti-insu-lin antibodies reduced the bioactivity substantially (Figure2C Table 2) but not as effectively as with drosophila ex-tracts (see DISCUSSION). W hen the skin ( outside ) wasseparated from the rest of the body ( inside ), there ap -peared to be somewhat more insulin in the skin.

DISCUSSIONThe specificity of the insulin radioimmunoassay has beencharacterized very extensively. To react in the assay, a nec-

essary (but not sufficient) condition is the presence of bothA- and B-chain joined by disulphide bridges; isolated A-and B-chains (sulphonated or carboxymethylated) evenfrom homologous species, alone or together, are nonreac-tive.18 20 Even molecules that contain the overall structure ofinsulin and that can react with insulin receptors to producethe characteristic biologic effects of insulin in vivo and invitro may be nonreactive in the radioimmunoassay; insulin-like growth factors I and II, whose A- and B-chain regionshave 50% of their amino acids in common with pork insulin,are totally nonreactive in this RIA, and guinea pig insulin,

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INSULIN IN INSECTS AND ANNELIDS

which has 65% homology with pork insulin, has trivial reac-tivity.21 Even insulins with biopotencies that are nearly thesame as pork insulin may be poorly reactive in the immun-oassay, e.g., fish insulins and avian insulins with about 70%and 90% homology, respectively, are much less reactive inthe pork insulin radioimmunoassay than in the bioassay forinsulin (Figure 3). No material has been found that reactsspecifically in this assay that is not very closely relatedstructurally to intact insulin.

That our material reacts specifically (experiments to ex-clude nonspecific effects are described in MATERIALS ANDMETHODS) in the radioimmunoassay for pork insulin sug-gests that it is very similar to pork insulin or proinsulin. Thatthe immunoreactivity is recovered in a peak in the sameplace as insulin suggests that its size-shape is very similarand unlikely to be proinsulin or proinsulin-like.

As suggested earlier in this DISCUSSION, the regions ofthe insulin molecule responsible for bioactivity are distinctfrom those required for immunoreactivity. That our materialis reactive in the bioassay suggests that it contains the spe-

1000 F

Chicken

B ony Fish

Guinea P ig

IGF I & II1.0 L Somatomed ins A +

: M SA

H a g f i s h ' *

Mouse

Neurospora

* Ear thworm ( I I

* .Drosoph ila Split Proinsulin

A Ear thworm (0)* Te t r a h y m e n a

Proinsulin

0.1 1.0 10 100 1000

IMMUNO REACTIVITY (% Relative to Pork Insulin)

FIGURE 3. The bioactivity (relative to pork Insulin) of Insulins andInsulin-related peptldes are plotted as a function of theirImmuno reactivity (relative to pork Insulin) In the pork insulinradioimmunoassay. The heavy diagonal line representsbioactlvlty/lmmunoreactlvlty equal to one. The upper dashed linerepresents blo/lmmuno equal to 3 and the lower dashed line

represents blo/lmmuno equal to V>. NGF and relaxln, despite theirstructural similarities to Insulin, are unreactive In both assay systems.Th e IGFs, somatomedlns, and MSA have Insulin bioactivity but aretotally unreactive In this Immunoassay. Note that all the naturallyoccurring Insulins have bio/lmmuno > 1, while pork proinsulin, splitproinsulin, and probably other Intermediates have blo/immuno < 1.The extracts that we have studied are designated by (*). These pointsshould more properly be drawn as lines parallel to the otherdiagonals through the designated points, because, In contrast to theother materials, we have only a ratio of the two activities with nomeasure of molar amounts for them. The extracts of the Inside andoutside of the earthworm are designated (I) and (0), respectively. Thedata for Tetrahymena and N eurospora are from ref. 11 . Note that wemay be overestimating the blo/lmmuno ratios of our extracts becausewe overestimate the bioactivity by using the total bioassay,uncorrected for the non-neutralized fraction, and because we areprobably underestimating the immunoreactivity by using values readdirectly from the pork Insulin standard curve.

cific regions needed for bioactivity, i.e., regions on the insu-lin molecule that are needed for bin ding to receptor ( affin-ity ) and regions required for activa tion of the target cell( intrinsic activity ). That anti-insulin antibody neutralizesthe bioactivity suggests that the immunoreactive site andbioactive site(s) are on the same molecular species.

There are substances other than insulins that can pro-duce responses in the insulin bioassays (proteases, oxidiz-ing agents, polyamines, heavy metals, etc.) but none ofthese co-migrate with insulin on Sephadex G-50, none reactspecifica lly in the pork insulin radioimmu noassay, and anti-insulin antibodies do not affect their bioactivity. Thus, wesuggest that the material in the extracts is very similar topork insulin.

In addition to the intestinal tract, insulin is present inmammalian brain cells and peripheral nerves, and there isevidence to indicate that at least some of this insulin isbeing produced locally by neural elements.9-10 Becauseboth insulin and specific receptors for insulin are widelydistributed throughout brain, and because peripheral nervestimulation seems to cause local release of insulin, 10-22 there

is the possibility that insulin in neural cells may be acting asa neurotransmitter of neuromodulator.

A popular hypothesis that the pep tide hormones of the in-testinal tract originate phylogenetically and ontogeneticallyfrom nervous tissue is supported by the finding that extirpa-tion of a particular nucleus of the brain of the blowfly mark-edly disturbed carbohydrate metabolism in this insect, thatextracts of the head contained material very similar to mam-malian insulins, and injection of the extracted material cor-rected the carbohydrate disturbance. 6 The authors suggestthat these neurosecretory cells of blowfly brain serve in thesame way as the /3-cells of the vertebrate pancreas.

We have raised the possibility that, in addition to gut-

derived and nerve-related cells, most cells make some in-sulin.13 This is based on the finding that many cells in mam-mals have insulin at concentrations similar to those found inbrain and several-fold higher than in plasma, and that theconcentration of insulin in these cells, as in brain, changedlittle or not at all with extreme changes in levels of plasmahormone.13-23 We also found insulin at similar concentrationsin cells grown in tissue culture, and the level of cellular in-sulin was not affected by depletion or supplementation of in-sulin in the medium in which the cells were grown.

The suggestion that many (if not all) cells make some in-sulin is supported by studies in guinea pigs in which we de-tected two distinct insulins.24 Typical guinea pig insulinwas present in very large amounts in the pancreas and wasthe only type of insulin detected in the blood. The secondinsulin, which very closely resembled pork insulin but wasquite different from guinea pig type-insulin, was present inbrain and in a wide range of extrapancreatic cell types in-cluding many unrelated to nerve or intestinal tract.

In the present study, we confirm that insects contain ma-terial that is very similar to mammalian insulin. Further, weshow similar material in annelids, which extends the distri-bution of insulin to another class of invertebrates. The con-centration of insulin (ng immunoreactive/g wet weight) thatwe found in heads of drosophila appears to us to be verysimilar to that reported for heads of blowfly.6 That the bodiesof the drosophila had only moderately less insulin than

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DEREK LEROITH. MAXINE A. LESNIAK, AND JESSE ROTH

the heads suggests that insulin in insects is not restricted toa single nucleus of the nervous system nor to the nervoussystem in general. Likewise, that the inside and outside ofthe worm had similar amounts of the material suggests,again, a very broad distribution of insulin rather thana distribution restricted to one region of brain or gut. (Thisconclusion assumes that the efficiency of recovery is similarfor the different parts, an assumption that is not yet proven.)Our earlier suggestion from previous data, and here fromthe flies and the worms, that many cell types (other than gutand nerve) make insulin is supported by our finding of ma-terial very similar to insulin in unicellular undifferentiatedeukaryotes such as Tetrahymena pyriformis, a ciliated pro-tozoa, and Neurospora crassa, a fungus. 11

The equivalence between the immunoreactivity andbioactivity of the drosophila extracts, when related to a porkinsulin standard, was surprising to us. Insulins isolated fromthe beta cells of most vertebrates typically have ratios ofbiologic to immunologic activities that are in excess of oneand vary over a range of several hundredfold (Figure 3). Theimmunologic reactivity of some nonmammalian vertebrate

insulins is orders of magnitude less than that of the mamma-lian insulins. Thus, our extract of material from drosophila iscloser to mammalian insulin than are insulins from orga-nisms that are closer phylogen etically to the mam mals (e.g.,fish and birds). A recent study from our laboratory, in whichextracts from guinea pig tissues were used, suggests thatthe evolution of insulin may in some cases be nonallelic.24

This may shed light on the discrepancy between the immun-ologic to biologic activity.*

The insulin bioactivity prepared from extracts of worms isonly partially neutralized by anti-insulin antibody. This sug-gests that, in addition to insulin, the worm extract containedinsulin-like growth factors analogous to the insulin-likegrowth factors and somatomedins described in mammaliansystems (or contained substances that stimulated the ratadipocyte to release insulin-like factors). Several well char-acterized insulin-like growth factors (somatomedins A andC, insulin-like growth factors I and II, and MS A mu ltiplica-tion stimulating activity) interact with insulin receptors toproduce insulin-like effects, but are not reactive with anti-in-sulin antibodies in the radioimmunoassay, and their bio-activity is unaffected by anti-insulin antibodies though theyare extremely similar to insulin in many aspects of theirstructure. Support for this speculation is provided by our ob-servation that effluent fractions closer to the void volume onthe Sephadex G-50 column (in the region typical of the insu-lin-like growth factor) show an even greater enrichment of

bioactivity relative to immunoreactivity than fractions in theinsulin region. Interestingly, other investigators have foundthat worms contain material that stimulates growth in hypo-

* Based on observations that a typical mammalian-type insulin is present inall invertebrates and most mammals; that the guinea pig has a typical mam-malian-type insulin in addition to its very unusual pancreatic insulin;25 an dthat many vertebrates have pancreatic insulins that differ from typical mam-malian insulin, we propose that the gene for typical mammalian-type insulinarose very early and has been very well conserved throughout invertebrateand vertebrate evolution. By analogy to the guinea pig, we speculate that allspecies in which the pancreatic insulin differs widely from typical mamma-lian insulins are expressing a different allele in their B-cells and also possessgenes for typical m amm alian insulins that they may or may not express in ex-trapancreatic tissues.

physectomized rats and material that reacts with growthhormone receptors in vitro.25*26

ACKNOWLEDGMENTSThe authors thank Ann Reed, Scott McFarlane, Andrew Fei-gin, Yaakov Glick, and Avi Ganan for performing dissec-tions and extractions early in the course of this work. Wewish to thank the Washington D. C , affiliate of the Am ericanDiabetes Association for supporting the above people. Wealso thank Bernice Samuels for the radioimmunoassays ofinsulin, Kathleen L. Baird and Lisa H. Underhill for adviceon the bioassays, and Pierre De Meyts, David M. Neville, Jr.,and Michele Muggeo for helpful discussions.

This paper is dedicated to Dr. Rachmiel Levine on theoccasion of his 70th birthday.

REFERENCES1 Falkmer, S., Emdin, S., Havu, N., Lundgren, G.. Marques, M., Ost-

berg, Y., Steiner, D. F., and Thomas, N. W.: Insulin in invertebrates and cy-clostomes. Am. Zool. 7 3:625-38, 1973.

2 Emdin, S. O., and Falkmer, S.: Physiology of insulin. Acta Paediatr.Scand. (Suppl.) 270:15-25, 1978.

3 Tager, H. S., Markese, J., Kramer, K. J., Spiers, R. D., and Childs, C.

N.: Glucagon like and insulin-like hormones of the insect neurosecretory sys-tem. Biochem. J. 756:515-20, 1976.4 P lisetskaya, E., Kazakov, V. K., Solititakays, L, and L eibson, L G.: In-

sulin producing cells in the gut of freshwater bivalve molluscs Anodonta cyg-nea a nd U nio pictorum and the role of insulin in the regulation of their carbo-hydrate me tabolism. Gen. Comp. Endo crinol. 35:1 33-4 5, 1978.

5 Meneses, P., and Ortiz, M. A.: A protein extract from Drosophilamelanogaster with insulin-like activity. Comp. Biochem. Physiol. 51AAS3-85 , 1975.

* D uve, H., Thorpe, A., and Lazarus, N. R.: Isolation of material displa y-ing insulin-like immunological biological activity from the brain of the blowflyCalliphera venitoria. Biochem. J. 784:221-27, 1979.

7 Fritsch, H. A. R., Van Noorde n, S., and Pearse, A. G. E.: Cytoch emicaland immunofluorescence investigations of insulin-like producing cells in theintestine of myfilus Edulis L (Bivalvia). Cell Tissue Res. 765:365 -69, 1976.

8 Davidson, J. K., Falkmer, S., Mehrotra, B. K., and Wilson, S.: Insulinassays and light microscopical studies of digestive organs in P rotostomianan d D euterostomian species and in coelenterates. Gen. Comp. Endocrinol.77:388-401, 1971.

9 Havrankova, H., Schmech el, D., Roth, J., and B rowstein, M. J.: Identi-fication of insulin in rat brain. Proc. Natl. Acad. Sci. USA 75:5737-41, 1978.

10 Uvnas, B., and Uvnas-W allenstein, K.: Insulinergic nerves to theskeletal muscles of the cat. Acta Physiol. Scand. 703:346-48, 1978.

11 Le Roith, D., Shiloach , J., Roth, J., and Lesniak, M. A. Evolutionaryorigins of vertebrate hormones: substances very similar to mammalian insu-lins are native to unicellular eu karyotes. Proc. Natl. Acad. Sci. USA 77:6184-88 , 1980.

12 Mirsky, I. A.: Insulin: purification and biochemical characterization.In Methods in Investigative and Diagnostic Endocrinology. Berson, S. A., Ed.New York, Elsevier, 1973, pp. 823-883.

13 Rosen zweig, J. L, Havrankova, J., Lesniak, M. A., Brownstein, M. J.,and Roth, J.: Insulin is ubiquitous in extrapancreatic tissues in rats andhumans. Proc. Natl. Acad. Sci. USA 77:572-76, 1980.

14 Yalow, R. S., and Berson, S. A.: Immunoassay of end ogeno usplasma insulin in man. J. Clin. Invest. 39:1157-7 5, 1960.

15 Gorde n, P., and He ndricks, C. M.: The measurement of plasma insu-lin and proinsulin like components. In New Techniques in Tumor Localization

and Ra dioimmunoassay. Croll, M. N., Brady, L. W., Honda, T., and Wa llner, R.J., Eds. New York, John Wiley and Sons, 1974, pp. 17-24.18 Rodbell, M.: Metabolism of fat cells. 1. Effects of hormones on glu-

cose metabolism and lipolysis. J. Biol. Chem. 239:375 -80, 1964.17 Moody, A. J., Stan, M. A., Stan, M., and Gliemann, J.: A simple free

fat cell bioassay for insulin. Horm. Metab. Res. 6:12-16, 1974.18 Freychet, P., Roth, J., and Neville, D. M., Jr.:.Insulin receptors in the

liver: specific binding of [125 l]insulin to the plasma membrane and its relationto insulin bioactivity. Proc. Natl. Acad. Sci. USA 68:1833-37, 1971.

19 Blundell, T., Dodson, G., Hodgkin, D., and Mercola, D.: Insulin: thestructure in the crystal and its reflection in chemistry and biology. In A d-vances in Protein Chemistry, Vol. 26. Anfinsen, C. B., Edsall, J. T.. and Rich-ards, F. M., Eds. New York Academic Press, 1972, pp. 280-402.

20 De Meyts, P., Van Obberghen, E., Roth, J., Wollmer, A., and Bran-denburg, D.: Mapping of the residues in the receptor binding region of insulinresponsible for the negative cooperativity. Nature 273:504-09, 1978.

21 Jukes, T. H.: Dr. Best, insulin, and molecular evolution. Can. J. Bio-chem. 57:455-58, 1979.

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22 Havrankova, J ., Roth, J . , and Brownstein, M .: Insulin r eceptors a rewidely d is t r ibuted in the central nervous system of the rat . Nature 272 :82 7-29 , 1978.

23 Havrankova, J . , Brownstein, M . J. , and Roth, J.: Concentrations of i n-sulin and of insulin receptors in the brain a r e independent of peripheral insu-lin levels. J . Cl i a Inves t. 64:636-42 , 1 9 7 9 .

24 Rosenzweig, J. L, Lesniak, M. A., Samuels, B. E., Yip, C. C, Zimmer-man, A. E., and Roth, J.: Evidence in the guinea p ig for a second (mamm a-

lian-like) insulin native to extrapancreatic tissues. Clin. Res. 28:557A, 1980.Abstract.

25 Tsushima, T., Friesen, H. G., Chang , T. W., and Raben, M. S.: Iden tifi-cation of sparganum growth factor by a radioreceptor assay for growth hor-mone. Biochem. Biophys. Res. Commun. 59:1062-68, 1974.

26 Steelman, S. L, Glitzer, M. S., Ostlind, D. A., and Mue ller, J. F.: Bio-logical properties of the growth hormone like factor from the plerocercoid ofspirometra manonoides. Rec. Prog. Horm. Res. 27:97-120, 1972.

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insulin in insects and annelids

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