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Functional homogeneity of pancreatic islets of aging rats
Metabolism Clinical and Experimental VOL. XXXI, NO. 9
PRELIMINARY
SEPTEMBER
1982
REPORT
Functional
Homogeneity E. Reaven,
of Pancreatic
R. Solomon,
S. Azhar,
Islets of Aging Rats
and G. Reaven
islets from different regions of pancreases of aging rats were compared for size and variations in response to glucose stimulation. The results show that pancreatic islets from the ventral-duodenal and splenic regions of 12-mo-old retired breeder Spraque-Dawley rats are comparable in all respects measured: thus, pancreatic regional differences cannot explain the age-associated reduction in beta cell secretory response noted in previous studies.
W
E HAVE PREVIOUSLY indicated that beta cells from collagenase-isolated islets of aging Sprague-Dawley rats secrete less insulin in response to glucose or leucine stimulation, than do islets from young rats.‘.‘.” Islets in these initial experiments were obtained from the whole pancreas of donor rats without regard to specific regions. More recent studies of young Wistar rats have suggested, however, that islets from different regions of the pancreas may not be identical, i.e., islets from the ventral-duodenal region of the pancreas differ in endocrine cell composition4.” and in insulin secretory response’ when compared to islets from the dorsal-duodenal, body or splenic regions of the pancreas. The present study was carried out to see whether such functional differences exist also in islets of aging, Sprague-Dawley rats.
MATERIALS
AND
regardless which
the
studies
of region. and to systematically tissues
from
on young
rats
uniquely
Sprague-Dawley rats. 2 and I2 mo-of-age (retired breeders), were directly from the Charles Rivers Co. (Wilmington, Mass.)
only
responses.
mo-old rats were confined
alternate
regions
showed
low secretory
wcrc
the sequence in
used. Since
ventral-duodenal
subsequent
these
islets
experiments
had
on 12.
to only two regions, i.e.. comparisons
made of islets taken from the ventral-duodenal
were
and splcnic regions of
the pancreas. The
pancreatic
islets
were
Lacy-Kostianovskycollagenase tissue was incubated 5.8 mg/ml N.J.) Fisher
operated
at setting
reflecting
by a moditication
In brief. minced pancreatic
at 37% with collagenase in spinner
Co.,
Pittsburgh.
Pa);
#7$
increased
ratsj.
(12-mo-old
stir plate (jumbo tissue
#7 (2-mo-old
This
CLSIV.
Co.. Vineland.
the pancreatic
setting
of the
(Worthington
flasks (25 ml, Bellco
for I4 min. at stir-plating
appreciably appearing
isolated method.’
at a rapid speed by a magnetic
Scientific
incubated min.
buffer)
spinner
islet yield. and consistently
size: was
rats) or I6
flask
produced
method healthy-
islets from a large range of sizes from aging rats faithfully the in vivo situation.’
out as previously
outlined’,6
7) was included
in
incubating
mM
(25.0
each experiment
METHODS
different
all inctances,
both
Insulin
secretion
except that HEPES preincubating
glucose)
were measured
solutions.
(2.X
studies were carried buffer mM
Non-incubated
for size’ and insulin
expressed per volume
(IO in M. pH glucose)
and
islets of
release was. in
islet.’
obtained and
maintained
2-month gms), The
as previously
old rats (220-260
were used to isolate isolated
described.’
gms),
islets from
islets were then
Pancreases
or six. I2-mo-old different
from
pancreatic
used to determine
nine.
rats (550-700 insulin
regions. secretory
response to glucose. In preliminary creatic
experiments
regions as defined
examined, regions.
by Orci.
i.e., ventral-duodenal, To study
individuals
worked
together;
to
Merabolfsm.
keep
the
starting
rats, islets from
et al.4 and Baetens,
dorsal-duodenal,
all pancreatic
to keep the islets well-gassed taken
on young
regions
an effort
from
four pan-
et al.5 were
body and splenic the same rats, two
was made to work quickly
and
with CO, and 0, at all times. Care was volume
Vol. 3 1, NO. 9 (September).
of
pancreas
1982
tissue
constant
From the Department of Medicine. Stanford Universit?, School c!/’ Medicine. and Veterans Administration Medical (‘enter. pair) alto, California 94304.
Rereivedfbr publication January 15, 1982. Supported in part by Grant No. ‘4G 1237, .f>om the Nationul institute of Aging. Address reprint requests to Dr. Eve P. Reaven. Veterans .4dministration Medical Center (18281. 3801 Miranda .4venue. Palo A/to. California 94304. rr)I982 bv Grune & Stratton, tnc. 002~~04b5/~2/3109~000/$1.00/0
859
860
REAVEN ET AL.
Table 1. Regional Comparisons
of Insulin Release From Islets of
2-mo versus 12-mo-old
Rats GllJXBe mMi-Stimulated lnsul1nRelease I@ Insulin/
125
Age of DOWX Rats
Numberof Experiments
m+4olume
PallCk?aS
isletl’
15
Ventral-duodenal
0.60
-r .07
[
15
Splenic
0.84
+ .12t
9
Ventral-duodenal
0.40
? .04
[
9
Splenic
0.41
+ .02
Z-m0
12-mo
Regionof
*Triplicate tubes containing 20 islets each Were preincubated
min at 37°C
for 30
in buffer containing 2.8 mM glucose: this solution was
subsequently replaced with buffer containing 25 mM glucose and the islets were incubated at 37°C
for 60 min. Subsequently the incubation
medium was assayed for insulin content. Average islet size (volume) was estimated
from measurements
of 40
non-incubated
islets in each
experiment and insulin release (mean of triplicate tubes) was expressed per mean islet volume for that experiment. tp < 0.05
as compared to insulin release from the ventral-duodenal
islets of the same aged rats.
RESULTS AND DISCUSSION
The results in Table 1 make the following points. First, islets from the ventral-duodenal region of the pancreas of young Sprague-Dawley rats secrete less insulin (approx. 25%) in response to glucose stimulation, than do islets from the splenic regions of the same rats. Although this decrease in secretion is somewhat less than that reported by Trimble and Renold6 in young Wistar rats, it appears that the observation holds for both species. No differences are noted in the size of the islets obtained from these two regions in young rats, i.e., mean islet volume = 3.35 + .22 versus 3.43 + .26 x 10” ~1~.respectively, for islets from the ventral-duodenal and splenic regions of 2-mo-old rats.
In contrast to the situation in 2-mo-old SpragueDawley rats, glucose stimulated insulin secretion by islets from the ventral-duodenal and splenic regions of 12-mo-old rats is identical (when adjusted for islet volume). Furthermore, this response is essentially half that found in the younger animals, as shown previously. ‘J,~ The average volume of islets from the 12-mo-old animals of this study is 7.15 _t .61 and 6.60 + .45 x 1Oh p3, respectively, for the ventral-duodenal and splenic regions-indicating that the islets are essentially double the size of those of the younger animals. However, there are no volume differences among islets from different regions of the same pancreas. These results indicate that regional variations are not seen when glucose-stimulated insulin release is measured in isolated islets from different regions of the pancreas of 12-mo-old, retired breeder, Sprague-Dawley rats. As such, these data show that regional differences cannot account for the reduced secretory response of islets from aging rats.‘,2,3 On the other hand, it may be that islets from aging rats share some common characteristic with the ventral-duodenal islet of the young rat which renders both categories of islets poor responders. A deficit in glucagon-producing cells may be such a factor as suggested by Trimble and Renold for ventral-duodenal islets,6 and current studies in our laboratory are aimed at examining this possibility.
ACKNOWLEDGMENT The authors wish to thank assistance in this study.
Helen
Ho for excellent
technical
REFERENCES I. Reaven EP, Gold G, Reaven GM: Effect of age on glucosestimulated insulin release by the beta cell of the rat. J Clin Invest 64591-599,
1979 EP, Gold G, Reaven GM: Effect of age on leucineinsulin secretion by the beta cell. J Gerontol 35:324-328,
2. Reaven
induced 1980
3. Reaven EP. Reaven GM: Structure and function changes in the endocrine pancreas of aging rats with reference to the modulating effects of exercise and caloric restriction. J Clin Invest 68:75-84, 1981 4. Orci L, Baetens
D, Ravazzola
M, et al: Pancreatic
polypeptide
and glucagon: non-random distribution in pancreatic islets. Life Sciences 19:1811-1816, 1976 5. Beatens D, Malaisse-Lagae A, Perrelet A, Orci L: Endocrine pancreas: Three dimensional reconstruction shows two types of Islets of Langerhans. Sciences 206: 1323-l 324, 1979 6. Trimble ER, Renold AE: Ventral and dorsal areas of rat pancreas: Islet hormone content and secretion. Am J Physiol 240:E422-E427. 198 1 7. Lacy PE, Kostianovsky M: Method for the isolation of intact Islets of Langerhans from the rat pancreas. Diabetes 16:35-39, 1967
PRELIMINARY
SEPTEMBER
1982
REPORT
Functional
Homogeneity E. Reaven,
of Pancreatic
R. Solomon,
S. Azhar,
Islets of Aging Rats
and G. Reaven
islets from different regions of pancreases of aging rats were compared for size and variations in response to glucose stimulation. The results show that pancreatic islets from the ventral-duodenal and splenic regions of 12-mo-old retired breeder Spraque-Dawley rats are comparable in all respects measured: thus, pancreatic regional differences cannot explain the age-associated reduction in beta cell secretory response noted in previous studies.
W
E HAVE PREVIOUSLY indicated that beta cells from collagenase-isolated islets of aging Sprague-Dawley rats secrete less insulin in response to glucose or leucine stimulation, than do islets from young rats.‘.‘.” Islets in these initial experiments were obtained from the whole pancreas of donor rats without regard to specific regions. More recent studies of young Wistar rats have suggested, however, that islets from different regions of the pancreas may not be identical, i.e., islets from the ventral-duodenal region of the pancreas differ in endocrine cell composition4.” and in insulin secretory response’ when compared to islets from the dorsal-duodenal, body or splenic regions of the pancreas. The present study was carried out to see whether such functional differences exist also in islets of aging, Sprague-Dawley rats.
MATERIALS
AND
regardless which
the
studies
of region. and to systematically tissues
from
on young
rats
uniquely
Sprague-Dawley rats. 2 and I2 mo-of-age (retired breeders), were directly from the Charles Rivers Co. (Wilmington, Mass.)
only
responses.
mo-old rats were confined
alternate
regions
showed
low secretory
wcrc
the sequence in
used. Since
ventral-duodenal
subsequent
these
islets
experiments
had
on 12.
to only two regions, i.e.. comparisons
made of islets taken from the ventral-duodenal
were
and splcnic regions of
the pancreas. The
pancreatic
islets
were
Lacy-Kostianovskycollagenase tissue was incubated 5.8 mg/ml N.J.) Fisher
operated
at setting
reflecting
by a moditication
In brief. minced pancreatic
at 37% with collagenase in spinner
Co.,
Pittsburgh.
Pa);
#7$
increased
ratsj.
(12-mo-old
stir plate (jumbo tissue
#7 (2-mo-old
This
CLSIV.
Co.. Vineland.
the pancreatic
setting
of the
(Worthington
flasks (25 ml, Bellco
for I4 min. at stir-plating
appreciably appearing
isolated method.’
at a rapid speed by a magnetic
Scientific
incubated min.
buffer)
spinner
islet yield. and consistently
size: was
rats) or I6
flask
produced
method healthy-
islets from a large range of sizes from aging rats faithfully the in vivo situation.’
out as previously
outlined’,6
7) was included
in
incubating
mM
(25.0
each experiment
METHODS
different
all inctances,
both
Insulin
secretion
except that HEPES preincubating
glucose)
were measured
solutions.
(2.X
studies were carried buffer mM
Non-incubated
for size’ and insulin
expressed per volume
(IO in M. pH glucose)
and
islets of
release was. in
islet.’
obtained and
maintained
2-month gms), The
as previously
old rats (220-260
were used to isolate isolated
described.’
gms),
islets from
islets were then
Pancreases
or six. I2-mo-old different
from
pancreatic
used to determine
nine.
rats (550-700 insulin
regions. secretory
response to glucose. In preliminary creatic
experiments
regions as defined
examined, regions.
by Orci.
i.e., ventral-duodenal, To study
individuals
worked
together;
to
Merabolfsm.
keep
the
starting
rats, islets from
et al.4 and Baetens,
dorsal-duodenal,
all pancreatic
to keep the islets well-gassed taken
on young
regions
an effort
from
four pan-
et al.5 were
body and splenic the same rats, two
was made to work quickly
and
with CO, and 0, at all times. Care was volume
Vol. 3 1, NO. 9 (September).
of
pancreas
1982
tissue
constant
From the Department of Medicine. Stanford Universit?, School c!/’ Medicine. and Veterans Administration Medical (‘enter. pair) alto, California 94304.
Rereivedfbr publication January 15, 1982. Supported in part by Grant No. ‘4G 1237, .f>om the Nationul institute of Aging. Address reprint requests to Dr. Eve P. Reaven. Veterans .4dministration Medical Center (18281. 3801 Miranda .4venue. Palo A/to. California 94304. rr)I982 bv Grune & Stratton, tnc. 002~~04b5/~2/3109~000/$1.00/0
859
860
REAVEN ET AL.
Table 1. Regional Comparisons
of Insulin Release From Islets of
2-mo versus 12-mo-old
Rats GllJXBe mMi-Stimulated lnsul1nRelease I@ Insulin/
125
Age of DOWX Rats
Numberof Experiments
m+4olume
PallCk?aS
isletl’
15
Ventral-duodenal
0.60
-r .07
[
15
Splenic
0.84
+ .12t
9
Ventral-duodenal
0.40
? .04
[
9
Splenic
0.41
+ .02
Z-m0
12-mo
Regionof
*Triplicate tubes containing 20 islets each Were preincubated
min at 37°C
for 30
in buffer containing 2.8 mM glucose: this solution was
subsequently replaced with buffer containing 25 mM glucose and the islets were incubated at 37°C
for 60 min. Subsequently the incubation
medium was assayed for insulin content. Average islet size (volume) was estimated
from measurements
of 40
non-incubated
islets in each
experiment and insulin release (mean of triplicate tubes) was expressed per mean islet volume for that experiment. tp < 0.05
as compared to insulin release from the ventral-duodenal
islets of the same aged rats.
RESULTS AND DISCUSSION
The results in Table 1 make the following points. First, islets from the ventral-duodenal region of the pancreas of young Sprague-Dawley rats secrete less insulin (approx. 25%) in response to glucose stimulation, than do islets from the splenic regions of the same rats. Although this decrease in secretion is somewhat less than that reported by Trimble and Renold6 in young Wistar rats, it appears that the observation holds for both species. No differences are noted in the size of the islets obtained from these two regions in young rats, i.e., mean islet volume = 3.35 + .22 versus 3.43 + .26 x 10” ~1~.respectively, for islets from the ventral-duodenal and splenic regions of 2-mo-old rats.
In contrast to the situation in 2-mo-old SpragueDawley rats, glucose stimulated insulin secretion by islets from the ventral-duodenal and splenic regions of 12-mo-old rats is identical (when adjusted for islet volume). Furthermore, this response is essentially half that found in the younger animals, as shown previously. ‘J,~ The average volume of islets from the 12-mo-old animals of this study is 7.15 _t .61 and 6.60 + .45 x 1Oh p3, respectively, for the ventral-duodenal and splenic regions-indicating that the islets are essentially double the size of those of the younger animals. However, there are no volume differences among islets from different regions of the same pancreas. These results indicate that regional variations are not seen when glucose-stimulated insulin release is measured in isolated islets from different regions of the pancreas of 12-mo-old, retired breeder, Sprague-Dawley rats. As such, these data show that regional differences cannot account for the reduced secretory response of islets from aging rats.‘,2,3 On the other hand, it may be that islets from aging rats share some common characteristic with the ventral-duodenal islet of the young rat which renders both categories of islets poor responders. A deficit in glucagon-producing cells may be such a factor as suggested by Trimble and Renold for ventral-duodenal islets,6 and current studies in our laboratory are aimed at examining this possibility.
ACKNOWLEDGMENT The authors wish to thank assistance in this study.
Helen
Ho for excellent
technical
REFERENCES I. Reaven EP, Gold G, Reaven GM: Effect of age on glucosestimulated insulin release by the beta cell of the rat. J Clin Invest 64591-599,
1979 EP, Gold G, Reaven GM: Effect of age on leucineinsulin secretion by the beta cell. J Gerontol 35:324-328,
2. Reaven
induced 1980
3. Reaven EP. Reaven GM: Structure and function changes in the endocrine pancreas of aging rats with reference to the modulating effects of exercise and caloric restriction. J Clin Invest 68:75-84, 1981 4. Orci L, Baetens
D, Ravazzola
M, et al: Pancreatic
polypeptide
and glucagon: non-random distribution in pancreatic islets. Life Sciences 19:1811-1816, 1976 5. Beatens D, Malaisse-Lagae A, Perrelet A, Orci L: Endocrine pancreas: Three dimensional reconstruction shows two types of Islets of Langerhans. Sciences 206: 1323-l 324, 1979 6. Trimble ER, Renold AE: Ventral and dorsal areas of rat pancreas: Islet hormone content and secretion. Am J Physiol 240:E422-E427. 198 1 7. Lacy PE, Kostianovsky M: Method for the isolation of intact Islets of Langerhans from the rat pancreas. Diabetes 16:35-39, 1967