Rapid Publications
Amelioration of Diabetes Mel I it us
in Partially Depancreatized Rats
by Poly(ADP-ribose) Synthetase Inhibitors
Evidence of Islet B-Cell Regeneration
YUTAKA YONEMURA, TORU TAKASHIMA, KOICHI MIWA, ITSUO MIYAZAKI, HIROSHI YAMAMOTO,
AND HIROSHI OKAMOTO
SUMMARY
Ninety percent depancreatized rats received daily intra-
peritoneal injection of 0.5 g/kg nicotinamide and 0.05
g/kg 3-aminobenzamide, potent inhibitors of islet
poly(ADP-ribose) synthetase. One to three months
after the partial pancreatectomy, urinary and plasma
glucose levels in nicotinamide- and 3-aminobenza-
mide-treated rats were markedly lower than those in
saline-treated control rats. Morphologic examination of
the remaining pancreata revealed that islets in the
poly(ADP-ribose) synthetase inhibitor-treated rats were
markedly enlarged and consisted largely of B-cells.
These results suggest that poly(ADP-ribose) synthe-
tase inhibitors induce islet B-cell regeneration, thereby
preventing or improving diabetes mellitus in partially
depancreatized rats. DIABETES 33:401-404, April 1984.
R
emoval of the insulin-producing pancreatic B-
cells from an animal induces a syndrome that fea-
tures many of the acute metabolic derangements
of diabetes as they are known in man. Mering and
Minkowski1 in 1890 were the first to produce this form of
diabetes by surgically removing the pancreata of dogs. Al-
loxan in 19432 and streptozotocin in 19633 were found to be
highly selective B-cytotoxins and thereafter have been
widely used to produce diabetes in experimental animals.
Recently, Okamoto and his colleagues4"10 have clarified that
alloxan and streptozotocin cause DNA strand breaks in pan-
creatic B-cells to stimulate nuclear poly(ADP-ribose) syn-
4hetase, thereby depleting the intracellular nicotinamide-ad-
enine dinucleotide (NAD) level and inhibiting B-cell functions
including proinsulin synthesis. We also reported that alloxan-
From the Department of Biochemistry, Toyama Medical and Pharmaceutical
University School of Medicine, Toyama, Japan; and the Department of Surgery,
Kanazawa University School of Medicine, Kanazawa, Japan.
Address reprint requests to Professor Hiroshi Okamoto, Department of Bio-
chemistry, Toyama Medical and Pharmaceutical University School of Medi-
cine, Toyama 930-01, Toyama, Japan.
Received for publication 15 December 1983.
DIABETES, VOL. 33, APRIL 1984
and streptozotocin-induced diabetes can be prevented by
inhibiting poly(ADP-ribose) synthetase of B-cells.8"10 In this
article we show that poly(ADP-ribose) synthetase inhibitors
prevent experimental diabetes caused by surgically remov-
ing the pancreata of rats. Morphologic evidence indicates
that regeneration or proliferation of the pancreatic B-cells
occurs in the poly(ADP-ribose) synthetase inhibitor-treated
rats.
MATERIALS AND METHODS
Male Wistar rats weighing 180-200 g were 90% depan-
creatized according to the technique described by Foglia11
and maintained on standard rat chow. From 7 days before
the partial pancreatectomy, nicotinamide, at a dose of 0.5
g/kg body wt in 2 ml saline, and 3-aminobenzamide • HCI,
at a dose of 0.05 g/kg body wt in 2 ml saline, were injected
intraperitoneally into each group of five rats every day. Nic-
otinamide and 3-aminobenzamide are inhibitors of pan-
creatic B-cell poly(ADP-ribose) synthetase; the inhibitory
ability of the latter is about 10-fold as potent as that of the
former.9 The injection was continued until the ninetieth post-
operative day. Five control rats received saline daily before
and after the partial pancreatectomy. The residual pan-
creatic tissue, referred to as the remaining pancreas, was
removed 3 mo after the partial pancreatectomy and fixed in
Bouin's solution. Hydrated 5-|xm sections of paraffin-embed-
ded pancreatic tissues were stained for insulin by the per-
oxidase-anti-peroxidase method12 using DAKO Pap Kit
(DAKO Co., California).
Urinary and plasma glucose levels were measured by the
glucose-oxidase method using Glucose Auto & Stat GA-
1110 (Kyoto Daiichi Instrument Inc., Kyoto, Japan). Statistical
significance of differences between rats treated with and
without poly(ADP-ribose) synthetase inhibitors was analyzed
using Student's t test.
RESULTS
As shown in Figure 1, the control rats exhibited glucosuria
1-3 mo after the 90% pancreatectomy. However, in rats
401
 SD, N = 105) and 0.143 ± 0.114 mm (mean ± SD, N =
89), respectively, while the value of normal untreated rat
islets was 0.112 ± 0.090 mm (mean ± SD, N = 28). The
number of islets per cm2 was 56.0 ± 23.1 (mean ± SD, N =
6) and 52.4 ± 43.1 (mean ± SD, N = 5) in nicotinamide-
and 3-aminobenzamide-injected rats. The contours of the
islets were somewhat irregular; loops and cords of insular
cells pushed their way out between the adjacent acini (see
Figure 3D). The size of each cell in these islets was normal.
Therefore, an increase in cell number was considered to be-
responsible for the increase in islet size in partially depan-
creatized rats treated with poly(ADP-ribose) synthetase in-
hibitors. When the remaining pancreata were immunohis-
tochemically stained, almost all the area of the enlarged islets
in nicotinamide- and 3-aminobenzamide-treated rats was
densely stained for insulin (Figure 3, C and D); only a scanty
number of islet cells were stained for insulin in the control
rats (Figure 3B). On the other hand, cells stained for glu-
cagon were localized only on the peripheries of enlarged
islets in the remaining pancreata of rats treated with nicotin-
amide or 3-aminobenzamide, and their number was almost
the same as that in islets of normal rats (data not shown).
The number of cells stained for somatostatin was also un-

••••

Time (month)
FIGURE 1. Urinary glucose excretion in partially depancreatized rats
with or without poly(ADP-ribose) synthetase inhibitor injection. One,
two, and three months after the 90% pancreatectomy, the urine of each 500
rat was collected for 24 h. Control rats (O); nicotinamide-injected rats
( • ) ; 3-aminobenzamide-injected rats ( • ) . Each point is the mean for
five different rats; vertical bars show SD when larger than the symbol
indicating the mean value. *P < 0.10, **P < 0.05, and *P < 0.025 ver-
sus control rats. The time after the partial pancreatectomy is shown on
the abscissa. ^ 400
receiving 0.5 g/kg nicotinamide or 0.05 g/kg 3-aminoben-
zamide daily, the urinary glucose excretion level decreased
markedly. As shown in Figure 2, plasma glucose levels, be-
fore and after an intravenous glucose load, in the rats re-
ceiving the poly(ADP-ribose) synthetase inhibitors were also
significantly decreased in comparison with those in the con-
trol rats. These results indicate that poly(ADP-ribose) syn-
thetase inhibitors can prevent or improve diabetes mellitus
in partially depancreatized rats.
We next examined morphologically the remaining pan-
creas stained with hematoxylin and eosin of the partially
depancreatized rats with and without poly(ADP-ribose) syn-
thetase inhibitor injection. To obtain the islet relative abun-
dance, the number was determined in at least five sectional
areas of 0.5 cm2.13 Three months after the partial pancrea-
tectomy, the islets of control rats without poly(ADP-ribose)
synthetase inhibitors were decreased in number (18.4 ± 0 5 30
9.8 islets/cm2 [mean ± SD, N = 7]; 47.3 ± 15.7 islets/cm2 Time (min)
[mean ± SD, N = 18] in normal untreated rats), small in
FIGURE 2. Plasma glucose levels during an intravenous glucose toler-
size, and had irregular contours (see Figure 3B). Fibrotic ance test. Three months after the partial pancreatectomy, glucose (0.5
degeneration and degranulation were frequently encoun- g/kg body wt) was injected into the femoral vein of rats that had
fasted for 24 h. A blood sample was taken from the subclavian vein 0,
tered. These observations were in agreement with those re- 5, and 30 min after the glucose load. The results are expressed as
ported by others.1415 However, the islets in the remaining mean ± SD (N = 5). Saline-injected control rats (O); nicotinamide-in-
pancreas of rats that received the nicotinamide and 3-ami- jected rats ( • ) ; 3-aminobenzamide-injected rats ( • ) . Shaded area
shows plasma glucose levels during the glucose tolerance test in nor-
nobenzamide injection were extremely large (see Figure 3, mal age-matched rats. **P < 0.05, *P < 0.025, * * P < 0.010, and
C and D); their diameters were 0.221 ± 0.141 mm (mean ± *P < 0.005 versus control rats.

402 DIABETES, VOL. 33, APRIL 1984

FIGURE 3. Immunohistochemical straining for insulin. (A) Pancreas from normal rat. (B) Remaining pancreas of control partially depancreatized
rat. (C) Remaining pancreas of nicotinamide-treated rat. (D) Remaining pancreas of 3-aminobenzamide-treated rat. Magnification x 825.
changed. These immunohistochemical findings indicate that
B-cells increased in the islets of the remaining pancreas of
poly(ADP-ribose) synthetase inhibitor-treated rats.
DISCUSSION
The present study demonstrated that poly(ADP-ribose) syn-
thetase inhibitors can prevent diabetes in partially depan-
creatized rats. Morphologic and immunohistochemical ex-
aminations of the remaining pancreas of poly(ADP-ribose)
synthetase inhibitor-treated rats showed a marked increase
in the B-cell population. Therefore, it is reasonable to assume
that poly(ADP-ribose) synthetase inhibitors induce pan-
creatic B-cell regeneration, thereby improving diabetes mel-
litus caused by partial pancreatectomy. The fact that
poly(ADP-ribose) synthetase inhibitors induce pancreatic B-
cell regeneration seems to mean that the enzyme may play
a role in restricting B-cell replication. This concept may be
reconcilable to the observations that poly(ADP-ribosyl)ation
suppresses DNA replication in rat liver nuclei16 and that nic-
otinamide stimulates DNA synthesis in HeLa cells.17
We have already shown that alloxan and streptozotocin
induce islet DNA strand breaks, and that poly(ADP-ribose)
DIABETES, VOL. 33, APRIL 1984
synthetase acts to repair the DNA breaks, consuming islet
NAD.4-7 This rapid and marked depletion of islet NAD has
been regarded as the primary molecular mechanism behind
the destruction of the B-cells.4718 Therefore, poly(ADP-ri-
bose) synthetase inhibitors can prophylactically prevent al-
loxan- and streptozotocin-diabetes by blocking the NAD
consumption.
Evidence in the present article suggests an alternative
function of poly(ADP-ribose) synthetase inhibitors in the im-
provement of surgical diabetes. In this case, poly(ADP-ri-
bose) synthetase inhibitors may relieve restriction of DNA
replication and so cause B-cell regeneration. Since a low
capacity for B-cell regeneration has been suggested as a
predisposition for the development of human diabetes,19 the
present study may provide a novel clue for the prevention
and treatment of human diabetes.
ACKNOWLEDGMENTS
We thank Susan Behague for her assistance with the man-
uscript. This work was supported in part by grants-in-aid for
Cancer Research and for Scientific Research from the Min-
istry of Education, Science and Culture, Japan.
403
AMELIORATION OF DIABETES MELLITUS BY POLY(ADP-RIBOSE) SYNTHETASE INHIBITORS
REFERENCES
1
Mering, J. von, and Minkowski, 0.: Diabetes mellitus nach Pankreas-
exstirpation. Arch. Exp. Pathol. Pharmakol. 1890; 26:371-81.
2 10
Dunn, J. S., Sheehan, H. L, and McLetchie, N. G. B.: Necrosis of
islets of Langerhans produced experimentally. Lancet 1943; 1:484-87.
3 injection of alloxan and streptozotocin on islet proinsulin synthesis. Diabetes
Rakieten, N., Rakieten, M. L, and Nadkarni, M. V.: Studies on the
diabetogenic actions of streptozotocin. Cancer Chemother. Rep. 1963; 29:91-
98.
4
Okamoto, H.: Regulation of proinsulin synthesis in pancreatic islets
and a new aspect to insulin-dependent diabetes. Mol. Cell. Biochem. 1981;
37:43-61.
5
Yamamoto, H., Uchigata, Y, and Okamoto, H.: Streptozotocin and
alloxan induce DNA strand breaks and poly(ADP-ribose) synthetase in pan-
creatic islets. Nature 1981; 294:284-86.
6
Yamamoto, H., Uchigata, Y, and Okamoto, H.: DNA strand breaks in
pancreatic islets by in vivo administration of alloxan or streptozotocin.
Biochem. Biophys. Res. Commun. 1981; 103:1014-20.
7 in severely diabetic dogs. Diabetes 1964; 13:60-70.
Okamoto, H., and Yamamoto, H.: DNA strand breaks and poly(ADP-
ribose) synthetase activation in pancreatic islets. In Proc. 13th Int. Symp. of
The Princess Takamatsu Cancer Research Fund, ADP-ribosylation, DNA Re-
pair and Cancer. Miwa, M., Hayaishi, O., Shall, S., Smulson, M., and Sugimura,
T., Eds. Tokyo, Japan Scientific Societies Press, 1983:297-308.
8
Yamamoto, H., and Okamoto, H.: Protection by picolinamide, a novel
inhibitor of poly(ADP-ribose) synthetase, against both streptozotocin-induced
depression of proinsulin synthesis and reduction of NAD content in pancreatic
islets. Biochem. Biophys. Res. Commun. 1980; 95:474-81.
9 19
Uchigata, Y, Yamamoto, H., Kawamura, A., and Okamoto, H.: Pro-
tection by superoxide dismutase, catalase, and poly(ADP-ribose) synthetase
404
inhibitors against alloxan- and streptozotocin-induced islet DNA strand breaks
and against the inhibition of proinsulin synthesis. J. Biol. Chem. 1982;
257:6084-88.
Uchigata, Y, Yamamoto, H., Nagai, H., and Okamoto, H.: Effect of
poly(ADP-ribose) synthetase inhibitor administration to rats before and after
1983; 32:316-18.
11
Foglia, V. G.: Caracteristicas de la diabetes en la rata. Rev. Soc.
Argent. Biol. 1944; 20:21-37.
12
Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J., and Meyer, H. G.:
The unlabeled antibody enzyme method of immunohistochemistry. J. Histo-
chem. Cytochem. 1970; 18:315-33.
13
Opie, E. L: On the histology of the islands of Langerhans of the
pancreas. Bull. Johns Hopkins Hosp. 1900; 11:205-209.
14
Martin, J. M., and Lacy, P. E.: The prediabetic period in partially
pancreatectomized rats. Diabetes 1963; 12:238-42.
15
Volk, B. W., and Lazarus, S. S.: Ultrastructure of pancreatic B cells
16
Burzio, L, and Koide, S. S.: A functional role of polyADPR in DNA
synthesis. Biochem. Biophys. Res. Commun. 1970; 40:1013-20.
17
Smulson, M. E., and Rideau, C : Nuclear polyadenosine diphos-
phoribosylation during restricted macromolecular synthesis of HeLa cells.
Biochim. Biophys. Acta 1972; 272:408-16.
18
Gunnarsson, R., Christian, B., and Hellerstrom, C: Cytotoxic effects
of streptozotocin and N-nitrosomethylurea on the pancreatic B cells with spe-
cial regard to the role of nicotinamide-adenine dinucleotide. Biochem. J. 1974;
140:487-94.
Hellerstrom, C , Andersson, A., and Gunnarsson, R.: Regeneration
of islet cells. Acta Endocrinol. 1976; 83 (Suppl. 205): 145-60.
DIABETES, VOL. 33, APRIL 1984