Mammalian pancreatic islet tissue in organ culture

Experimetffal ('ell Research 41, 365 375 (1066)

3~5

MAMMALIAN PANCREATIC ISLET TISSUE IN ORGAN C U L T U R E II. INSt;I~IN ( ] O N T E N T S O F T I S S U E S AN1) CIII,TIH/E MEI)IUM I, R. M U R R E L L ,

C. R. M O R G A N and A. L A Z A R O W

Department of Anatomy, University of Minnesota, Minneapolis, Minn. 55455, U.S.A.

Received June 30, 1965 TmxT mammalian p a n c r e a s is e a l m h l e o f s u r v i v a l a n d d i f f e r e n t i a t i o n in o r g a n c u l t u r e h a s b e e n k n o w n s i n c e 1954 [3]. R e c e n t d a t a f r o m this l a b o r a t o r y [14] i n d i c a t e t h a i f e t a l r a t p a n c r e a s , a s j u d g e d b y m o r p h o l o g i c e r i t e r i a , undergoes specilie endocrine cyto-differenliation under certain culture conditions. The i)rescnt paper eonsidcrs selected aspects of insulin production in o r g a n c u l t u r e s ()f f e t a l r a t p a n e r e a t i e islets. A r e v i e w o f t h e r e l e v a n t l i t e r a t u r e on p a n c r e a t i c t i s s u e in e u l t u r e w a s i n e l u d e d in t h e p r e v i o u s r e p o t { in t h i s s e r i e s . S o m e o f t h e p r e s e n t d a t a h a v c a p p e a r e d in a b s t r a c t [15]. METHODS

P a n c r e a t i c explants, 1 . l > 1).5 ram, were i)repared from 17-day fetal Spraguel ) a w l e y - S i m o n s e n rats a n d cultured at 35 :+:_0.5'(; on the surface of a semi-defined m e d i u m in a w a t e r - s a t u r a t e d a t m o s p h e r e of !)5 per cent (h- 5 per cent CO,. E a c h culture c o n t a i n e d 30 to 40 explants, representing the tissues of an entire litter, less one fetus per l i t t e r r e t a i n e d as a control. Culture m e d i u m (8() per cent CMRL 1066 1)lus 20 per cent chick e m b r y o e x t r a c t plus 20.0 mg h y d r o c o r t i s o n e per liler) was replaced a t 48 + t hr intervals t h r o u g h up to I0 days of culture. Methods of culture and m e d i u m p r e p a r a t i o n were identical to those r e p o r t e d in detail p r e v i o u s l y [ 14 [. After two, four, six, eight~ or 10 days in vitro, cultures were t e r m i n a t e d , a n d t h e entire tissue mass of each culture was transferred to a microhomogenizer, frozen in liquid nitrogen a n d stored at - 2 0 " ( ; until e x t r a c t e d . Pancreatic tissue from n o r m a l control fetal or p o s t n a t a l rats of 17, 18, 19, 21, " 2 3 " , " 2 5 " and "27" d a y s were also collected and frozen for subsequent extraction. (All ages given as days post-fertilization; ages of p o s t n a t a l animals given in q u o t a t i o n marks.) F o r e x t r a c t i o n , tissues were suspended in triple-distilled water, m e c h a n i c a l l y homogenized, and the tissue l)roteins were p r e c i p i t a t e d hy adding 10 per cent (w/v) trichloroacetic acid (TCA) equal in volume to the water used for homogenization. Experimenltd Cell Research 41

366

L. R. Murrell, C. R. Morgan and A. Lazarow

Following centrifugation, the TCA supernatant was discarded and the trichlorprecipitable fraction (TCAp) was extracted with two successive 200 ~1 aliquots of acid alcohol (15 ml 12 M HC1 per liter 70 per cent [v/v] cthanol). Each aliquot of acid alcohol remained in contact with the suspended TCAp for 60 to 90 min. The two acid alcohol fractions from each culture were pooled and designated acid alcohol soluble fraction (ASF). That portion of the TCAp not soluble in alcohol was dissolved in 0.1 N NaOH and designated trichlor-precipitable residue (TPR). The entire extraction, carried out at 5 ~ to t0~ is summarized in Fig. 1. Duplicate 25 /d aliquots of each fraction (TCAp, ASF and TPR) were used for estimation of tissue proteins by a modified Lowry method [11]. The extraction method was identical for cultured tissue (Fig. 1) and normal pancreatic tissue, except that the volume of water used for suspension of the older control tissues was increased so that the protein concentrations in the TCAp fractions were similar to those of the TCAp fractions of cultured tissues. Normal pancreatic homogenates of 250/d were used for extraction. Duplicate 100 ~d aliquots of each cultured or normal tissue ASF fraction were diluted to 1.0 ml with one per cent bovine serum albumin in a borate buffer at pH 8.4 (1 per cent BSA) and their insulin contents determined by the immunoassay method of Morgan and Lazarow [12, 13]. Duplicate 0.5 ml aliquots of each medium sample (removed from cultures at 48-hr intervals through 10 days of culture) were similarly diluted to 1.0 ml with 1 per cent BSA and their insulin levels determined. Tissue ASF fractions and medium were occasionally assayed at greater dilutions than those stated above. Five to 10 samples of medium not exposed to cultured tissues were included with each series of insulin assays. Ninety-six samples, including bovine insulin standards, were assayed at one time. In addition, occasional ASF fractions prepared from nonpancreatic tissues were assayed. RESULTS

Normal pancreas Results of i m m u n o a s s a y s of acid alcohol soluble ( A S F ) tissue protein fractions o b t a i n e d f r o m n o r m a l fetal or postnatal rat p a n c r e a t a are s u m m a r i z e d in Fig. 2. Regression analyses (insulin content versus age) indicated, at a confidence limit of p ~< 0.005 (bo.995) that tissue insulin content i n c r e a s e d linearly with age f r o m 17 t h r o u g h 19 fetal (lays, a n d f r o m 21 t h r o u g h " 2 5 " fetal (lays. (Regression data a p p e a r in Tables I a n d II.) T h e insulin content of n o r m a l p a n c r e a s , expressed as # U b o v i n e insulin equivalents per leg TCA precipitable tissue protein (TCAp), d e c r e a s e d significantly (p ~< 0.001) between 19 a n d 21 (lays. No significant c h a n g e in insulin content o c c u r r e d between " 2 5 " a n d " 2 7 " days (0.6 > p > 0.5).

Cultured pancreatic islets Results of insulin assays of A S F fractions f r o m p a n c r e a s explanted on the 17th fetal d a y a n d cultured for periods f r o m 48 hr to 10 (lays, expressed as

Experimenlal Cell Research 41

Insulin synthesis in organ culture. II CULTURED ISLET TISSUE ( 3 0 - 4 0 Expkants) w o s h In GK~I (2 x 10 ml) - ~

~

(150

pl

,iil

,I.~CARp =,KIN

H.O)

prec~pitete qc:

- 2.0 i

tissue

homogenize

~

367

prolelns rC&

t:,~

I[

Is

It~

21

"2~ . . . .

"23'

B~R1H

27'

f,estatlo~

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N

,,r

15

12

q

0.12 0.(9

I, M 1%05 rLNr i)1

TCip

.

":

II t 2 " - ~ 1 1 r4 %,~ ,r

c

-: 9 ,,

~l, ~nii' i ul

~2 LF~ ASF

f

I

~z Jte t

O" '~ NO')H 'PSO#[)

2 "2ml

I,.'

L,

2

4

(,

(17 day fetal pancreas)

,llSSO~Ve in

,J!

e
W7

0L~

r

i'2

iprotein I

~.

1.50

0C~

~2

I

.

:,s

Fig. 2.

' 2 ' , :5 ul

.

,JSI

:, ,4

,,i

resuspend ( ~ , ~1 r?,'.

(2

;Jf,r

' 2 , 25 pl)

2or N

13

6

J? S.E.M.

0.12 0.05

2.1 0.3

6 2.4 0.2

6 1.4 0.2

6 1.5 0.2

7 1.32 0.05

Fig. 3.

in @r s t ' e z ~

@ i1!,

T

, T~q

I

OlSS~ lye

Lpro~

( 2 5 N ' .1 \ k , - : l , )

ASF

i.o

=

2ro~,5

0

l:ig~ 1.

2

4

N

30

24

.f

1.5 0.1

1.01 0.07

S.E.M.

G

18 0.78 0.08

8 Ooys ol

12 0.80 0.09

J0 culture

6 1,03 0,03

Fig. 4, Fig. l.--l':• of cullured islet tissue. GI,:N is an isotonic, iso-osmotie solution of glucose, potassium and sodium chlorides [141. Other abbreviations used are explained in the text. Fig, 2. I n s u l i n contt, nts of n o r m a l h'lal or postnatal rat pancreas, ba~ed on a n a l y s e s of A S F fractions. Vertimd l i n e s r e p r e s e n t s.r:.~l. In t h i s a n d Figs. 3 and 4, N is the m n n b e r of duplicate d e t ( ' r m i n a / h m s , 2 is t h e m e a n v a k w :tnd sJ,:.~t is the ~tandm'(? e r r o r of t h e m o a n , cr/~,/.\'.

Fig. 3. --lnsttlit~ ('~ttLct~ts of orgatt ctdtttved islet. Pancreatic explants prepared from 17-day fetal rats and c u l t u r e d for the tlttIut)er of d a y s indicated on the horizontal axis. []used on a n a l y s i s (ff A S F tissue ft'aelions. Fig. 4 . - - I n s u l i n released into the culture medium per 48 hr culture interval. Note that the uledit|m was totally replaeed at 48:L-I hv intervals Ihrou~h 10 days of culture. Thus, total a m o u n t of~ insulin released through t (lays of culture is obtained h y adding the values for the 2 and 4 day periods. The values arc expressed as ttl" bovine insulin equivalents released p e r / t g TCAp cultured lisstte protein. Experimetdal Cell Research 41

L. R. Murrell, C. R. Morgan and A. Lazarow

368 #U

b o v i n e i n s u l i n e q u i v a l e n t s p e r # g TCA~, a r e g i v e n in Fig. 3. F o r e a c h o f

the culture intervals studied, "organ cultured islet" contained significantly m o r e i n s u l i n t h a n w a s p r e s e n t i n n o r m a l p a n c r e a s at t h e ag e o f e x p l a n t a t i o n ( p ~< 0 . 0 0 1 ) . I n d e e d , a f t e r o n l y 48 h r in vitro, " c u l t u r e d i s l e t s " c o n t a i n e d 18 t i m e s as m u c h i n s u l i n as c o u l d b e d e t e c t e d in c o n t r o l p a n c r e a s at t h e a g e o f explantation. I n t h e c u l t u r e i n t e r v a l s f r o m t w o to f o u r , a n d f r o m 6 to 10 d a y s , t h e t i s s u e i n s u l i n l e v e l s d i d n o t c h a n g e s i g n i f i c a n t l y ( p = 0.5). A d e c r e a s e i n t i s s u e i n s u l i n l e v e l ( p ~< 0 . 0 1 ) o c c u r r e d b e t w e e n f o u r a n d 6 (lays o f c u l t u r e . Direct comparison

of insulin

contents

of " c u l t u r e d

islet"

and

normal

TABLE I. Normal fehd rat pancreas, 17 to 19 days postgeslalion. Regression of insulin content (Y) against age (X). The linear regression equation, calculated from the data of Fig. 2, is: ~z = 0.632 + 0.540 (X - 17.82). The analysis in the table below confirms the linearity of the relationship at a confidence limit of F0.995 (p ~<0.005).

Within groups Regression About regression Total

Sum of squares

d. [.

2.1370 6.272 0.032

31 1 1

6.239

2

0.032 F = --- = 0.47 0.069

Mean square 0.069 0.032

Fo.995(1, 31) = 9.14

T a b l e II. Normal rat pancreas, 21 to "'25" days poslgestation. Regression of insulin content (Y) against age (X). The linear regression equation, calculated from the data of Fig. 2, is: Yx = 0.965 + 0.184 (X - 23.343). The analysis in the table below confirms the linearity of the relationship at a confidence limit of Fo.995 (p ~<0.005). Sum of squares Within groups Regression About regression Total F=

Experimenlal Cell Research41

0.059 0.017

d./.

Mean square

0.535 3.115 0.059

32

0.017

3.174

2

=3.52

1 1

Fo.995 (1, 32)- 9.10

0.059

Insulin sgnthesis in organ culture. I I pancreas

of corresponding

contained

relatively

These were

studies

between

a moditied

total age indicated

(p < 0.02) more

are

based

four and

Lowry

on

cultures

containing

six #g TCAp protein

It l ] m i c r o p r o t e i n

(Table

insulin through

369

Ii1) that cultured

islet

6 days of culture. 30 40

per explant,

explants.

There

as determined

by

method.

Insulin in culture medium Insulin from

immunoassays

cultures

Table

after

of 0.5

48 hr

ml

exposure

aliquots to tissues

of culture are

medium

summarized

removed in

Fig.

4.

I I I . Insulin contents of cultured and normal r(d pancreas, compared bg

Shtdenl's l-lest. The ranges (columns 2 and 4) represent standard errors of the mean. All the cultured tissues were cxplanted on the 17th fetal day. Cultured pancreas

Norulal pancreas

Days of culture

puling TCAp

Insulin

Age in days

ttU/pg TCAp

Illsulin

P (<)

'2 4 6 8 10

2.1 + 0.3 2.4 + 0.2 1.4 • O.2 1.5 _+ I).2 1.32 + 0.05

19 21 "23" "25" "27"

1.2 +0.1 0.60• 0.81+0.04 1.30• 1.27 + 0.05

0.02 (t.02 0.02 -0.05

T A B L F I V . Insulin content o[' cullure medium, ~8 hr intervals from 2 through

6 d(lgs o[' culture. Regression of insulin content (Y) against time (X). The linear regression equation, calculated from the data of Fig. 4, is: Y,--1.16 0.188 (X-3.67). The analysis in the table below confirms the linearity of the relationship at a confidence limit of F0.9.,J5 (p "50.005).

Within groups Regression About regression Total F=

0.2787 t).2492

Sum of squares

d. 1.

17.194(I 6.5596 (I.2787

69 1 1

6.8383

2

1.119

Mean square 0.2492 0.2787

Fo.9,):,(1, 69)_~8.4

Experimenlal Cell Research 41

370

L. R. Murrell, C. R. Morgan and A. Lazarow

Medium freshly prepared or incubated without tissue ( " O " days of culture in Fig. 4) contained no demonstrable insulin by our assay technique. During the first 48 hr of culture, the tissues released 1.5 _+ 0.1 #U insulin per #g TCAp tissue protein. The rate of release decreases linearly (p ~<0.005, Table IV) through the sixth (lay of culture, then remains essentially constant through l0 days. (The slight increase between 8 and 10 days, by Student's t-test, has p = 0.05. We do not regard this as a statistically significant change.)

Control studies Extraction procedure.--hnInunoassays reported above indicated that insulin appeared in the ASF fraction of both cultured and control pancreatic tissue. To obtain a measure of the distribution of insulin in the various fractions, an experiment was conducted in which exogenous bovine 131ITABLI,: V. Recovery of exogenous insulin-t31I during extraction of cultured islet tissue. Tracer insulin was bovine insulin-131I, Abbott Laboratories lot no. 19739. The sample contained no measurable free iodide (water c h r o m a t o g r a m by manufacturer); 96 % of the radioactivity was precipitable by specific insulin immunoprecipitation. 32.5 # U (0.234 pC; specific a c t i v i t y = 18 mC/mg) in 10 pl 1% BSA were added to the tissue.

CPM a

Per cent total counts recovered

348

2.4

Second TCA s u p e r n a t a n t (after resuspension TCAp in 5 % TCA)

31

0.21

First acid alcohol soluble fraction

12,263

84.3

1,223

8.4

206

1.4

Tissue fraction

First TCA s u p e r n a t a n t

Second acid alcohol soluble fraction Third acid alcohol soluble fraction Trichloracetic acid precipitable residue

380

Total counts recovered

14,451

2.6 (100 %)

a Net counts, expressed as CPM/tota] fraction. Each reading is the average of duplicate determinations.

Experimental Cell Research 41

Insulin .s'!tnlhesi,s itt organ cullure. I I

371

insulin was a d d e d to a saml)le of cultured p a n c r e a t i c tissue prior to extraction. The extraction was similar to that routinely used (Fig. t) except that: ( . ) duplicate aliquots of each fraction were c o u n t e d for 1311; (b) three successive extractions (200 ffl each, at 4~ for 90, 45 a n d 45 rain respectively) ,'t ? ', ,',

:-'C

,

I L

'

a Pancrl~a~

He,3rt

Ki,lney

Li~er Pqncreas He,]rt Kidney Liver

I

F'ancre,]s A, id -,piant*~ ~r A h:~,hi31 ,,r< tetu, ~ l ~ y , rssue~ ~,gar :ulrured

17 day fetq~ tlSS,ues

Neo ,et,~ tissues (B:rth ~ , , c,d ,ja)s}

~ da,~

Fig. 5.--Specificity of insulin assay. Imnmnoassay of acid alcohol soluble (ASF) fractions of various normal rat tissues and cultured pancreas, expressed as flU bovine insulin equivalents per /*g TCAv tissue protein. The values for normal and cultured pancreas are transferred from Figs. 2 and 3. were perfl,rmed, and (c) no protein d e t e r m i n a t i o n s were done on a n y fractions. T h e distribution of 13xI activity, s u m m a r i z e d in T a b l e V, i n d i c a t e d that over 90 per cent of e x o g e n o u s l y a d d e d tracer insulin was r e c o v e r e d in the first two acid alcohol fractions. On the basis of this experiment, as well as the k n o w n acid alcohol solubility of insulin, it was a s s u m e d that the m a j o r i t y of e n d o g e n o u s insulin extracted by the outlined p r o c e d u r e wmfld a p p e a r in the A S F fractions. Specificit 9 of assag. Varim~s other fetal a n d n e o n a t a l tissues were extracted by the p r o c e d u r e r,mtinely used for p a n c r e a t i c tissue. T h e results (Fig. 5) indicate the high degree of specitk'ity of the assav technique. We also c o n d u c t e d a series ,~f analyses in w h i c h culture m e d i u m or A S F tissue fractions were assayed at various dilutions to 1 : 1 0 4. The results indicated that dilution effects or " c o m p l e m e n t " artifacts of the type r e p o r t e d elsewhere [13j were not present in these experiments.

DISCUSSION

l m m u n o a s s a y of acid alcohol extracts of n o r m a l 17-day fetal rat p a n c r e a s indicated insulin contents of 0.12_+ 0.05 ff.U insulin per fig TCAp tissue 25 - 661801

Experimental Cell llesearch 41

372

L. R. Murrell, C. R. Morgan and A. Lazarow

protein. Assuming that protein represents 10 to 20 per cent of tissue weight, this corresponds to 0.012 to 0.024 units insulin per gram pancreas--a value 5 to 10-fold lower than the insulin content of 17-day pancreas found with an epididymal fat pad insulin assay by Dixit et al. [61. Differences of this order of magnitude are commonly observed in insulin bioassays by these two techniques I2, 9, 181. The epididymal fat pad assay provides results as "insulin-like activity". It is noteworthy that our immunoassay provides insulin values as bovine insulin equivalents, since rat insulin standards were not available for calibration. Thus, an absolute error may be present in the insulin values reported, however any such error does not affect the comparison between normal and cultured tissue (and culture medium) insulin levels. Our immunoassay technique is preferred since it provides a rapid, reproducible and sensitive estimate of bovine insulin equivalents in large numbers of samples of normal and cultured tissue extracts, and culture medium. It is also of interest that our experiments, like those of Dixit et ah I6], detected insulin in pancreata of 17-day fetal rats. Extrapolation of the values obtained from animals ranging from 17 through 21 days suggests that neither technique would detect insulin in pancreata of animals at earlier developmental ages. The period at which there would be no insulin storage (Y~ = 0), calculated from the linear regression equation (Table I) of insulin against age (17-19 days) is 16.71 days. A similar calculation from the regression equation provided by Dixit et al. (for animals from 17 to 21 clays), gives the age at which no insulin would be stored as 17.03 days. This agreement is remarkable, considering the differences in assay techniques used in the two groups of experiments. Pancreatic islets in the rat are morphologically recognizable by 13-fetal clays [7], possibly earlier I19]. In a previous report I141, we noted, in agreement with other investigators I6, 8, 19] that aldehyde fnchsin positive beta cell staining (under the conditions used, an estimate of stored insulin) did not occur prior to 17- or 18-fetal days. The present studies support this histological evidence of the onset of insulin storage. After 48 hr in vitro, "cultured islets" had l:roduced a total of 3.6 #U insulin per btg TCAp tissue protein. Of this amount, 1.5 #U were released into culture medium and 2.1 #U were stored in the tissue. After 10 days of culture, a total of 6.4/zU insulin had been produced by each/zg of cultured tissue protein. Of this amount, 5.1 #U were released into the medium during the 10-day period, and 1.3 #U remained stored in the tissue. Thus, after 10 days of culture (during which the medium was changed at 48 hr intervals), each /~g TCA~ tissue protein produced over 50 times the amount of insulin Experimental Cell Research 41

Insulin synlhesis in org~tn cullure. H

373

present at the beginning of lhe culture period. (The 0.12 /tU insulin per :tg TCAp tissue protein present al Ihe t,eginning ,,f culture have I,een included in these net tissue insulin eah'ula/ions. The approximation does not materially all'cot our conclusions.) l)uring Ihe Iirst four clays of culture, relatively more insulin can he rec()vere(t fr(>m lhe cultured islets than (luring the later culture periods. Insulin continues to he reh, ase(I into lhe medium during the sixth through 10th clays a period in which Iheinsulin content o f l h e t i s s u e s remains essentially constant. Therefore, insulin svnlhesis eonlinues throughout the lO-day culture period. Complele l>.rtrayal of insulin produclion necessarily inw>lves consideration of synthesis, storage, reh'ase and degradation of the hormone, l)elerminations of tissue insulin levels (in both cultured anti normal pancreas) provide a measure <)f insulin storage; determinations of insulin in the culture medium provide a measure of insulin released under these culture conditions. Tissue plus medium insulin levels, when compared [o insulin contents of normal 17-day fetal pancreas, provide a measure of net insulin synthesis during culture. Such determinations supl)ly only minimal insulin levels: no account is taken of the occurrence or extent of insulin degradation. The cultt,re medium used in these experiments contains at)l>roxilnately the same amounts ot; glucose present in normal rat hh>od (101)_+ 10 ing%, determined hy glucose oxidase). Preliminary unl>uhlished experiments suggest that the glucose content of Ihe medium remains within normal i)hysio logical limits during Ihe lwo days fol" which it is normally exposed lo tissues. In a given Iwo-day culture inlerval, the am(~unls of insulin reh'ase(I into the mediunl arc comparable lo lhe amounts stored in "cultured islet" at the end of lhe l)reeeding two day culture interval. This suggests that the tale of insulin turnover is high when heta cells are cultured under lhese conditions. Experiments in progress, involving varialions of medium glucose concentration, and of [he interval hetween medium changes, may provide more detailed infer,nation concerning Ihe interrelationshii)s of insulin synthesis, storage and release by "eulture
374

L. R. Murrell, C. R. Morgan and A. Lazarom

It has been suggested, on morphologic criteria, that cultured fetal rat pancreas I19] or "cultured islet tissue" I14] might contain insulin not present at the beginning of culture. Protein and/or insulin synthesis has been shown during short-term incubati(m of m a m m a l i a n pancreatic slices I10, 171 or fish islet tissuc [1] with radioisotope labeled amino acids. The present report represents unequivocal demonstration of insulin synthesis, release and storage during organ culture of m a m m a l i a n islet tissue. Relatively more insulin can be recovered from cultured islet than from normal pancreas of comparable total age by our methods of extraction and assay. This lends functional supi)ort to our previous hypothesis (based on morphoh)gic evidence) of selective endocrine differentiation in fetal rat pancreatic explants cultured under the specified conditions.

S UMMARY

Pancreatic explants obtained from 17-day fetal rats were cultured in a 95 per cent 0 2 - 5 per cent C()~ atmosphere on a semi-defined liquid culture medium, as previously described. Insulin immunoassays of whole culture medium and tissue extracts were performed. Tissues explanted from 17-day fetal rats anti cultured for 2, 4 or 6 days contained more insulin (p ~<0.02) than did uncultured control pancreata of comparable ages (i.e., 19, 21 anti 23 fetal days). Assays of tissue and medium indicated continuous synthesis and release of insulin (luring 10 days of culture. Total insulin recovered from tissue plus m e d i u m (medium changed each 48 hr) was over 50 times that recoverable from uncultured 17-day fetal pancreas. These data support our hypothesis of selective (lifferentiation and maintenance of metabolically active pancreatic beta cells in explants of fetal rat pancreas cultured under the specified conditions. The senior author is the grateful recipient of a Research Fellowship from the American Diabetes Association (1964-66). Dr Morgan was Research Fellow of the American Diabetes Association (1963-64); his present address is Department of Anatomy, Indiana University Medical Center, Indianapolis, Indiana. This study was supported in part by the U.S. Public Health Service Grants No. AM-06517 and 5-T1-AM-5127, from the National Institutes of Health.

Experimental Cell Research 41

I n s u l i n s y n t h e s i s in o r g a n c u l l u r e , l l

375

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