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18. P. DeMeyts, in, HI, M. J. Roth, D. M. Neville,J.R. Gav- 23. J.Gliemann,Diabetologia 3,382(1967). Assays
18.
P. DeMeyts,
in, HI, M.
J.
Roth,
D. M.
Neville,J.R.
Gav-
23.
J.Gliemann,Diabetologia
3,382(1967).
Assays
jected mice and animals
receiving
equal
A. Lesniak,Biochem.
Biophys. Res.
were
performedbyM.
Moule oftheBantingand
Commun. 55, 154 (1973);
Bianco,J.Roth,J.Biochem.
P.
DeMeyts,
A.
C.
BestDepartmentofMedicalResearch,Universi-
volumes of citrate buffer were controls.
251,
1877(1976).
tyofToronto.
Blood samplesforglucosedetermination
19.
R.J.Haslam
and S.Goldstein,Biochem. J.
144,
24.
Technical
assistance was
provided
by
E. K.
253 (1974); W. B.
Benjamin, S.T.
Fish,
I.Sing-
Rosenbloom and E. Moerman.
Supported
by
a
were collected in heparin-treated
pi-
er, Biochem. Soc. Trans.
2,
920
(1974);
M.
D.
facultydevelopment
award from the
University
pettes by orbital sinus puncture and as-
Hollenberg
and P. Cuatrecasas, J. Biol. Chem.
ofFlorida,
Medical Research Council
ofCana-
250,3845(1975).
da,
the
Canadian Diabetic Association Founda-
sayedas described (9).Sampleswere ob-
20.
J. T.
Cooper
and 6. Goldstein, Lancet 1973-H,
tionFund,andtheC. H. BestFoundation.
673(1973).
tained before each of the five daily in-
*
Presentaddress:
Department
ofPediatrics, Uni-
21.
J.M.
OlefskyandG. M. Reaven,J.Clin.Invest.
versity ofFlorida College ofMedicine,
Gaines-
jections and
at frequent
intervals
54, 1323(1974).
ville32610. AddressreprintrequeststoA.L.R.
22.
J. L.
Hamlin
and E. R. Arquilla,J.Biol. Chem.
249,21(1974).
28November 1975;revised27April1976
afterward until animals were killed 6
days after the last injection. Four mice
were also killed 12, 16,and 25 daysafter
the completion ofthe SZ injections. The
unpairedt-test(10)was usedinstatistical
analyses. Pancreatic tissue obtained at
Streptozotocin-InducedPancreaticInsulitis:
death was fixedforlightand electronmi-
Downloaded from http://science.sciencemag.org/ on November 10, 2017
New ModelofDiabetesMellitus
Abstract. Multiple small injections ofstreptozotocin in mice produce pancreatic
insulitis, withprogression tonearly complete beta celldestruction anddiabetesmel-
litus. The timingandappearance oftheinflammatoryisletlesionssuggestbutdo not
prove that streptozotocin acts by initiatinga cell-mediatedimmune reaction. Ultra-
structuralevidence ofabundant type C viruses within beta cellsoftreatedmice sug-
gests that streptozotocin may activate murine leukemia virus in vivo in susceptible
hosts.
croscopicstudyas described(11).
Plasma glucose values for SZ-injected
micewere significantlyelevated afterthe
fourth injection (12) and increased sub-
stantially during the subsequent 6 days
(Fig. 1) (13). Examination by light mi-
croscopy (Fig.2)revealedlargenumbers
of lymphocytes, moderate numbers of
macrophages, and rare neutrophils sur-
rounding and permeating the islets of
Streptozotocin (SZ) is a broad-spec-
trum antibiotic possessing antitumor (1),
SZ may initiate a cell-mediated immune
Langerhans, with distortion of archi-
tecture and beta cell necrosis. Surviving
oncogenic (2),and diabetogenic(3)prop-
beta cells were variably degranulated
erties.The lastactionismediatedbypan-
reaction directed against the beta cells.
The relevance, if any, of the increased
numberoftypeC virusparticlestothein-
creatic beta celldestruction and iswide-
lyused as a method forinduction ofdia-
betes in experimental animals and for
clinical treatment of malignant beta cell
and the islets were generally smaller. Is-
let inflammation gradually diminished in
animals killed 12, 16, and 25 days after
the completion of injections [mean
plasma glucose values of396 mg/100 ml
tumors. Forinductionofexperimentaldi-
abetes, SZ is conventionally adminis-
flammation and beta cell destruction is
unknown.
Forty adult male mice (Charles River
CD-I strain),allowedfreeaccess tofood
and water, received five daily intra-
(N = 12), 525 mg/100 ml (N = 7), and
venous or intraperitoneal injections of
657 mg/100 ml (N = 7),
respectively],
teredas a singleinjection(3). Afterrapid
clearance of SZ from the bloodstream
[serum half-life is 15 minutes (4)], light
SZ (40 mg per kilogram ofbody weight)
(8) dissolved in a citrate buffer,pH 4.2,
just before injection. A total of43 unin-
microscopic evidence ofbetacell necro-
sis is apparent within 24 hours (5). Beta
and the remaining islets were small and
composed almost exclusively of alpha
anddeltacells.
Ultrastructural studies of the islets in
ten mice killed 6 days after the com-
cell necrosis, however, is detected by
500r-
conventional ultrastructural examination
after 2 to 4 hours (5), and intra-
membranous particle depletion of beta
cellplasmamembranes isobservedwith-
pletion ofinjections revealed occasional
necrotic beta cells and numerous infil-
3K
- 400
trating lymphocytes and macrophages.
Unexpected, however, was the presence
E
0
}(5)
oflargenumbers oftypeC virusparticles
0
in 45 minutes in freeze-fracture studies
(6). Dissolution and phagocytosis ofne-
I-
(12)
E 300
(14)within many intact, partiallydegran-
ulatedbetacells(Fig.3B). Alphaanddel-
croticcellsisrapid,with virtuallynoevi-
i (12)
4n
0
dence of debris or inflammation visible
ta cells were normal. The pancreatic is-
letsofuninjected mice and mice injected
after 3 days (3, 5). In a correspondingly
to 200
with citrate buffer appeared normal
*
f (12)
i
rapidmanner, bloodglucosevaluespeak
(13) (12)
when examined by lightand electron mi-
E
(12)
I to 2 days after SZ administration and
(U
a
remainelevatediftheappropriatequanti-
o Dr
* P<0.001
tyoftheagentisgiven(7).
We presentevidenceherethatSZ,giv-
croscopy. Only an occasional virus par-
ticle was observed within the usually
well-granulated beta cells (Fig. 3A).
Quantitative morphometric studies of
en intravenously or intraperitoneally to
n
U-
o ti t
t
Dyt 5s
6
7
8
9
10
laboratory mice in multiple subdiabeto-
Days
genic doses, the usual method ofclinical
thenumber ofvirusparticlespercelland
the frequency of cells harboring viruses
were not performed. Viruses were ob-
administration (1, 4), induces (i) pro-
nounced pancreatic insulitis, with even-
tual destruction ofinsulin-secreting beta
Fig. 1. Plasma glucose response in Charles
River CD-1 mice
afterfive intraperitoneal
in-
servedneitherinthealphaand deltacells
norintheinflammatorycells.
jections ofSZ (40
mg/kg).
The mean
glucose
valuewas
significantlyincreasedoverthepre-
cells and diabetes mellitus, and (ii) en-
injectionvalue(day0)afterthefourthdose
of
hanced replication oftype C virus parti-
SZ and continued to increase
progressively
until termination ofthe
experiment.
Virtually
cleswithinpancreaticbetacells.Thetim-
identical results were obtained
in 24
mice
ing and appearance ofthe inflammatory
isletlesions suggestbutdo notprovethat
given intravenous injections of SZ (data not
shown).
In an effort to ascertain the timing of
theappearance ofthe inflammatory cells
in and around the pancreatic islets, ani-
mals were killed at daily intervals after
having received one, two, orthree intra-
peritoneal injections of SZ (40 mg/kg).
30 JULY 1976
415
One injection of SZ induced transient, mildbetacelldegranulation;insulitisand plasma glucose elevation were absent. The islets of mice
One injection of SZ induced transient,
mildbetacelldegranulation;insulitisand
plasma glucose elevation were absent.
The islets of mice receiving two and
three SZ injections had moderate beta
celldegranulation duringtheinitial4to5
daysafterthelastinjection. Insulitiswas
firstrecognized6and5daysafterthelast
injection in the mice that received two
and three injections, respectively. In-
flammation was more pronounced after
three injections, and these animals also
evidenced mild hyperglycemia (mean
plasmaglucose,214mg/100ml)7daysaf-
Downloaded from http://science.sciencemag.org/ on November 10, 2017
tercompletionofinjections.
The same dose schedule (8), routes of
administration, and timing of killing
failed to produce similar lesions in 21
Charles River rats despite the fact that
these animals become markedly hyper-
glycemic after a single large injection of
Sz.
Fig. 2. (A and B) Light microscopic photomicrographs ofpancreatic isletsfrom mouse injected
with citratebuffer. The well-delineated isletsare enmeshed inthe surroundingacinarcellsofthe
exocrine pancreas. Well-granulated beta cells stain intensely with aldehyde fuchsin (black in
photograph) indicating an abundance ofstored insulin. (A) Hematoxylin and eosin, x 144; (B)
aldehyde fuchsin, x 222. (C and D) Inflamed isletsfrom mouse killed6days afterreceivingfive
injections of SZ. The interior and periphery ofthe islets are permeated with large numbers of
mononuclear inflammatory cells (identified as lymphocytes and macrophages by electron
microscopy), which distort the isletarchitecture and extend into the adjacent exocrine tissue.
Substantial beta cell degranulation is evident (D) and is consistent with the presence of
hyperglycemia. (C)Hematoxylin and eosin;(D)aldehydefuchsin; both x 144.
Incontrastwiththeone-injectiontech-
nique of SZ-induced diabetes, wherein
beta cell necrosis occurs within 4 hours
and hyperglycemia is achieved rapidly,
multiple subdiabetogenic injections of
SZ induced gradual elevation of plasma
glucose in all animals tested, with maxi-
mum values realized 1 week orlongeraf-
terthelastinjection. Microscopic exami-
nation revealed mononuclear inflamma-
tory cells, including large numbers of
lymphocytes,inandaroundthepancreat-
ic islets in all mice studied, in contrast
with the virtually inflammation-free islet
lesions observed after a single large in-
jection.The timerequiredforthefirstap-
pearance of inflammatory cells (5 to 6
days after the last injection) is compat-
ible with a cell-mediated immune reac-
tion conceivably directed against beta
cells modified by the administration of
SZ. A nonspecific inflammatory re-
sponse to low-grade beta cell injury in-
ducedbySZ hasnotbeenexcluded,how-
ever. In further support for an immuno-
A;< Z:'v8^
Fig. 3. (A) Electron micrograph
of
logic pathogenesis, the reduction inbeta
pancreatic beta cell from untreated
t
a
*** <;-;j
0
Sr?
mouse with numerous secretory gran-
ules (arrowheads).
Two
immature
cell numbers and plasma glucose eleva-
tion become progressively more pro-
nounced during the 10 to 25 days after
type C virus particles are visible. A
thelastinjection,longafterSZ iscleared
typical, immature type C virus (single
straight arrow) is adjacent to
a long
from the bloodstream (4) and the short-
filamentous form (double arrow). Vi-
lived SZ beta cytotoxic action com-
ruses were identified with
difficulty
in
> m; the uninjected and buffer-treated
ani-
pleted. Itistherefore tempting to specu-
late that the mononuclear inflammatory
mals;N, nucleus;M, mitochondrion;
23,000. (B) Beta cells from mouse
cells (lymphocytes and macrophages)
x
killed 6 days after receiving five in-
are responsible for the progressive beta
jections of SZ. Secretory granules are
celldestructionandtheresultingincreas-
A5|+ fj4
virtually absent and the usual cy-
toplasmic organelles are replaced by
inglyseverehyperglycemia.
The possibility of an immunological
numerous
cisternae of the
smooth
and
rough
endoplasmic
reticulum
role ofthe type C virus particles within
containing many typical and filamen-
tous (cylindrical) immature type C viruses; x 14,800. (C) A portion (B) at higher magnification
revealing the varied appearance of the intracisternal viruses; x 49,300. (Inset) Two immature
type C virus particles with characteristic viral envelopes and inner ring-shaped nucleocapsids.
Mature particlesand virusesbuddingatthecellsurfacehave not been identified; x 69,000.
the beta cells ofSZ-treated mice should
be considered. Virus particles were not
observed buddingatthebetacellplasma
416
membranes, and the cells in which they
SCIENCE, VOL. 193
were locateddidnotusuallymanifestde- 6. L. Orci, M. Amherdt, W. Stauffacher, A. A. 349 (1972); H. Munterfering, Virchows
were locateddidnotusuallymanifestde-
6.
L. Orci, M.
Amherdt,
W. Stauffacher,
A. A.
349
(1972);
H.
Munterfering, Virchows Arch. A
Like, C. Rouiller,
A.
E. Renold, Diabetes 21
356,207(1972).
generative changes. It is conceivable,
(Suppl. 1), 326 (1972); L. Orci,
F. Malaisse-
17.
W. Gepts,Diabetes 14,619(1965).
nevertheless, that interactions between
Lagae,
M.
Ravazzola,
W. J.
Malaisse,
A.
18.
P. M.
LeCompte
and M. A.
Legg, ibid. 21, 762
Perrelet, A. E. Renold, Lab. Invest. 34, 451
(1972).
virus and cell membrane may
have in-
(1976).
19.
A.
E. Renold, J. S. Soeldner, J.
Steinke,
Ciba
ducedtheformationofaccessible,abnor-
7.
A. Junod, A. E. Lambert, W. Stauffacher,A. E.
Endocrinol.
15,
122(1964);P. E.
Renold,J.Clin.Invest.48,2129(1969).
Found. Coloq.
Lacy and P. H.
Wright,Diabetes
14,634
(1965);
mal beta cell immunogenic proteins re-
8.
Each
ofthe fivedailyinjections consisted of25
J. Logothetopoulos and E. G.
Bell,ibid. 15,205
percentofthedoseofSZ(160mg/kg)requiredto
(1966);G.FreytagandG.
Klbppel,Beitr.Pathol.
sponsiblefortheinitiationofacell-medi-
induce severe and
lasting
hyperglycemia when
Anat.Allg.Patthol.39, 138(1969).
atedimmune response(15).
Pancreaticinsulitiswas reportedinex-
administeredasasingleinjection.Streptozotocin
20.
For a
comprehensive review, see: P. S. Sarma
wasfromlotNo.
10508-GGS-37A,agiftofW.
E.
and A. F.
Gazdar, Curr.
Top. Microbiol.
DulinoftheUpjohnCompany.
Immunol.
68,
1(1974).
perimental virus-induced diabetes (16),
9.
A. A.
Rossini,
M.
Berger,
J.
Shadden, G. F.
21.
A.
A. Like and W.
L. Chick, Diabetologia 6,
Cahill,Jr.,Science 183,424(1974).
216(1970).
injuvenilediabeticsautopsiedshortlyaf-
10.
R. G. D. Steel and J. H.
Torrie,
in
Principles
22.
D.
R. Lowy, W. P. Rowe, N. Teich, J. W.
and Procedures of Statistics
ter onset ofclinical symptoms (17), and
(McGraw-Hill,
Hartley,
Science
174,
155
(1971);
V.
Klement,
New York, 1960),p. 173.
M.
0.Nicolson,R.J.Huebner,Nature(London)
inrareexamplesofmaturity-onsetdiabe-
11.
A. A.
Like and L. Orci,Diabetes 21 (Suppl. 2),
NewBiol.234, 12(1971).
511 (1972).
23.
R.
J. Huebner and G. J. Todaro,
Proc. Natl.
tes(18).A chronicinflammatorycellinfil-
trate within the isletsofLangerhans was
also described after unsuccessful at-
12.
Results of intravenous and
intraperitoneal
in-
Acad. Sci. U.S.A. 64, 1087(1969);G.
J. Todaro
jections
were
essentially equivalent.
Only
the
and R.
J. Huebner, ibid. 69, 1009
(1972); C. E.
data from animals
given intraperitoneal
injec-
Whitmire, R. A. Salerno, L. S.
Rabstein, R. J.
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tionsarepresented.
Huebner,H. C.Turner,J.Natl.CancerInst.
47,
temptstoproduceanimmune-typediabe-
13.
Mean plasma glucose values for
mice receiving
1255
(1971); A. Hellman and A. K.
Fowler,
citrate buffer alone were 175 to 185
mg/100 ml;
Nature(London)NewBiol.233, 142(1971).
tes(19).
thoseformice receivinga singleinjectionofSZ
24.
We
thankG. F. Cahill,Jr.,forhishelpfuladvice
Ofindependent significance, however,
(200 mg/kg) were greater than 500
mg/100
ml
duringthesestudiesandforcriticalreviewofthe manuscript and PaulaErlandson,
within48hours.
SerenaDavis,
istheobservationthatSZ,abetacelltox-
14.
We thank A. F. Gazdar ofthe National Cancer
Instituteforadvice and invaluable assistance in
the ultrastructural identification of the type C
viruses.
Claudia
Berger,
and David
Glynn
for
expert
technical assistance. Supported in part by re-
in,isapparently responsible forthe acti-
vation ofvirus replication inmouse beta
cells. The genome of the mouse type C
search grants AM 19155, AM
15191, and AM
05077 from the Public Health
15.
G.J.Todaro,Am. J.Pathol. 81,590(1975).
from the Juvenile Diabetes
Service, a grant
Foundation, and a
16.
J. E.
Craighead
and M. F.
McLane, Science
grantfromtheUpjohnCompany.
virus is present inmost ifnot all strains
162, 913
(1968);
K. F.
Wellmann,
D. Amster-
dam, P. Brancato, B. W.
Volk,Diabetologia 8,
11February 1976
oflaboratoryandwildmice(20).Further-
more, structuresresemblingtypeC virus
particles have been observed inthebeta
cells ofcertain inbred mice (21). 5'-Bro-
modeoxyuridine and 5'-iododeoxyuri-
dine induce viral genome activation in
AlternativeTransformationBehaviorinSulfides:
vitro(22),andactivationofmouse leuke-
DirectObservationsbyTransmissionElectronMicroscopy
mia virus isinduced invivoby x-irradia-
tion, chemical carcinogens, and steroid
hormones (23). This report ofvirus acti-
Abstract.Structuralphase transformationsinNi7S6andCu7S4havebeenobserved
dynamically by in situ experiments in a transmission electron microscope. In this
vation in vivo by SZ is important be-
cause of the drug's occasional clinical
way itispossible todemonstrate thepossibilityoftwofundamentally differenttypes
ofbehavior:(i)theidealtransformationfrom thestablehigh-temperatureform to the
use in the treatment of neoplastic dis-
stablelow-temperatureform and vice versaand(ii)alternativentetastableprocesses
ease. This finding is ofmore than theo-
retical importance when one considers
theoncogenicpotentialofSZ (2).
whichoperatewhen theformationofthelow-temperaturestateisimpeded.
The process ofinversion from a high-
ARTHUR A. LIKE
DepartmentofPathology,
temperature to a low-temperature form
is ofconsiderable importance for an un-
this report I describe experiments in
which both the ideal and the alternative
behavior can be observed dynamically
UniversityofMassachusetts
derstandingofthebehaviorofcrystalline
MedicalSchool, Worcester01605
and the course of the transformations
can be directly controlled by variations
ALDO A. RossINI
JoslinResearchLaboratory,
HarvardMedicalSchool, and
PeterBentBrighamHospital,
phases. In particular, the situation can
arisewhen thekineticsoftheprocessare
such that the formation of the low-tem-
intheexperimentalconditions.
Structural phase transformations in a
perature form is impeded and hysteresis
is introduced, or in the extreme case
number of sulfides occur at rates which
make direct observation possible in a
Boston, Massachusetts02115
when the low-temperature form is in-
accessible. Although the existence of a
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417

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Downloaded from <a href=http://science.sciencemag.org/ on November 10, 2017 Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus AA Like and AA Rossini Science 193 (4251), 415-417. DOI: 10.1126/science.180605 ARTICLE TOOLS REFERENCES http://science.sciencemag.org/content/193/4251/415 This article cites 33 articles, 10 of which you can access for free http://science.sciencemag.org/content/193/4251/415#BIBL PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions Use of this article is subject to the Terms of Service Science (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. 2017 © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. The title Science is a registered trademark of AAAS. " id="pdf-obj-3-6" src="pdf-obj-3-6.jpg">

Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus

AA Like and AA Rossini

Science 193 (4251), 415-417. DOI: 10.1126/science.180605

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Use of this article is subject to the Terms of Service