DAVID E. HARRISON,. AND. LOREN. G. LIPSON1. *The ]ackson Laboratory, Bar Harbor, Maine 04609, USA; tDivision of Sciences, University of New Brunswick ...
Aging
and glucose
male
homeostasis
mice
EDWARD H. LEITER,’ LOREN G. LIPSON1 *The ]ackson St. Jo/zn,
Laboratory,
New
of Medicine,
Brunswick,
Los Angeles,
FRANCIS Bar Harbor, Canada; CA,
PREMDAS,T
Maine
04609,
and 1Division
changes in glucose specific pathogen-free
homeostasis C57BL/6J
were male
with lower nonfasting plasma glucose levels and improved clearance of either an oral or an i.p. administered glucose load in comparison with young, mature (4-5-month-old) males. The almost twofold increase in islet size correlated with a twofold increase of glucosestimulated insulin secretion from perifused islets from 25-month-old males compared with 5-month-old males. These aging male mice did not become obese, and there were no fibrotic changes associated with the hyperplastic islets observed in the old males. Thus, the findings that glucose tolerance did not deteriorate with age, coupled with the lack of evidence for impaired i3 cell responsiveness to glucose in old males, suggest that deterioration in glucose homeostasis is not an inevitable consequence of aging in the mouse. LEITER, E. H.; PREMDAS, F.; HARRISON, D. E.; LIPS0N, L. G. Aging and glucose homeostasis in C57BL/6J male mice. FASEBJ 2: 2807-2811; 1988. mice#{149} aging
USA;
tDivision
glucose tolerance
islets
insulin
mass
(10).
associated with a progressive impairment in the disposal of a glucose load, whereas circulating insulin levels are generally maintained or increased with advancing age (1). Indeed, this glucose intolerance of aging is reflected in the United States by the fact that 8% of the people over 65 have glucose intolerance disorders severe enough for a diagnosis of diabetes (2). The majority of these maturityonset cases have a type II (noninsulin dependent) diabetes mellitus characterized by insulin resistance, which in turn is associated with obesity in more than 80% of cases (3). However, it remains unclear to what IS SOMETIMES
0892-6638/88/0002-2807/$01
University
University
of New
of Southern
Brunswick,
Ca4fornia
School
resistance
secondary
.50. © FASEB
It has
been
inferred
normally
that
to glucose
the
inability
on aging
an
intrinsic loss of an adaptive mechanism In contrast to the many studies of rats, been few reports describing age-related
of i3
represents (11). there have changes in
glucose homeostasis in specific pathogen-free (SPF) colonies of inbred mice without recognized diabetes or obesity genes. Analysis of normal (+1+) mice in the Aston stock (maintained by breeding mice heterozygous for the obese (ob) gene on chromosome 6) showed that disposal best in
extent the age-associated insulin sic aging process or results from
of Sciences,
cells to respond
modest IN HUMANS
AND
changes such as increased adiposity, decreased physical activity, or dietary alterations. Since deterioration of glucose tolerance is not characteristic of all aging individuals, genetic components may also be implicated. Obesity has strong genetic components, but increasing adiposity may not be the primary cause of age-related glucose intolerance in humans (4). Considerable literature describes pathophysiological changes at the level of the pancreatic islets of rats (1, 5). Islets from older rats are larger and contain more cells, with more insulinper (3cellthan islets from younger rats (6, 7). Yet these (3 cells often exhibit glucose insensitivity (8) and other secretory anomalies (9). The decline in insulin secretion per f3 cell in perfused pancreas preparations was not modifiable by environmental (dietary) manipulation, although the dietary environment modified the size of the pancreatic endocrine
(12). AGING
E. HARRISON,
of Geriatric Medicine,
mice. Increased islet size and pancreatic insulin content in old (21-25-month-old) mice were associated
Key Words: secretion
DAVID
USA
ABSTRACT Age-dependent assessed in
in C57BL/6J
It
of a glucose young adult
load was poor in weanling (10-wk-old) mice, and
deterioration
in older
is important
that
adult
plasma
(30-60 insulin
mice, showed
wk) levels
mice (both
basal and glucose-stimulated) increased, but the hypoglycemic response to insulin decreased significantly with age, which suggests progressive impairment of tissue sensitivity to insulin. Not all inbred strains exhibited age-dependent deterioration in glucose handling.
Inbred C57BL/KsJ mice showed severe glucose intolerance only in the early preweaning period (13); a normal pattern established by 3 wk was maintained as these mice matured. The isolated perfused pancreases of 18-month-old NMRI male mice showed age-related adult
increases
in islet
volume,
in glucose-stimulated
insulin
is an intrin-
age-related
‘To whom
reprint
requests
should
be addressed.
2807
killed by cervical dislocation and the pancreases were bisected longitudinally, with half fixed in Bouin’s fluid, paraffin-embedded, and the (3 cells stained with aldehydefuchsin; the other half was extracted in acid:ethanol for determination of insulin content per mg protein (16). The mean islet area as well as the mean area of the islet occupied by aldehyde-fuchsin stained beta cells of each pancreas, was estimated using a modified linear scan technique employing an image analyzer (Optomax, Burlington, MA) (16). The tissue block was sectioned to a depth of 1 mm (level 1; proximal end of block); the next 10 serial 4-tm sections were mounted and stained, and the areas of all islet profiles greater than 50 tm in diameter were recorded. The block was then sampled as mentioned previously at two consecutive 1-mm linear scan distances (level 2, middle of block; level 3, distal end of block). This three-level sampling procedure ensured that each islet analyzed for area was different from those measured on a previous sectional profile and, thus, that the islet area distribution was representative of the entire pancreas. The image analyzer automatically quantified the percentage of isletarea occupied by granulated (aldehyde fuchsin staining) /3 cells by grey level estimation of stained vs. unstained areas within the islets.
secretion, and in pancreatic insulin content, whereas islet numbers remained relatively constant (14). In the present study, young and old male C57BL/6J mice were studied for age-related changes in glucose tolerance and pancreatic f3 cell function to test whether deterioration in glucose tolerance was an intrinsic consequence of the aging process.
MATERIALS
AND
METHODS
Mice The husbandry and specific pathogen-free status of the aging colony of barrier-maintained C57BL/6J (B6) mice at The Jackson Laboratory has been fully described elsewhere (15). The mean life span of B6 males in this colony is 815 ± 16 days. Mice were maintained within a controlled environment of 12 h light, 12 h dark, and a mean temperature of 22 ± 2#{176}C, with three or four mice caged per side in double-pen polycarbonate cages (13 x 15 x 28 cm). They were allowed free access to food (formulation 96WA, Emory Morse Co., Guilford, CT) and chlorinated drinking water. One week before glucose tolerance testing, mice were transferred from the barrier colony to a research colony, where they were handled daily to acclimate them to the handling stress involved in glucose tolerance testing. Radioimmunoassay of insulin in plasma and in the pancreas was performed as described elsewhere (16). For the glucose tolerance tests, mice were fasted from 4 PM to 8 AM, when half of the mice within an age group received a glucose load (1.5 g/kg body weight) by i.p. injection of a 15% solution (w/v) and the other half received the same dosage per os by gavage. Two weeks later, the same two groups were retested but the route of administration was reversed. Nonfasting plasma glucose levels on the same groups (sampled between 8 and 10 AM before the initiation of glucose tolerance testing) were also determined with a glucose analyzer (Beckman Instruments, Fullerton,
CA). Thus,
the same mice received
i.p. and per os, with an intervening tl of blood from the retroorbital into heparinized capillary tubes ment (time 0) and at 30, 60, and treatment. At the ages shown in
TABLE
a glucose
Islet
For each set of determinations, islets isolated simultaneously from one young (5-month-old) and one old (25-month-old) mouse were used. The mice were killed by cervical dislocation, the pancreas removed, trimmed free of adipose tissue, and minced in Hanks’ solution. Islets were isolated by collagenase digestion; mediumsized islets were collected by micropipet, and sensitivity to glucose-stimulated insulin release was determined as previously described (17). Medium-sized islets isolated from aged B6 pancreases were at least 1.5 times larger than medium-sized islets from young pancreases (as seen under the dissecting microscope). Duplicate batches of 20 islets apiece from the young and the aged pancreas were placed atop filters (Sartorius Corp., Burlingame, CA) in four conical leucite perifusion chambers of 0.07-mi capacity and perifused simultaneously at 37#{176}C. The perifusion medium [modified Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM HEPES and 0.5% bovine serum albumin] was
loading
interval of 2 wk. 50 sinus was withdrawn before
glucose
perifusion
treat-
120 mm after glucose Table 1, mice were
1. Comparison of parameters associated with glucose homeostasis in agingB6 male mice” (mean ± SEM) Insulin
Age, months 4-5
Body wt, g 28.6
± 0.5
Plasma
9.74
glucose,
mM
Plasma,
± 0.22
U/ml
28 ± 2
Pancreat
ic, pg/mg
Islet area, (no. of pancreases,
2.22 ± 0.18
0.0163
mm’ no. of islets)
± 0.0013
(5, 127) 8-9
34.6 ± 0.9”
6.88
21-25
38.9 ± 2.4”
7.06 ± 0.22”
±
045b
“Means (± SEM) are from 10-12 mice per age group except different from 4-5-month-old group at P 0.01. ‘Significantly
2808
for
32 ± 3
2.65
±
34 ± 3
3.79
± 0.40”
islet
different
0.0191
± 0.0009” (6, 55) 0.0248 ± 0.0018”” (5, 104)
017b
analysis. Nonfasting plasma glucose values from 8-9-month-old-group at P 0.01.
are
shown.
b Significantly
LEITER El AL.
warmed at 37#{176}C and equilibrated with 95% oxygen and 5% CO2 before being pumped into the chambers. Dynamic response of these four batches of islets to glucose was analyzed by shifting the glucose concentration (after an initial equilibration period of 33 mm) from 2.8 mM glucose to 16.7 mM glucose for 27 mm. Perifusate was collected at 1-mm intervals from all four chambers simultaneously with separate fraction collectors. Medium insulin content was determined by immunoassay with rat insulin as a standard (17). Statistical comparisons of data from perifusions and of glucose tolerance tests were performed using one-way analysis of variance; Students’ t test for unpaired samples was used to compare significance of differences between other parameters measured.
2.8mM
16.7mM
Ui
ISO +1 C 0 I,
E -
25
V V
.
100
z 0
75 0 Ui 0)
-J C’,
z
RESULTS
25
Data in Table 1 show age-related changes in parameters associated with glucose homeostasis in B6 males. Body weights of both older groups were significantly higher than the youngest group (P 0.01). Nonfasting plasma glucose was significantly higher (P 0.01) in the youngest group (4-5 months) than it was in either of the older groups, neither of which differed from the other. Plasma insulin level remained relatively constant over the age range examined (differences not significant). In contrast, data in Table 1 show a continued, significant increase in pancreatic insulin content and in average islet size. Compared with the 4-5-month-old group, the 21-25-month-old group showed a 71% increase in mean pancreatic insulin content and a 52% increase in mean islet size. These increases between the oldest group compared with the youngest group remained when normalized for the body weight differential. Pancreatic islet structure in the 8-month-old group sampled was unremarkable, but islet sizes were skewed toward the larger end of the normal distribution spectrum compared with the 5-month-old group. Although no histopathological lesions in islets from the 25month-old age group were observed, hyperplastic islets were present, some obviously representing fusions between adjacent islets. j3 cells within these islets were heavily granulated (aldehyde fuchsin stain), with an increased number of intra-islet capillaries occasionally observed in the large islets. Mean percentages of granulated /3 cells per islet in the 5-month-old vs the 25-month-old age groups were similar (71 vs. 80%), with the increase in the older males being significant (P < 0.001). In contrast to the structural integrity of the islets, age-related lesions in the exocrine parenchyma were noted in all five pancreases sampled; these included foci of leukocytic infiltrates and zones of exocrine cell autolysis, in some cases with extensive replacement by fatty tissue. No fibrosis was observed around islets; however, leukocytes emanating from ad-
jacent
ducts or vasculature
were occasionally
noted
at
the perimeter of an islet. Comparison of 3 cell sensitivity to glucose stimulation by perifusion of islets from 5-monthvs 25-monthold males is shown in Fig. 1. Mean insulin release rates AGING
AND
GLUCOSE
HOMEOSTASIS
IN MALE
MICE
25
30
35
40
TIME
45
50
55
60
(minI
Figure 1. Age-associated increases in biphasic insulin secretion in response to glucose. Each point represents the mean ± SEM for replicate perifusions of batches of 20 islets from seven pancreases per group. #{149}) Islets from 5-month-old males; 0) islets from 25-month-old males.
at low (2.8 mM) glucose between 26 and 33 mm (basal release) were significantly elevated in islets from the 25-month-old mice compared with those of islets from the 5-month-old mice (16.1 ± 2.0 Vs. 7.2 ± 0.7 pg insulin/(islet mm), P < 0.003). Stimulating (16.7 mM) glucose elicited pronounced first- and second-phase insulin release from both groups of islets (Fig. 1). The first phase of the biphasic release profile was equally rapid and sharply delineated in both age groups. Over the 27-mm stimulatory period, the total amount of insulin released by islets from 25-month-old pancreases was approximately twice the amount released by islets from 5-month-old pancreases (3169 ± 205 vs. 1509 ± 110 pg insulin/islet). This approximate twofold increase was highly significant (P < 0.001) and correlated well with the observed increases in mean islet size and insulin content (Table 1). When the insulin secretion data for each islet batch from the respective age groups were normalized for differences in baseline release by calculation of a glucose stimulation index [(glucose stimulated secretion basal secretion) #{247} (basal secretion)], the index was nearly identical in both groups (mean index for old 7.15 ± 1.0 vs. young 7.23 ± 0.7). This indicated no loss of glucose sensitivity by 3 cells in the islets of the 25-month-old donors. Comparison of both i.p. and oral glucose tolerance tests between 5-monthand 25-month-old males (Fig. 2) permitted further assessment of the physiological significance for the increases in age-related changes in .
-
=
mean
islet
size,
=
insulin
content,
and
response to glucose challenge found There were no significant differences
insulin
release
in
in older B6 males. in fasting (0 time) 2809
16.5
.
A
11.0
5.5 LU U) o C.)
0.0
-I
0 4
B
16.5
Cl)
4
-J
a. 11.0
5.5
0.0
0
30
60 TIME
90
120
(mm)
Figure 2. Age-associated improvements in clearance of a glucose load administered (A) orally or (B) i.p. Mice were given either an i.p. or oral test at the first age point, and 2 wk later received glucose by the alternate route. Number of males tested are shown in parentheses; data are mean ± SEM. 0) 4.5- to 5-month-old males; #{149}) 24.5- to 25-month-old males.
glucose levels between the two age groups. Regardless of route of glucose administration, the older groups cleared glucose more rapidly. Thirty mm after glucose loading, the 25-month-old, but not the 5-month-old, males held mean plasma glucose to 16 mM or below, indicating a more efficient glucose clearance (Fig. 2).
DISCUSSION
ported that insulin-secretory responsiveness of perfused pancreas-to-glucose stimulation was strongly correlated both with age-associated increases in islet area and insulin content. Although recent studies with SpragueDawley rats indicated that aging did not result in uncoupling of insulin synthesis and secretion (20), a decline in insulin secretion per /3 cell may nevertheless have occurred in these rats inasmuch as glucosestimulated insulin secretion increased twofold, whereas islet mass increased threefold (21). An age-related defect in islet sensitivity to glucose rather than a diminished capacity to secrete insulin was suggested by a study with isolated perfused male Wistar rat pancreases (22). In the present study, a nearly identical glucose stimulation index obtained from the isolated penfused islets from young and old mice failed to indicate an age-related decline in /3 cell sensitivity to glucose. A major species difference that distinguishes normal mice from normal rats is that in the former, maximum weight gain is usually attained by 8 months of age, whereas in the latter, body weight often continues to increase with age. In the present study, the increase in insulin release from glucose-stimulated, perifused islets correlated very well with age-related changes in islet area, insulin content, and accelerated glucose clearance in the whole animal. Thus, in nonobese B6 males maintained under our husbandry conditions, there was no basis for assuming diminished function per /3 cell. These findings are consistent with the finding of no age-related decline in sensitivity to trophic hormones of B6 male pituitary and testes (23). B6 mice were similar to most other inbred strains studied in this laboratory insofar as the highest nonfast-
ing plasma glucose levels were observed around weaning (at 4-5 wk of age) with a subsequent decline to lower levels thereafter. However, genetic differences between inbred strains exist that can interact to produce abnormal glucose homeostasis in males. This is especially true of certain C3H hybrids and congenics; an example is the so-called Wellesley hybrid (an Fl hybrid produced by crossing C3Hf females with I/Ln strain males) that develops a transient glucose intolerance associated with hyperinsulinemia and islet hyperplasia
(24). This syndrome has been repoduced in C3H.SW/SnJ males (an H-2 congenic resistant stock); in this strain, adrenalectomy
Studies of laboratory animals have not always been in agreement as to whether glucose homeostasis deteriorates as an intrinsic feature of the aging process. In studies using rats, several extrinsic factors may have influenced the results obtained, including obesity, the presence of fibrotic lesions around the islets of older rats (18) (possibly a function of the SPF-status of the colony), and variation in the collagenase digestion time sometimes required for isolation of islets from young vs. old rats (19). Data obtained in the present study of C57BL/6J [SPF] males (genetically standardized mice from a standard source colony) indicated that pancreatic /3 cell secretory dysfunction was not necessarily an intrinsic part of the aging process. Similarly, with strain NMRI male mice, Bonnevie-Nielsen, Skovgaard, and Lernmark (14) re2810
was found
implicated
the antagonism
absorption secretions
or of glucose rather than
to block
the syndrome,
of insulin metabolism an
intrinsic
which
action on glucose by other endocrine /3 cell aging phe-
nomenon (25). Indeed, perturbations in glycemic control exhibited by B6 mice were achieved by outcross to C3H/HeJ mice (26), and age-related declines in glucose homeostasis have been observed in one mouse colony
intolerold age. Because glucose homeostasis is under polygenic control, age-related deterioration may be a reflection of perturbations at multiple control levels, and need not be limited to age-associated reductions in insulin secretion from individual /3 cells. For example, age-associated differences in glucose-stimulated secretion of insulin by ance
(12). Our
is
not
an
data
demonstrate
inevitable
that
consequence
glucose of
LEITER ET AL.
rat /3 cellshave been attributed to alterationsin islet paracnine control mechanisms, such as somatostatin and glucagon secretion (27). This
publication
was
DK27722
from
National
the
supported
by
Institutes
grants
DK17631
and
of Health,
Bethesda,
MD.
The authors gratefully acknowledge the skilled of Harry Chapman and David Lewis.
technical
hormone 15.
16.
assistance
content
in the isolated
perfused
mouse
creas. Endocrinology 112: 1049-1056; 1983. HARRISON, D. E.; ARCHER, J. R. Physiological
for biological age in mice: relationship renal function, and longevity. Exp. 245-251; 1983. LE, P. H.; LEITER, E. H.; LEYENDECKER,J. control of susceptibility to streptozotocin
panassays
of collagen, Aging Res. 9:
R. Genetic diabetes in
effect of testosterone and H-2 haplotype. 116: 2450-2455; 1985. M. M.; PREMDAS, F. H.; LIPs0N, L. G. Insu-
inbred
mice:
Endocrinology 17.
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LERNand
Received for publication February 5, 1988. Accepted for publication May 12, 1988.
2811
