Dietary restriction and glucose regulation in aging rhesus
monkeys: a follow-up report at 8.5 yr
Theresa A.
Gresl1,2,
Ricki J.
Colman1,
Ellen B.
Roecker3,
Thomas C.
Havighurst3,
Ze
Huang5,
David B.
Allison6,
Richard N.
Bergman7, and
Joseph W.
Kemnitz1,4
1 Wisconsin Regional Primate Research Center, Madison 53715;
Departments of 2 Nutritional Sciences, 3 Biostatistics
and Medical Informatics, and 4 Physiology, University of
Wisconsin- Madison 53706; and 5 Veterans Administration
Geriatric Research, Education and Clinical Center, Madison,
Wisconsin 53715; 6 Obesity Research Center, Columbia University
College of Physicians and Surgeons, New York, New York 10025; and
7 Department of Physiology and Biophysics, University of
Southern California, Los Angeles, California 90033
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ABSTRACT |
In a longitudinal study of the
effects of moderate (70%) dietary restriction (DR) on aging, plasma
glucose and insulin concentrations were measured from semiannual,
frequently sampled intravenous glucose tolerance tests (FSIGTT) in 30 adult male rhesus monkeys. FSIGTT data were analyzed with Bergman's
minimal model, and analysis of covariance revealed that restricted (R)
monkeys exhibited increased insulin sensitivity (SI,
P < 0.001) and plasma glucose disappearance rate
(KG, P = 0.015), and reduced fasting plasma
insulin (Ib, P < 0.001) and insulin response to
glucose (AIRG, P = 0.023) compared with control
(C; ad libitum-fed) monkeys. DR reduced the baseline fasting
hyperinsulinemia of two R monkeys, whereas four C monkeys have
maintained from baseline, or subsequently developed, fasting hyperinsulinemia; one has progressed to diabetes. Compared with only
the normoinsulinemic C monkeys, R monkeys exhibited similarly improved
FSIGTT and minimal-model parameters. Thus chronic DR not only has
protected against the development of insulin resistance in aging rhesus
monkeys, but has also improved glucoregulatory parameters compared with
those of otherwise normoinsulinemic monkeys.
glucose metabolism; insulin; minimal model; energy restriction
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INTRODUCTION |
DIETARY RESTRICTION
(DR) has been shown to slow the rate of physiological decline and the
development of age-associated diseases in rodents and to extend life
span in a variety of species (42). We are studying 30 male
rhesus monkeys longitudinally to characterize changes in indexes of
glucose regulation with age and long-term moderate DR. Because the
rhesus monkey is prone to the spontaneous development of obesity and
diabetes in midlife (25, 26, 37), it is an excellent
animal model for studying the progression of metabolic changes that
occur before the onset of type 2 diabetes. This progression is similar
to that observed in humans (18). Although it remains
unclear whether it is age itself or the changes in body composition
that occur with aging that underlie the frequently reported impairments
in glucose tolerance in aging subjects, it is likely that the latter,
particularly the increase in visceral fat, may play an important role
in age-related disease (2).
We have previously reported that, after 2.5 yr of DR, these monkeys had
increased insulin sensitivity, whereas fasting plasma insulin and
glucose levels were reduced compared with controls' values.
(27). Other studies of DR in both rodents and nonhuman primates (rhesus and cynomolgus monkeys) have produced similar findings. In general, energy restriction markedly increases insulin sensitivity (6, 8), reduces fasting or mean 24-h insulin concentration (6, 20, 29, 32), and, in some
(29, 32) but not all cases (6, 8, 20),
significantly reduces fasting or mean 24-h glucose concentration
compared with control animals. Taken together, these data suggest that
DR acts to enhance glucoregulatory health in aging animals, thus
retarding the development of insulin resistance and type 2 diabetes.
Whether the aging process itself is slowed in conjunction with altered
glucose and insulin concentrations is not yet known. It has been
suggested that the reduced exposure to insulin over time
(34) or the reduction of oxidative damage (40) may be related to the life-span-extending effects of
DR. In this report, we describe the glucoregulatory-related effects of
DR and ad libitum feeding in adult male rhesus monkeys after 8.5 yr of
regular, standardized assessments.
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RESEARCH DESIGN AND METHODS |
Experimental design and subjects.
The experimental design and methodology have been described in detail
previously (12, 27, 28). Briefly, 30 adult male rhesus
monkeys (Macaca mulatta), born and raised at the Wisconsin Regional Primate Research Center, have been used in this ongoing, multidimensional study of the effects of moderate DR on aging. The
monkeys were 8-14 yr of age at the beginning of the study, and
after 8.5 yr, the mean age was 17.8 yr. The median life expectancy of
the rhesus monkey in captivity is ~26 yr, with some of the monkeys in
this colony living into their late 30s (11).
Animals were individually housed to control access to food and to allow
accurate food intake measurements. Animals had continuous access to tap
water. Room temperature was maintained at ~21°C, and the animals
were maintained on a 12:12-h light-dark cycle with lights on between 6 AM and 6 PM. The animals underwent a 2-mo prebaseline period for
adaptation to the environment before the start of the experiment.
Semiannual assessment periods included measurements of energy
expenditure, body composition, and glucose metabolism, as described below.
Diet.
Monkeys were fed a defined, pelleted diet (Teklad, Madison, WI)
comprised of 15% protein as lactalbumin, 10% fat as corn oil, and
~65% carbohydrate as sucrose, starch, and dextrin, by weight. All
monkeys were fed in the morning, and 6-8 h later, the remaining or
spilled food was removed and weighed, and the animals were given a
piece of fresh fruit. Food intake was calculated as previously described (28).
DR was 70% of individual baseline intake. This reduction in energy
intake was achieved by random assignment of animals to a treatment
group after a 3- to 5-mo period of baseline assessment, during which
food intake of the experimental diet was determined for individual
monkeys. Food intakes of the monkeys assigned to DR (R;
n = 15) were reduced from their baseline period
averages by 10%/mo for 3 mo and then maintained at this level. The
control animals (C; n = 15) continued to have free
access to food during the 6- to 8-h daily feeding period, and if all
food provided was consumed in that time, subsequent food allotments
were increased as necessary.
Body size and composition.
Semiannual body weight measurements were made while the monkeys were
anesthetized with ketamine HCl (10 mg/kg body wt im). Body mass index
(BMI) was calculated as body weight (kg) divided by the square of crown
rump length (m2), measured with the animal in lateral
recumbency by use of a calibrated rule with a fixed head rest.
Abdominal skinfold measurements were taken as previously described
(12). Beginning with 1 yr after initiation of DR, body
composition was measured annually through 5 yr with dual-energy X-ray
absorptiometry (DEXA; model DPX-L, Lunar, Madison, WI) while the
monkeys were sedated with ketamine HCl (10 mg/kg body wt im), followed
by ketamine HCl-xylazine (7 mg/kg body wt ketamine HCl, 0.6 mg/kg body
wt xylazine im) for additional muscular relaxation and anesthesia.
Glucose regulation.
Frequently sampled intravenous glucose tolerance tests (FSIGTT) were
performed semiannually according to the tolbutamide-modified minimal-model protocol (4). After an overnight (~16 h)
fast, monkeys were anesthetized with ketamine HCl (15 mg/kg body wt im)
and diazepam (1.25 mg/kg body wt im). Sedation was maintained with
additional ketamine administration (5-10 mg/kg body wt im) as
needed. A central venous catheter was positioned for blood sampling and
administration of glucose (300 mg/kg body wt at 20 min) and tolbutamide
[5 mg/kg body wt (Orinase Diagnostic, provided courtesy of Pharmacia & Upjohn, Kalamazoo, MI)] during the procedure.
Plasma glucose concentration was measured by the glucose oxidase method
(Yellow Springs Instruments, Yellow Springs, OH). Plasma insulin
concentration was measured by a double-antibody RIA. From baseline
through the 5.0-yr assessment period, this assay measured a combination
of both insulin and proinsulin (and cross-reactivity with proinsulin
was ~30%) [Binax, Portland, ME; inter- and intra-assay coefficients
of variation (CVs): 5.56% and 4.50%, respectively]. From 5.5 through
8.5 yr, the assay reagents used were more specific for insulin (Linco
Research, St. Charles, MO; inter- and intra-assay CVs: 6.39% and
3.29%, respectively). Triglycerides were measured in serum by a
spectrophotometric assay. Total glycated hemoglobin was measured using
a Glyco-Teck affinity column method (Helena Labs, Beaumont, TX).
Plasma glucose and insulin concentrations were analyzed using the
minimal-model method, which describes the dynamics of insulin and
glucose during a 3-h FSIGTT. The minimal model (version 3.0, R. N. Bergman) provides estimates of insulin sensitivity (SI) and
glucose effectiveness (SG), as well as an integrated
measure of suprabasal insulin secretion assessed as acute (0-10
min) plasma insulin response to glucose (AIRG)
(3). SI reflects the effect of insulin to
promote glucose uptake and to inhibit hepatic glucose production.
SG reflects the ability of glucose to enhance its own
uptake and to suppress hepatic glucose production independently of an
increase in insulin above a basal (Ib) level. Furthermore, SG includes an insulin-independent component: glucose
effectiveness at zero insulin (GEZI), calculated as GEZI = SG
(SI · Ib)
(23). Fasting plasma glucose (Gb) and
Ib were calculated as the average of four prechallenge
plasma values (
15,
10,
5, and
1 min). Glucose disappearance
rate (KG) was calculated as the slope of the log-linear
regression of glucose concentration above Gb between 10 and
19 min.
Separation of data and statistical analysis: criterion of
prediabetic status.
Guidelines for impaired glucose tolerance in humans are not appropriate
to use for rhesus monkeys, because monkeys tend to have higher insulin
and lower glucose plasma concentrations compared with humans. However,
the progression through prediabetic and diabetic stages in rhesus
monkeys has been documented cross-sectionally (18) and
longitudinally (19). The first identifiable changes included an increase in insulin response to glucose administration and
a rise in Ib. In the present study, the 90th percentile of the Ib distribution at baseline was taken as the point
above which the monkeys were considered relatively hyperinsulinemic and
insulin resistant and were possibly "at risk" for subsequent
worsening of glucose tolerance. This baseline value was also used
through the 5-yr assessment period and then recalculated for the change to a more specific insulin RIA at 5.5 yr; these values were used as a
guideline to identify other potentially at-risk animals. At baseline,
two monkeys in each treatment group exhibited fasting hyperinsulinemia
on the basis of this criterion; these values also fell within the
Ib range of the early prediabetic hyperinsulinemic stages
described by Hansen and Bodkin (18). SI values
of these monkeys were also relatively low (mean ± SE: 1.85 ± 0.37 × 10
5 · min
1 · pM
1
compared with group mean ± SE of 5.91 ± 0.80 at
baseline). Because of the variability of SI,
Ib may be a more useful indicator of insulin sensitivity
than the SI index itself in less insulin-sensitive individuals (36). Because the two variables have very
different curves over time, however, this may apply when individuals
are in the very early stages of the progression toward diabetes, before the reduced insulin secretion observed in the latter stages.
Individuals in the highest quartile of fasting insulin concentration
are more likely to develop impaired glucose tolerance and diabetes
within several years than individuals in the lowest quartile
(17). Both at-risk C monkeys exhibited either continual or
episodic hyperinsulinemia from baseline, and two additional C monkeys
were identified as hyperinsulinemic and relatively insulin resistant for prolonged periods. Data of these four C monkeys (referred to as
"hyperinsulinemic" for simplicity), were removed from one round of
the repeated-measures longitudinal analyses, as described below. The
hyperinsulinemia exhibited at baseline by the two R monkeys, however,
was ameliorated within 2 yr of DR.
Statistical analyses.
Statistical comparisons of treatment groups across time points after
baseline were made by repeated-measures analysis of covariance (ANCOVA)
using SAS Proc Mixed (SAS Institute, 1989, release 6.09). These
analyses were also performed when data of the four hyperinsulinemic C
monkeys were removed due to the effect on the distributions. All
longitudinal analyses reported contrast in both the R vs. all C monkeys
and R vs. normoinsulinemic C monkeys. In the latter comparison,
baseline values were included as a covariate to adjust for any
imbalance due to the exclusion of four C monkeys at baseline. Only the
figures showing Ib, AIRG, and SI
include the data of both all C and normoinsulinemic C monkeys. Thirteen
estimates of SI, primarily among R monkeys, were
significantly elevated above the 95th percentile of the distribution of
values over the 8.5 yr. We truncated these values to 33 × 10
5 · min
1 · pM
1,
roughly the 95th percentile of this distribution, to prevent influencing the estimate of the mean (38). When the
time-by-treatment group interaction was significant (P < 0.05), indicating that the treatment group differences varied
significantly across time, treatment group comparisons at each time
were tested by Fisher's protected least significant difference
procedure (33). No longitudinal statistical analyses were
performed with the hyperinsulinemic C animals as a group due to their
small number. Within-treatment group differences at baseline and 8.5 yr, shown in Table 1, were tested using
the Wilcoxon signed-rank test (for paired data), whereas differences
between R and C at each time point were tested using the Wilcoxon
two-sample test (for unpaired data) with significance at P
< 0.05. Finally, Spearman's rank correlation analysis was used
to examine the associations among estimates of adiposity (i.e., percent body fat, abdominal circumference, abdominal
skinfold thicknesses, BMI), serum triglyceride content, KG,
and SI. The Wilcoxon and Spearman's analyses were
performed using JMP statistical software (SAS Institute, 1994, version
3.2).
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Table 1.
Baseline and 8.5-yr characteristics of body composition and
FSIGTT/minimal-model variables for control and restricted monkeys
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RESULTS |
Energy intake.
Figure 1 shows energy intake over 8.5 yr
among all C, normoinsulinemic C, and R groups. Although the intended
level of restriction was 30%, 6-mo averages have ranged from 23 to
37% of C intake (mean ± SE: 29 ± 1) from 2 through 8.5 yr.
Due to a voluntary reduction in food intake by controls in the initial
months of the study, food allotment was reduced for all R monkeys at
1.5 and 4.5 yr to reestablish the ~30% difference in group intake (27). Between 6 and 8.5 yr, however, food allotments for
seven restricted monkeys were increased incrementally to maintain a minimal level of 5% body fat. In addition, if a C monkey consistently consumed all food provided on average over a 3-mo period, the food
allotment was increased by 20 g.

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Fig. 1.
Daily energy intake (kcal/day) through 8.5 yr for all
control (C; n = 13-15), normoinsulinemic control
(NIC; n = 9-11), and restricted (R;
n = 12-15) groups. Due to a voluntary reduction in
energy intake among C monkeys after baseline, food allotment among R
monkeys was further reduced at 1.5 and again slightly at 4.5 yr
(arrows) to maintain the intended 30% difference between treatment
groups. Food allotment was marginally increased during 1 time point
between 6.0 and 8.5 yr for 7 of the R monkeys based on individual need
to maintain a minimum body fat of ~5% for health reasons. All values
shown are means ± SE. Scale of graph may obscure SE bars in some
cases.
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Eight and one-half years after initiation of DR, the animals ranged in
age from 16 to 22 yr (mean ± SE: 17.8 ± 0.3). Both C and R
animals appeared healthy, although two deaths in each group occurred
since our last report on this topic (27). One death in
each group was anesthesia related; the other C group death was due to
herniation of the colon, and the R group death was due to asymptomatic cardiomyopathy.
Table 1 shows group characteristics and indexes of body composition and
glucose metabolism at baseline and 8.5 yr. Univariate analyses
reavealed that Gb, Ib, AIRG,
glycated hemoglobin, and serum triglycerides were significantly lower,
and KG and SI were higher in R vs. C monkeys at
8.5 yr. The treatment group difference in glycated hemoglobin remained
marginally significant (P = 0.073) after the data from
one monkey with diabetes were removed from the analysis. Ib
and triglyceride levels were also reduced, and SI was
significantly elevated from baseline (or 5.0 yr for triglycerides) in R
monkeys. Among C monkeys, KG, SG, GEZI, and
SI were significantly reduced from baseline values. R
monkeys were leaner than C monkeys, and this observation is confirmed
by the significant cross-sectional differences observed at 8.5 yr in
body weight, BMI, percent body fat, and abdominal circumference found
between groups (all P < 0.001). Longitudinal changes
through 8.5 yr in body weight, body fat, abdominal circumference, and
lean body mass were also apparent between R and C groups (Fig.
2, A-D,
all P < 0.001). These changes in body composition were
reported from 1.0 through 7.5 yr (12). Furthermore, at 8.5 yr and despite the loss of body weight and fat with this diet, R
monkeys exhibited a significant increase from 1.0 yr in lean tissue
mass (P = 0.044).

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Fig. 2.
Body weight (kg; A), body fat (%;
B), abdominal circumference (cm; C), and lean
body mass (kg; D) among all C (n = 13-15) and R (n = 11-15) groups through 8.5 yr after initiation of dietary restriction (DR). Dual-energy X-ray
absorptiometry data for %body fat were available annually, beginning
at 1 through 5 yr and semiannually through 8.5 yr. The
time-by-treatment interaction was significant for all 3 parameters
(P < 0.001) for R vs. C groups; it was significant for all
but lean mass for R vs. NIC (not shown). Significant cross-sectional
treatment group differences using Fisher's protected least significant
difference were seen between both R vs. C (and R vs. NIC groups) at
each time point from 1.0 yr for body weight, body fat, and abdominal
circumference, and from 2.0 yr for lean body mass (all
P < 0.05). All values shown are means ± SE.
Scale of graph may obscure SE bars in some cases.
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Figure 3, A and B,
shows Gb and Ib over time. When all C were
compared with R monkeys, there was a significant time-by-treatment interaction for Ib (C > R, P < 0.001) but not Gb (P = 0.139). However, when the
four hyperinsulinemic monkeys were removed from the analysis (i.e., R
vs. normoinsulinemic C), the time-by-treatment interactions for both
were significant (Ib, normoinsulinemic C > R, P
< 0.001; Gb, normoinsulinemic C > R, not shown,
P = 0.028). Likewise, regardless of whether the comparison
was between R and all C monkeys or R and normoinsulinemic C monkeys,
there were significant time-by-treatment interactions for
AIRG (Fig. 4, R < C,
P < 0.023; R < normoinsulinemic C, P < 0.001) and for SI (Fig. 5,
C < R, and R < normoinsulinemic C, both P < 0.001).

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Fig. 3.
Fasting plasma glucose (Gb; A) and
fasting plasma insulin (Ib; B) among all C
(n = 13-15) and R (n = 11-15)
groups through 8.5 yr after initiation of DR. A: neither the
time-by-treatment interaction (P = 0.139) nor the main
effect of treatment (P = 0.132) for glucose was
significant for R vs. C groups. However, when the hyperinsulinemic
controls (including the diabetic monkey) were removed from the
analysis, the time-by-treatment interaction was significant between R
and NIC (not shown; P = 0.028) with significant
cross-sectional treatment group differences using Fisher's protected
least significant difference evident at 2.0, 2.5, 3.5, 6.0, and
7.0-8.5 yr (P < 0.05). The elevated glucose means
among C monkeys is due almost entirely to the monkey that developed
diabetes. B: time-by-treatment interaction for insulin was significant
when comparing both C vs. R and NIC (n = 9-11) vs.
R groups (both P < 0.001), with significant
cross-sectional treatment group differences evident at all points from
2.0 yr (P < 0.05). The same longitudinal results were
seen for comparisons between C vs. R and NIC vs. R when this analysis
was carried out from baseline through 5.0 yr only, before the change in
insulin RIA kit. All values shown are means ± SE. Scale of graph
may obscure SE bars in some cases.
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Fig. 4.
Acute integrated (0-10 min) insulin response to
glucose administration (AIRG) during a frequently sampled
intravenous glucose tolerance test (FSIGTT) among all C
(n = 13-15), NIC (n = 9-11),
and R (n = 11-15) groups through 8.5 yr after
initiation of DR. The time-by-treatment interaction was significant for
R vs. C groups (P = 0.023) as well as for R vs. NIC groups
(P < 0.001). Significant cross-sectional treatment group
differences using Fisher's protected least significant difference for
R vs. C groups were evident from 2.0 yr and for R vs. NIC groups at
2.5, 3.0, and 4.5-8.5 yr (P < 0.05). All values
shown are means ± SE. Scale of graph may obscure SE bars in some
cases.
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Fig. 5.
Insulin sensitivity index (SI) derived from
minimal modeling of FSIGTT data among C (n = 10-15, without the type 2 diabetic monkey from 7.0 yr on), NIC
(n = 8-11), and R (n = 11-15)
groups through 8.5 yr after initiation of DR. The time-by-treatment
interaction was significant for both R vs. C groups and R vs. NIC
groups (both P < 0.001) through 8.5 yr. Significant
cross-sectional treatment group differences using Fisher's protected
least significant difference were evident between R vs. C groups at
2.0-3.0 and 4.5-8.5 and for R vs. NIC groups at 2.5, 3.0, and
4.5-8.5 yr (all P < 0.05). A significant
time-by-treatment interaction was evident for R vs. C groups
(P = 0.002) as well as for R vs. NIC groups
(P < 0.001) when the analysis of covariance was
carried out from baseline through 5.0 yr, before the change in insulin
RIA kit. All values shown are means ± SE. Scale of graph may
obscure SE bars in some cases.
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The time-by-treatment interaction for KG (Fig.
6) reached statistical significance when
all C were compared with R (P = 0.015), but this was only
marginally significant when R were compared with normoinsulinemic C
monkeys (P = 0.064). Treatment groups did not differ over
time with respect to either SG (R vs. normoinsulinemic C,
P = 0.314; R vs. all C, P = 0.319) or
GEZI (R vs. normoinsulinemic C, P = 0.572; R vs. all C,
P = 0.478).

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Fig. 6.
Glucose disappearance rate (KG) calculated
from 10- to 19-min FSIGTT data for all C (n = 13-15) and R (n = 11-15) groups through 8.5 yr after initiation of DR. The time-by-treatment interaction was
significant for R vs. C groups (P = 0.015) but only
marginally significant for R vs. NIC (not shown; P = 0.064) through 8.5 yr. Significant cross-sectional treatment group
differences using Fisher's protected least significant difference were
evident for R vs. C groups at 3.0, 3.5, and 7.5-8.5 yr (P
< 0.05).
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Associations between the estimates of adiposity and serum triglyceride
level vs. KG and SI within both C and R groups
at the 8.5-yr assessment period did not achieve statistical
significance, with the following exceptions. In C monkeys only, serum
triglyceride level was associated inversely with SI
(r =
0.62; P = 0.042) and marginally
associated with KG (r =
0.48;
P = 0.094). In addition, among C monkeys there was a
marginally significant association between abdominal circumference and
KG (r =
0.52; P = 0.070).
In addition, serum triglyceride level was not associated with any
estimate of adiposity in either group, whereas KG was
significantly associated with SI in both C
(r = 0.62; P = 0.040) and R
(r = 0.66; P = 0.026) groups.
Because we substituted a more specific insulin RIA at 5.5 yr (Linco)
for the one we had used through the 5-yr assessment (Binax), we
performed a small study to examine its effect, if any, on FSIGTT and
minimal-model parameters (data not shown). From FSIGTT plasma samples
taken from another group of rhesus monkeys on the same purified diet,
we measured insulin concentration with both Binax and Linco kits, and
each data set was analyzed with the minimal model. Although the
Linco-measured insulin concentrations were generally lower than the
corresponding Binax-measured values throughout the FSIGTTs and fasting
insulin concentration differed significantly (P = 0.046), there was no evidence that group means differed for any other
FSIGTT and minimal-model estimates (e.g., SI,
SG, AIRG, and GEZI, all P > 0.05).
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DISCUSSION |
We have previously reported that 2.5 yr of adult-onset DR in
rhesus monkeys resulted in reduced body weight and lower central adiposity, as well as reduced fasting insulin and glucose
concentrations, glucose-stimulated insulin responses, and enhanced
insulin sensitivity compared with age-matched, ad libitum-fed controls
(27). After 8.5 yr, as the animals were advancing into
middle age, these changes were still apparent. Glucose tolerance was
maintained similar to baseline levels in R monkeys, whereas among C
monkeys it had begun to decline. These results are consistent with the
DR-induced changes in glucose regulation seen in other studies of aging
nonhuman primates (6, 8, 29, 30), among humans subjected
to brief (10, 31) and chronic reduced energy intake
(43) and, in general, with the disease-retarding effects
of DR observed in other species (42).
The R animals were lean and appeared healthy after 8.5 yr. They had
lost ~14% of their adult body weight from the onset of the study and
>50% of the fat mass present at 1 yr. Despite the loss of body weight
and fat mass, lean mass was ~9% greater at 8.5 yr than at 1 yr, and
abdominal circumference was not different from the baseline value. In
contrast, and although they also appeared to be in good health, body
weight and abdominal circumference of C monkeys had gradually increased
from the beginning of the study, consistent with the observed increase
in adiposity of aging rhesus monkeys (22, 37) and humans
(39). It is worth noting that, for the R monkeys, this is
not simply a weight (or fat) reduction study. Weight and fat reduction
were expected outcomes in these animals, but in contrast to many or
most human weight reduction studies, our data support maintenance or an
increase in fat-free mass (12, 13), suggesting an
adaptation to chronic moderately restricted energy intake. The wide
range of body fat after 8.5 yr among monkeys subjected to a similar
level of DR supports the notion that body composition, particularly
total body fat, is not indicative of the level of restriction and
suggests that energy efficiency varies greatly in this group.
Furthermore, it is not clear that DR exerts its effects through the
loss of fat mass.
Development of hyperinsulinemia among four control animals.
At baseline, two R and two C monkeys exhibited Ib above the
90th percentile of the Ib baseline distribution
(n = 30). These animals were considered relatively
hyperinsulinemic and insulin resistant and were potentially at risk at
that time for worsening of glucose tolerance. Ib levels of
the two baseline hyperinsulinemic R monkeys were reduced to levels of
other R monkeys by 2.0 yr (R group mean ± SE at 2 yr: 189 ± 27 pM), whereas the SI levels were increased, in the case
of one animal >10-fold to 14.95 × 10
5 · min
1 · pM
1.
Insulin responses to glucose, in turn, were also reduced.
Ib values of the two baseline hyperinsulinemic C monkeys,
in contrast, remained elevated most if not all of the time from
baseline, whereas their SI levels remained low (at 8.0 yr:
SI = 0.54 × 10
5 · min
1 · pM
1).
By 4 yr, two additional C monkeys had developed hyperinsulinemia for
prolonged periods, while SI remained reduced. One of these latter two C monkeys subsequently developed diabetes mellitus and
received daily insulin therapy. Although this animal was not hyperinsulinemic at baseline as two other C monkeys were, it progressed relatively quickly to type 2 diabetes, indicative of the individual variability of the development of impaired glucose tolerance in monkeys
(19). The monkeys in this study ranged in age from <10 to
17 yr, corresponding to young adulthood and early middle age in humans
(11), when changes in glucose metabolism began to emerge
and diabetes was diagnosed (19).
Longitudinal analyses.
Differences in Ib and Gb began to emerge only
after an adjustment in food allotment at 1.5 yr, which reestablished
the ~30% difference in intake between R and C groups
(27). After 8.5 yr, these differences over time continued
to be significant between the R monkeys and either all C or
normoinsulinemic C monkeys. In agreement with our preliminary findings
(28), 1 yr of DR in cynomolgus monkeys did not result in
reduced Gb despite a similar (~34%) level of restriction
(8). Lane et al. (29) found Gb in
rhesus monkeys to be reduced only after 3-4 yr of DR but noted that Gb had increased in young adult R monkeys after 7 yr,
resulting in only a small difference between the groups. Although that
study lacked an older adult R group with which to compare, the
Gb levels of older C monkeys appeared to increase over the
same time period as well. It is possible that small but significant
DR-induced reductions of Gb in younger adulthood may be
attenuated with increasing age in nonhuman primates. It is not clear
whether age or differences in feeding protocol explain the findings of
Bodkin et al. (6), who reported no reduction in
Gb after ~9 yr of weight clamping (i.e.,
maintaining body weight at a constant level by titrating energy intake).
Ib levels were markedly reduced for R monkeys; in contrast,
Ib of the normoinsulinemic C group increased and then
appeared to level off for a few years, consistent with findings
reported by Lane et al. (29). The decline in
Ib in both C and R groups at the 5.5-yr assessment is
likely due to the change to a more specific insulin RIA at that time;
however, among C monkeys, the mean Ib level subsequently
rose to pre-5-yr levels, whereas in the R monkeys, Ib
remained at lower levels. When the lower Ib levels are
taken into account after the change in insulin assay in both groups,
these data suggest an increase in Ib with age among ad
libitum-fed normoinsulinemic monkeys with perhaps little, if any, rise
observed among the R animals.
As observed in other chronically restricted monkeys (29),
the acute plasma insulin response to glucose was also elevated among C
vs. R monkeys. This difference was evident both before and after the
change in insulin assay. The apparent drop in insulin response values
among R monkeys after this change may be responsible, in part, for the
elevated SI levels from 5.5 yr on. However, the apparent
reduction in insulin response values among controls at 5.5 yr did not
result in a sustained enhancement of SI. Furthermore, because the substantial increase in SI among R monkeys is
evident 6 mo earlier (i.e., at 5 yr), it is more likely that this was due to the small reduction in food allotment following the 4.5-yr assessment period. Likewise, an increase in SI was seen
after the reduction in food allotment in R monkeys after 1.5 yr. It is
reasonable to speculate that SI levels may also reflect an adaptation to chronic DR.
In agreement with our findings that SI was markedly
increased and insulin responses were reduced in R monkeys within 1 yr of reestablishing ~30% difference in energy intake between treatment groups, Cefalu et al. (8) also reported SI to
be significantly improved after only 1 yr of DR in adult cynomolgus
monkeys. Likewise, Bodkin et al. (6) reported enhanced
maximally insulin-stimulated glucose uptake as measured during a
hyperinsulinemic euglycemic clamp procedure. Although this measure is
not directly comparable with the minimal-model-derived SI,
they are highly correlated (4, 15). Agreement of these
findings from studies with such diverse methodologies and among animals
of varied ages and species further suggests a protective effect of
energy restriction on the development of insulin resistance with
increasing age. Moreover, the elevated fasting insulin and insulin
response levels among ad libitum-fed C monkeys suggests a potentially
greater exposure of these animals to insulin over time.
Hyperinsulinemia itself, independent of hyperglycemia, has recently
been proposed to be a major contributor to oxidative damage with age
(14). Taken together, these data are consistent with a
proposed theory that DR may exert its disease-retarding and
life-extending benefits by reducing exposure to insulin over time
(34).
The results from our longitudinal analyses are also consistent with the
work of Hansen and Bodkin (20), demonstrating that prevention of obesity by weight clamping prevented the development of
insulin resistance and type 2 diabetes in older rhesus monkeys. DR
reduced total body fat over time in R monkeys, whereas monkeys in the C
group gained a substantial amount of fat (12), including an increase in centrally located fat mass. Clearly, increasing adiposity is associated with hyperinsulinemia and insulin resistance (5, 9, 16, 35) and likely contributed to or resulted from
the observed reduction in insulin sensitivity over time among some C
monkeys. Insulin resistance, however, frequently occurs in the absence
of overweight or obesity (1), and the question of whether
the loss of fat mass, particularly visceral fat mass, with DR is
requisite for improved insulin sensitivity is not resolved. The loss of
body fat or prevention of fat gain does not appear to entirely explain
DR-induced improvement of glucose metabolism, because chronic DR has
had this effect in younger, lean adult monkeys (29).
Short-term DR among older adult monkeys has also improved insulin
levels and insulin response to glucose before a detectable change in
body composition (30). Furthermore, when obese humans with
type 2 diabetes are briefly energy restricted, most individuals exhibit
rapid improvements in glucose metabolism while maintaining (or before a
loss of) a considerable level of adiposity (21, 24, 31,
41). Only some of our R monkeys exhibited a reduction in
abdominal circumference from baseline. Our observation that abdominal
circumference at 8.5 yr was not different from baseline also provides
support for the premise that DR may not exert its effects on glucose
regulation through a reduction in central adiposity in all animals.
Finally, elevated serum triglycerides are a feature of obesity and the
insulin resistance syndrome. Although insulin sensitivity was lower and
BMI, percent body fat, and triglyceride levels were greater in C vs. R
animals at 8.5 yr, the relationship among these variables was not
clear. Despite the evidence that increasing centrally located fat mass
may play a role in the observed decline in SI with age
(9), our correlation analyses performed at 8.5 yr revealed
that in neither group was SI significantly associated with
measures of adiposity. This may have been due to the variability of
SI and adiposity estimates among the small number of
animals. Only among C monkeys did the inverse association between
SI and triglycerides reach statistical significance. In
humans, the level of SI is an important determinant of
serum triglyceride levels, with the greatest triglyceride levels
observed among individuals within the lowest SI tertile,
independent of obesity (7). Our data support this observation.
In summary, we performed tolbutamide-modified FSIGTTs semiannually for
8.5 yr in male rhesus monkeys, one-half of which were subjected to
chronic, moderate DR, and we analyzed the data with the use of
Bergman's minimal model. Changes in indexes of glucose metabolism were
apparent within 0.5-1 yr after successfully reestablishing ~30%
difference in treatment group energy intake, and after 8.5 yr, as the
animals had entered middle age, DR protected against the development of
insulin resistance and type 2 diabetes with no apparent adverse effect.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the technical assistance of Scott Baum,
Lori Mason, Kelly Gari, and Julie Adriansjach and the veterinary and
animal care staff, as well as the suggestions and comments of Drs. Jon
Ramsey and Greg Cartee.
 |
FOOTNOTES |
National Institutes of Health Grants R01-AG-07831, PO1-AG-11915, and
P51-RR-00167 supported this study. This is publication number
40-026 of the Wisconsin Regional Primate Research Center.
Address for reprint requests and other correspondence: J. W. Kemnitz, Wisconsin Regional Primate Research Center, 1220 Capitol Court, Madison, WI 53715 (E-mail:
kemnitz{at}primate.wisc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 December 2000; accepted in final form 30 May 2001.
 |
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