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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 58:B598-B610 (2003)
© 2003 The Gerontological Society of America

Dietary Restriction and Beta-Cell Sensitivity to Glucose in Adult Male Rhesus Monkeys

Theresa A. Gresl1,4, Ricki J. Colman1, Thomas C. Havighurst2, David B. Allison5, Dale A. Schoeller4 and Joseph W. Kemnitz1,3

1 Wisconsin National Primate Research Center, Madison.
2 Department of Biostatistics and Medical Informatics, University of Wisconsin–Madison.
3 Department of Physiology
4 Department of Nutritional Sciences
5 Department of Biostatistics, University of Alabama–Birmingham.


   Abstract
Top
 Abstract
Research Design and Methods
 Results
 Discussion
 References
 
We examined the effects of dietary restriction (DR) and age on ß-cell function and peripheral insulin sensitivity in rhesus monkeys. A semipurified diet was provided either ad libitum for ~8 hours/day to controls (C) or as ~70% of baseline intake to restricted (R) animals for 10 years. The minimal model of C-peptide secretion and kinetics and the labeled 2-compartment minimal model of glucose kinetics were identified using plasma glucose, C-peptide, and insulin concentrations during an intravenous glucose tolerance test. R monkeys had less body fat, lower basal ß-cell sensitivity to glucose (Øb), greater insulin sensitivity, and lower first-phase plasma insulin response. DR did not significantly affect first-phase and second-phase ß-cell sensitivity to glucose. Indices of body fatness were highly predictive of the effect of DR on Øb, fasting insulin concentration and insulin responses to glucose. Enhanced peripheral insulin sensitivity among R monkeys was strongly correlated with lower Øb.

DIETARY restriction (DR) without malnutrition has been shown consistently to increase life span and to delay the onset, lessen the severity, and decrease the incidence of diseases of later life in various species (1). Several laboratories, including our own, have reported that moderate DR in both rhesus or cynomolgus macaques reduced fasting insulin and insulin responses to glucose, as well as enhanced insulin-stimulated glucose disposal compared with ad libitum-fed age-matched controls (2–7). In addition, fasting glucose is reduced among restricted animals (4–6), although this finding is less consistent among nonhuman primate studies (2,3,8). The magnitude of the reduction in fasting glucose is considerably less in monkeys than that observed in rodents (9,10), perhaps due to the lower level of restriction used for nonhuman primates (~20%–30%) versus in rodents [often >=40%; see also (11)]. This may have important implications for life-span extension given the emerging role of insulin in oxidative stress with age (12) and the association among elevated insulin levels, insulin resistance, and risk for development of metabolic disorders (13).

Three studies currently under way in the United States are investigating the effects of DR in rhesus monkeys using different methodologies. Ours, begun at the University of Wisconsin in 1989, is a multifaceted examination of many areas of health assessment, including body composition and glucose regulation (14).

Glucose tolerance, a function of both insulin secretion and sensitivity, is often observed to be reduced in late adulthood (15). Confounding the interpretation of this observation is the age-related increase in body fat, especially centrally deposited fat, in both monkeys (16–18) and humans (19). There is abundant evidence linking body fat or its distribution to dysregulation of glucose homeostasis by alterations in both insulin secretion and action, although there is no clear cause/effect relationship (e.g., 16,20–25). Whether fat mass, fat distribution, or some metabolite that either changes in parallel with it or that is secreted by adipocytes, alters insulin-related variables remains an important question.

Rhesus monkeys exhibit a progression in the development of impaired glucose tolerance (IGT) and type 2 diabetes similar to that observed in humans and so serve as a useful model for studying the dysfunction of glucose homeostasis in humans (23,26). The estimates of insulin sensitivity that we have reported previously in this group of animals (4,5,27) were based on minimal modeling (28) of plasma insulin and unlabeled glucose from the tolbutamide-modified frequently sampled intravenous glucose tolerance test. The insulin sensitivity index from the model of Bergman and colleagues provides an estimate of the effects of insulin both to inhibit hepatic glucose production and to stimulate glucose uptake.

The goal of the present study was to further characterize whole-body glucose regulation and, specifically, ß-cell function of these animals. Therefore, at the 10-year assessment period, we performed intravenous glucose tolerance tests using stable isotope-labeled glucose and, with minimal model analysis employing a 2-compartment model of glucose kinetics (29), estimated peripheral tissue insulin sensitivity. We simultaneously assessed indices of ß-cell secretion using the minimal model of C-peptide kinetics and secretion (30) as described in this article. Furthermore, since loss of fat mass is a reliable effect of DR and is a strongly associated improvement of glucose regulation in both animals and humans, we examined the relationship between body fat and group differences in insulin secretion.


   RESEARCH DESIGN AND METHODS
Top
 Abstract
 Research Design and Methods
Results
 Discussion
 References
 
The research design and general methodology of the study have been previously described (5,27). Briefly, semiannual assessments, including measures of body composition and glucose regulation, were performed annually or semiannually for 10 years in a group of adult male rhesus monkeys. Half of the animals (restricted, R) were fed approximately 70% of their individual baseline consumption levels of a pelleted, purified diet (Teklad, Madison, WI). Age-matched controls (C) were allowed ad libitum intake of the same diet 6–8 hours each day, after which any wasted or remaining food in the cages was removed. Macronutrient content of the diet consisted of ~65% carbohydrate, 15% protein, and 10% fat by weight. A micronutrient mixture was included in the diet. The diet of the R monkeys was additionally supplemented by 30% with the vitamin and mineral mixture. All animals also received a piece of fruit daily. The findings reported here reflect data collected only at the 10-year assessment period of this continuing study.

The animals were anesthetized with ketamine HCl (15 mg/kg body weight, i.m.) and diazepam (1.25 mg/kg body weight, i.m.) for the intravenous glucose tolerance tests (IVGTTs). Additional ketamine HCl was used to maintain sedation as needed during procedure. A central venous catheter was positioned for blood sampling and administration of glucose (300 mg/kg body weight). At the 10-year assessment period, one tenth of this dosage was administered as [6,6-2H2]glucose for estimation of insulin sensitivity and glucose effectiveness indices in the 2-compartment minimal model (2CMM). Three prechallenge 2.5-mL blood samples were drawn, glucose was administered at 0 minutes over ~45 seconds, and additional blood samples were drawn at 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 60, 80, 100, 120, 140, 180, 210, and 240 minutes. Samples were centrifuged and the plasma separated into 3 aliquots and stored at -20° C for later measurement of total glucose, [6,6-2H2]glucose, C-peptide, and insulin concentrations. Only data through the 180-minute sample were used to model C-peptide and insulin secretion described below in the minimal model of C-peptide kinetics and secretion (CPMM). Data through 240 minutes were used to model labeled glucose kinetics, some of the results of which are reported here.

Approximately 4 weeks prior to the day of the IVGTT, body weight and size measurements were made while the monkeys were anesthetized with ketamine HCl (10 mg/kg body weight, i.m.). Body mass index (BMI) was calculated as body weight (kg) divided by the square of crown rump length (m2), measured with the animal in the supine position using a calibrated rule with a fixed head rest. Abdominal skinfold measurements were taken as previously described (31). Body composition was measured by dual-energy x-ray absorptiometry (DXA) (Model DPX-L, Lunar Corp., Madison, WI) while the monkeys were sedated with ketamine HCl (10 mg/kg body weight, i.m.), followed by ketamine HCl/xylazine (7 mg/kg body weight ketamine HCl, 0.6 mg/kg body weight xylazine, i.m.) for additional muscular relaxation and anesthesia (31).

Plasma glucose concentration was measured by the glucose oxidase method (YSI, Yellow Springs, OH). Labeled glucose was measured using a pentaacetate derivitization followed by gas chromatography mass spectroscopy (GCMS) analysis (32). Plasma C-peptide and plasma insulin concentrations were measured by double-antibody radioimmunoassay (Linco Research, St. Louis, MO, and Diagnostic Products Corp., Los Angeles, CA, respectively). The interassay and intraassay coefficients of variation (CVs) were 6.39% and 3.29%, respectively, for insulin, and 8.25% and 4.32%, respectively, for C-peptide. Unlabeled plasma glucose and C-peptide data from the IVGTT were submitted to the CPMM to estimate secretion parameters. The model was run using the Simulation and Analysis Modeling software, SAAM II (version 1.1.1 for Macintosh, SAAM Institute, Seattle, WA). Fasting plasma levels of glucose, C-peptide, and insulin were calculated as the mean of 3 plasma values prior to the glucose bolus. Area under the curve (AUC) was calculated for suprabasal plasma C-peptide and insulin concentrations in the first (0–10 min; C-peptide: CP0–10; Insulin: AIRG [acute insulin response to glucose]) and second (10–180 min; C-peptide: CP10–180; Insulin: Phi2) phases using the trapezoidal rule. AUC was also calculated during these phases for the glucose level above the CPMM-estimated threshold glucose concentration, h. The fraction of insulin extracted by the liver in the basal state was calculated as the difference between fasting plasma C-peptide and insulin levels, divided by fasting plasma C-peptide.

Total glycated hemoglobin was measured using a Glyco-Teck affinity column method (Helena Laboratories, Beaumont, TX). Triglycerides were measured in serum using a spectrophotometric assay from a fasted blood sample taken on a separate day during the same assessment period.

The insulin molecule of the rhesus monkey is identical to human insulin, and rhesus and human C-peptide molecules differ by only 1 amino acid at position 5 (33). C-peptide and insulin are released in equimolar amounts into the portal vein upon stimulation of the ß-cells by plasma glucose (34). C-peptide is cleared relatively slowly by the kidney and negligibly by the liver (35). By contrast, insulin exhibits a much shorter half-life in large part because of rapid extraction by the liver (36). We take advantage of the different avenues of C-peptide and insulin clearance when we model C-peptide and glucose data.

Minimal Models
We used a simplified version of model for the CPMM (Figure 1), model M3 as described by Tofolo and colleagues (30). The model equations are:


where CP1 and CP2 are the C-peptide concentrations (pmol) in compartments 1 and 2, respectively; t = time (min); k12 and k21 (min-1) are transfer rates between compartments 1 and 2, k01 (min-1) is the rate of irreversible loss from compartment 1; SR is the secretion rate (pmol/min/L) normalized to the C-peptide distribution volume of compartment 1. This model allows for a short delay in the provision (Y) of additional C-peptide for release through X (pmol), the amount of C-peptide in the ß-cells (37). Y decays rapidly and is stimulated by glucose concentration above a threshold level h, through ß:


Since the kinetics of the rhesus system are rapid, the secretion response is calculated as


The first phase ß-cell sensitivity to glucose is calculated as


where X0 is the amount of C-peptide stored in the system prior to the glucose bolus and {Delta}G is the difference between peak glucose and steady state glucose. Second phase ß-cell sensitivity to glucose is estimated by the model as ß.


Basal ß-cell sensitivity to glucose calculated as


where SRb is the basal secretion rate from the model and GSS is the end-test steady state glucose level. SRb is calculated as


where CPb is the end-test basal plasma C-peptide concentration.



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  		Figure 1. The minimal model of C-peptide kinetics and secretion (CPMM) shown as a 2-compartment representation. CP1 and CP2: C-peptide concentrations in accessible and peripheral compartments, respectively. k01, k12, and k21 are kinetic parameters. Secretion rate, SR(t), is proportional to the C-peptide content of ß cells, X(t), and the secretion rate constant, m. X(t) is refilled via the provision Y(t), stimulated by glucose concentration above a threshold level, h, through ß, which measures glucose stimulation of secretion. X0 is the amount of C-peptide initially available for secretion. {Delta}G is the maximal increment in glucose concentration above steady state (GSS). CPSS = C-peptide steady-state concentration

 
Because there is very limited information on C-peptide kinetics in rhesus monkeys and because of limitations on frequency of anesthesia and other invasive procedures in these monkeys, we estimated the kinetic parameters K01, K12, and K21 based on the work of Van Cauter and colleagues with human data (38). Therefore, results of this study must be interpreted in light of this assumption. These estimates utilize information on both age and adiposity of the subjects. At 18 to 24 years of age, the monkeys in this study were considered middle-aged and we used 40 years as an approximate human age for all animals. While several of the animals appeared overweight, we chose 30% body fat as a cut-off point for designation as "obese" and below that level as "normal" because of the natural division in the data distribution at that point. Using this criterion, 7 animals were considered obese, all of which were C, while 13 were considered normal, 3 of which were C. The transfer rate K12 was calculated and designated a "fixed" parameter in the model, 0.0487 for normal weight and 0.0490 for obese. Likewise, K21 was calculated and fixed as 0.0539 for normal weight and 0.0611 for obese similar to the human values described by Van Cauter and colleagues (38). In addition, we provided some flexibility to the modeling process by allowing the secretion rate constant, m, to be a Bayesian parameter, and the fractional clearance rate of C-peptide, K01, to be either adjustable or Bayesian. All samples in the data sets submitted to the model were weighted to describe the rising portion of the C-peptide concentration. Apart from the kinetic parameters designated as fixed in the model, we used the human values in the model as starting parameter values the first time the data were run. These values were subsequently adjusted based on the mean values after several rhesus data sets were modeled.

Weighted residuals were calculated as the differences between weighted observed values and the model predictions. All but 4 data sets (2 C and 2 R) ran well in this model; both model fit and precision of estimates were taken into account to make this determination. For 3 monkeys (2 R and 1 C), the data could not be resolved by the model, and therefore there were no data generated. For 1 C monkey, the data were resolved by the model, but the extreme values of the parameters themselves, the poor precision of parameter estimation, and the pattern of residuals rendered these data unacceptable for inclusion. It is not clear why the model could not be adequately resolved with the data from these monkeys.

The 2-compartment minimal model of glucose kinetics (2CMM) has been described elsewhere (32). Briefly, we used labeled ([6,6-2H2]glucose), unlabeled glucose, and insulin data from the same IVGTT, in combination with the model (also run in SAAM II) described by Vicini and colleagues to estimate parameters of insulin sensitivity and glucose effectiveness related to glucose uptake (29). The insulin sensitivity index (SI2) describes the effect of insulin to enhance the uptake of glucose, while glucose effectiveness (SG2) describes the effect of glucose itself to enhance its uptake, independent of a rise in insulin concentration above basal. Basal plasma clearance rate (PCR) and basal hepatic glucose production (HGRb) were calculated from SG2 as:


where {alpha} is 0.465, a rate constant which represents a portion of glucose uptake in the basal state. This value is derived from previous studies with two (2CMM) model versions identified on the data of 14 normal humans (unpublished data); and


Segregation of Control Group Animals and Other Considerations
Over the previous several years on the study, some of the C monkeys exhibited either chronic or episodic fasting hyperinsulinemia based on our previously reported criteria (4). Briefly, if for two or more consecutive assessment periods, the fasting plasma insulin level was above the 90th percentile (~55 µU/mL) of its distribution at the 5-year assessment period, we considered that animal hyperinsulinemic (hyperinsulinemic controls, HIC). There were 4 C animals in this subgroup. For some of the regression analyses, we separated the data of these from other C monkeys to allow comparison of R monkeys to the normoinsulinemic controls (NIC).

Data from the single animal with type 2 diabetes in this study were not modeled in the CPMM and its data were excluded from other C group data. Likewise, since data from 4 of the animals were not well described by the CPMM, we also did not include non-CPMM data from these 4 animals.

Statistical Methods
Comparison of treatment groups at 10 years was made by analysis of variance (ANOVA) using JMP (Version 3.2.2, SAS Institute, Cary, NC). ANOVA was carried out, first, with all C versus R monkeys and, secondly, when the C group was segregated into the HIC and remaining NIC monkeys. When the overall model F statistic for the latter ANOVA was significant (p <.05) indicating that the treatment group differences varied significantly, NIC versus R, HIC versus R, and NIC versus HIC treatment group comparisons were tested individually by Fisher's protected least significant difference procedure (39).

Because basal state ß-cell sensitivity to glucose, fasting plasma C-peptide and insulin, and insulin responses during the IVGTT differed between C and R monkeys, we examined the contribution of body fat (Z) as a mediator (40) of the relationship between DR (X) and each of these (Y) variables. DR was treated as a dichotomous variable. Briefly, first we examined correlations of all possible X–Y, X–Z, and Y–Z combinations. For each Y–Z pair, only if all correlations were significant (p <.05), we compared the slopes of 2 equations: the slope of the regression of Y on X and the slope of the same regression when the body fat variable was added as a second independent variable (Y on X + Z). We used a bootstrap analysis performed in S-PLUS (version 3.4, Insightful Corp., Seattle, WA) to estimate the variance of the differences in these slopes from 10,000 simulated data sets for each Y–Z pair, where data were randomly selected with replacement. Finally, we performed a t test to determine whether the mean of these 10,000 values was different from zero. A p value <.05 provided evidence that the fat variable altered the slope of the original Y = X regression and, thus, mediated the effect of DR on the Y variables. The percent change in slope with and without the fat variable was calculated to quantitate the effect of fat on this relationship.


   RESULTS
Top
 Abstract
 Research Design and Methods
 Results
Discussion
 References
 
Characteristics of Animals After 10 Years of DR
Characteristics for all animals (age ~19 years) whose data were modeled using the CPMM are summarized in Table 1. Apart from fasting hyperinsulinemia, HIC monkeys exhibited significantly elevated triglycerides, body fat, and abdominal circumference, and reduced peripheral insulin sensitivity. These characteristics in conjunction with hypertension and type 2 diabetes and cardiovascular disease have been referred to collectively as "syndrome X" (13,41).


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  		Table 1. Characteristics of Animals at 10-Year Assessment.

 
IVGTT Glucose, Insulin, and C-Peptide Concentration Profiles
Profiles of plasma glucose, insulin, and C-peptide during the IVGTT are shown in Figure 2A–C. Glucose disappearance rate (KG, not shown) did not differ between groups. First phase (0–10 minutes) insulin (AIRG), but not first phase C-peptide area under the curve (AUC), was greater among C versus R monkeys (AIRG: C, 10.6 ± 3.0 nM * min; R, 4.7 ± 1.0 nM * min, p =.034; first phase C-peptide AUC: C, 19.0 ± 4.0 nM * min; R, 13.7 ± 2.1 nM * min, p =.332). Second phase (10–180 minutes) insulin (Phi2) and C-peptide AUC were greater in C versus R monkeys (Phi2: C, 42.7 ± 14.2 nM * min; R, 2.5 ± 3.1 nM * min, p =.007; second phase C-peptide: C, 144.1 ± 39.5 nM * min; 15.4 ± 23.6 nM * min; p =.012). NIC monkeys also exhibited marginally greater first-phase insulin (AIRG, p =.102) and second-phase C-peptide (p =.062), and greater second-phase insulin (Phi2, p =.038) values than R monkeys.



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  		Figure 2. Plasma glucose (A, showing only control and restricted groups), insulin (B), and C-peptide (C) concentration profiles during the intravenous glucose tolerance test at the 10-year assessment period, with inset graphs of the first 20 minutes showing mean ± SE for all controls, normoinsulinemic controls, and restricted monkeys in B and C. Some SE bars are not shown due to scale

 
Plasma Glucose Stimulus, CPMM Secretion Parameters, Plasma C-Peptide and Insulin Levels, and Reconstructed Insulin Secretion Rate
Basal state glucose stimulus, Øb, and plasma levels of C-peptide and insulin are shown in Figure 3A–C. First-phase plasma glucose stimulus as AUC above h, Ø1 and plasma of C-peptide, and insulin response AUC are shown in Figure 4A–C. Second-phase variables are shown in Figure 5A–C. The reconstructed insulin secretion rate for all C, NIC, and R monkeys is shown in Figure 6. Weighted residuals, indicating model fit, are shown in Figure 7 for both C and R groups. CPMM parameter mean ± SE, including secretion parameters, are shown in Table 2. The Øb values from our middle-aged C monkeys ranged from 14–67 (109 * min-1) (with high–end HIC values) and 5–20 (109 * min-1) for R monkeys. The Ø1 values for C monkeys ranged from 65–620; R monkey values ranged from 74–342. Ø2 values from the C monkeys ranged from 10–92 (109 * min-1) and 16–50 (109 * min-1) for R monkeys.



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  		Figure 3. Basal plasma glucose concentration (A), basal ß-cell sensitivity to glucose (Øb, B), and basal plasma C-peptide and insulin concentrations (C). Values expressed as mean ± SE; some SE bars are not shown due to scale

 


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  		Figure 4. First-phase (0–10 min) plasma glucose stimulus above h, the glucose concentration threshold above which insulin secretion is stimulated (A), C-peptide minimal model of kinetics and secretion first-phase ß-cell sensitivity to plasma glucose (Ø1, B), and first-phase area under the curve plasma C-peptide and insulin responses above basal (C). Values expressed as mean ± SE; some SE bars are not shown due to scale

 


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  		Figure 5. Second-phase (10–180 min) plasma glucose stimulus above h, the glucose concentration threshold above which insulin secretion is stimulated (A), C-peptide minimal model second-phase ß-cell sensitivity to glucose (Ø2, B), and second-phase AUC plasma C-peptide and insulin responses (C). Values expressed as mean ± SE; some SE bars are not shown due to scale

 


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  		Figure 6. Reconstructed prehepatic insulin secretion rate in all controls (C), normoinsulinemic controls (NIC), and restricted (R) monkeys. Inset shows clearer view of same data through first 20 minutes. Values expressed in pmol/L/min as mean ± SE; some SE bars are not shown due to scale

 


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  		Figure 7. C-peptide weighted residuals (difference between the weighted observed values and the model predictions) for C (n = 10) and R (n = 10) monkeys

 

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  		Table 2. C-Peptide Minimal Model Parameters.

 
Body Fat as a Mediator
Because the fasting C-peptide and insulin concentrations, the insulin response variables, and basal ß-cell sensitivity to glucose estimated from the CPMM differed between R and C groups, we assessed the role of percent body fat, abdominal circumference, and total body fat as mediators (independent variables, Z) in the relationship between DR (independent variable, X) and these parameters (dependent variables, Y) (Table 3). All 3 body fat variables tested appeared to alter the initial Y on X slope for most Y variables. However, this effect was not simply due to DR (reflected in the p value for the slope of X), because when the body fat variable was added to the regression, the contribution of DR was never significant, indicating that the effect of DR on the Y variables was not independent of body fat. In contrast, the effect of body fat remained significant (p value for Z) when added to the regression equations for ß-cell sensitivity to glucose, fasting plasma insulin, and the second-phase insulin response AUC indicating that the effect of fat may be independent of DR. The percent change in slope quantified the effect of body fat to alter the relationship of the Y on X; this effect appeared to be strongest for second-phase insulin response AUC (>110%).


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  		Table 3. Effect of Fat as a Mediator (Z) in the Relationship Between Dietary-Restricted (X) and Y Variables.

 
Individual HIC Secretion Parameters
Individual CPMM secretion parameters Ø1 and Ø2, and the 2CMM insulin sensitivity index and body fat indices, are shown in Table 4 for the HIC monkeys. Two of the 3 exhibited the greatest Ø1 values of all C and R animals, while their SI2 values were also the lowest of the group.


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  		Table 4. Individual C-Peptide Minimal Model Parameters.

 

   DISCUSSION
Top
 Abstract
 Research Design and Methods
 Results
 Discussion
References
 
In the present study, we used a relatively noninvasive IVGTT procedure accompanied by mathematical modeling to examine the effect of ad libitum and chronically energy-restricted diets on pancreatic ß-cell secretion in middle-aged rhesus monkeys. Examination plasma glucose, C-peptide, and insulin levels in conjunction with the calculated secretion parameters provided a view of each phase of secretion from both pre- and posthepatic perspectives, and insight into the mechanism by which DR reduces fasting plasma insulin levels and by which plasma insulin responses may be modulated. One of the most important findings from this study is that in the fasting state, R monkeys secreted less insulin than age-matched ad libitum-fed C monkeys because their ß-cells were less sensitive to the plasma glucose stimulus. Secondly, ß-cell sensitivity following an incremental glucose stimulus was highly variable, especially among C monkeys, and while a difference between groups was not detected, some of the C monkeys clearly were more sensitive to plasma glucose than others. Finally, there is some evidence that differences in secretion between groups, as well as within the C group, are related to differences in body fatness.

Because we had no prior knowledge of C-peptide kinetics in rhesus monkeys and needed initial estimates of these parameters to run the CPMM, we calculated standard values based on the work of Van Cauter and colleagues in humans (38). Consistent with our observation that the second-phase plasma insulin response to i.v. glucose tends to peak earlier in rhesus monkeys than in humans, perhaps due to central glucose administration in our preparation, the version of the CPMM that worked best with rhesus monkey data allowed for slightly faster kinetics. In addition, because we also introduced some flexibility to the model by allowing one of the kinetic parameters (k01) to be adjustable, compensations likely arose when both kinetic and secretion parameters were estimated simultaneously. Thus, the precision of parameter estimation was somewhat compromised (30). This effect was more pronounced with first-phase than second-phase sensitivity. Although monkeys appear to have greater ß-cell sensitivity than humans, at least in the basal and second phases of secretion, rhesus monkey CPMM parameters did not differ greatly from those reported for young healthy humans. CPMM parameter values from young adult humans have been reported in the ranges of 3–6 (109 * min-1) for Øb, 50 to 422 (109) for Ø1, and 8–15 (109 * min-1) for Ø2. Greater basal sensitivity of ß-cells to glucose may be reflected in the rhesus monkeys' generally higher fasting insulin (~240 pM) and lower glucose concentrations (~3.6 mM) compared to humans.

Basal State Insulin Secretion
In the basal state, plasma glucose was not different between groups, and ß-cell sensitivity was significantly enhanced among all C and NIC monkeys (with no observable difference between these C subgroups), indicating that ß-cells of C monkeys were secreting more insulin per unit glucose stimulus than those of R monkeys. The lower ß-cell sensitivity among R monkeys, in turn, was reflected in their lower plasma C-peptide levels. All C monkeys' mean plasma insulin levels were also enhanced relative to R monkeys' levels, suggesting that in all groups a significant portion of the insulin secreted into the portal vein was extracted on first pass through the liver, as we confirmed (Table 1). Earlier in the study we reported that, at several assessment periods, R monkeys exhibited slightly (~10%), but significantly, lower mean fasting plasma glucose levels versus C monkeys (4,5). At 10 years no difference in fasting glucose between groups was evident. Had we detected a difference, a lower glucose level for R monkeys would have provided one explanation for lower plasma C-peptide and insulin levels, independent of whether ß-cell sensitivity was also lower. Because plasma glucose was not lower, however, the lower plasma C-peptide and insulin levels were the result of lower ß-cell sensitivity and not due to differences between groups in hepatic extraction. Basal hepatic insulin extraction was, in fact, nearly identical and although it appears relatively high at ~85%, a wide range of basal hepatic insulin extraction levels (53%–94%) has been reported elsewhere (42).

Dynamic State Insulin Secretion
ß-cell sensitivity to glucose did not differ in either the first or second phase of secretion. During the first phase of secretion, only a marginal difference in the glucose stimulus and no difference between groups in plasma C-peptide concentration could be detected. At the same time, plasma insulin levels were different between groups, suggesting a difference in hepatic insulin extraction. A cursory examination of the differences in plasma C-peptide and insulin levels cannot be interpreted, however, to indicate a particular level of hepatic insulin extraction under dynamic conditions, because differences in their half-lives and changing secretion rates may not be directly reflected by plasma concentrations (43,44). Since C-peptide is cleared more slowly (long half-life of ~30 min) than insulin (<15 min), it could be argued that plasma C-peptide levels measured within 10 minutes of the glucose bolus mainly reflect its secretion, not clearance, unlike later in the IVGTT. Because insulin but not C-peptide concentrations differed, insulin extraction in this early stage of the IVGTT may have been greater in the R group, lower in the C group, or both may have occurred to some degree. Although we have no evidence for a greater hepatic extraction among R, there is some evidence that additional body fat among C monkeys may have reduced hepatic extraction in that group.

Abdominal obesity, exhibited by many of the C monkeys, has been associated in dogs with hepatic insulin resistance and elevated portal fatty acid levels (45); the latter have been shown to reduce hepatic insulin extraction in vitro (46) and in vivo (47). In vitro studies with rat hepatocytes have also demonstrated that fatty acids reduce insulin binding, degradation, and function (48). Furthermore, in obese rats, hepatic triglyceride levels are inversely associated with extraction (49). Surgical removal of visceral fat in rats and the subsequent increase in hepatic insulin sensitivity suggest the importance of increased centrally deposited fat as an initiating event in insulin resistance (50), and the same has been proposed to occur in humans (20,51). Reduced insulin extraction would effectively expose peripheral tissues to excess insulin over time, increasing the risk for development of several metabolic disorders (13). Hyperinsulinemia has been proposed to be detrimental to healthy aging in general (52) and, specifically, it has been implicated in the increasing levels of oxidative stress with age (12). Thus, the flexibility in the liver's capacity to extract newly secreted insulin may be an important mechanism by which it regulates the amount of insulin to which peripheral tissues are exposed (20,51). Animals that are more insulin resistant, such as the C monkeys in the present article, require greater peripheral insulin levels to overcome insulin resistance and maintain glucose tolerance, assuming there is a primary defect in the peripheral tissues versus the ß cell. R monkeys, with lower levels of abdominal fat and greater peripheral (and possibly hepatic) insulin sensitivity, by contrast require less peripheral insulin. From a different perspective, DR may have a primary effect to decrease secretion and, in response, peripheral insulin sensitivity is increased. Limiting exposure to insulin over a lifetime may contribute to the life-span-extending effects of DR observed in a variety of species (1,53).

During the second phase of secretion, the plasma glucose stimulus was significantly lower among R monkeys likely due to greater insulin sensitivity in this group. The lower C-peptide concentration among R monkeys was due to the lower glucose stimulus, because the ß-cell sensitivity to glucose did not differ between groups. While it is not possible to determine hepatic insulin extraction under these conditions based simply on plasma C-peptide and insulin levels, we speculate that extraction may have been reduced among some of the C monkeys at this time. In support of this, we found that indicators of body fat, especially abdominal circumference, had strong mediating effects on the statistical relationship between energy intake and second-phase plasma insulin response.

Contribution of HIC to All C Group
During the dynamic phases of the IVGTT, only 2 of the 3 (obese) HIC animals' secretion parameters were greater than NIC values. However, at similar levels of glucose stimulation, these animals also exhibited some of the highest plasma C-peptide and (first-phase) insulin responses (Table 3), suggesting that they were hyperinsulinemic because of greater ß-cell sensitivity. This is consistent with the work of Kautzky-Willer and colleagues who found that hypersecretion, not reduced insulin extraction, was responsible for elevated peripheral insulin levels (54). However, this group also reported that among the obese individuals there was a strong correlation between waist-to-hip ratio and hepatic insulin extraction. This observation supports the possibility that lower insulin extraction may have contributed in part to the significantly elevated plasma insulin levels of these animals.

The fact that all 3 HIC animals exhibited relatively low SI2 values is also consistent with greater ß-cell sensitivity. The animal with the lowest SI2 showed the greatest ß-cell sensitivity during both dynamic secretion phases, perhaps, as a compensatory mechanism to maintain (marginal) glucose tolerance (KG = 1.38 %/min), the lowest of all animals in this group. Alternatively, ß-cell hypersecretion may be a primary defect leading to reduced SI2 (55,56). The importance of the hyperbolic relationship between insulin secretion and peripheral sensitivity was first described by Bergman and colleagues (57) and expanded on by Kahn and colleagues (58). Referred to as the disposition (or ß-cell compensation) index, the product of secretion and sensitivity for each individual is a constant, i.e., a reduction in insulin sensitivity is compensated for by an increase in insulin secretion to maintain glucose tolerance. If insulin secretion were elevated but still inadequate given the reduced insulin sensitivity, glucose tolerance would be compromised. Consistent with the low SI2 values and high ß-cell sensitivity indices of 2 of the 3 HIC monkeys at 10 years, we previously reported that this index was reduced during the course of this study among some of the same HIC monkeys (59). Furthermore, the single HIC monkey whose first-phase ß-cell sensitivity was not as elevated as the others', during the previous few years, had exhibited insulin sensitivity values that were somewhat elevated compared with much earlier levels, although still very low compared to those of other C monkeys. Thus, for this animal, highly elevated ß-cell sensitivity was not necessary to maintain adequate glucose tolerance. Furthermore, had insulin secretion of these HIC monkeys been even greater, glucose tolerance also may have been greater.

Elevated ß-cell sensitivity to glucose and plasma insulin levels in these HIC monkeys is, however, not inconsistent with the notion that secretory capacity may have been compromised. It has been proposed that obesity, possibly through a mechanism involving fatty acids, elevates uncoupling protein 2 (UCP2) levels in pancreatic ß cells. The increase in UCP2 ultimately leads to ß-cell dysfunction and, in turn, promotes development of type 2 diabetes. Islets of ob/ob mice, a model in which type 2 diabetes is induced by excessive obesity, have greater UCP2 mRNA and protein levels; UCP2-deficient ob/ob mice exhibit marked improvements in insulin secretion while body weight does not differ from "normal" ob/ob mice (60).

Influence of DR on Adiposity and Insulin Secretion
The mechanisms by which chronic DR exerts its effects on glucose regulation are not yet fully understood, but they may involve the loss in fat mass that accompanies long-term energy restriction (61). Whether fat mass, its distribution, or some product secreted from adipocytes alters insulin secretion (and/or insulin sensitivity) remains an important question. DR consistently results in both loss of body weight and fat and reduces circulating levels of insulin in rodents (10,62) and monkeys (2,5,6).

The mediating effect of body fat on the relationship between DR and several plasma insulin and CPMM variables in this study highlights the contribution of lower body fat content and/or a more favorable fat distribution to the effects we would otherwise attribute to DR. The greatest effect (change in slope of 125%) was observed with abdominal circumference as a mediating influence on the relationship between energy intake and second-phase insulin response, linking centrally deposited fat with alterations in insulin secretion and/or hepatic insulin extraction. This finding supports the observation that fatty acids released from a highly lipolytically active fat depot may be an important regulator of one or both of these processes (63). Specifically, free fatty acids (FFA) are important for normal potentiation of glucose-stimulated insulin secretion, yet prolonged elevation can lead to impaired ß-cell function (64). Fatty acyl coAs, the active intracellular version of FFAs, have numerous functions in the ß cell (65), and their increased concentrations have been implicated in hypersecretion of insulin through production of intermediates such as diacylglycerol (66). Cultured islets incubated with long-chain fatty acids have been shown to hypersecrete insulin by enhancing sensitivity of the secretory process through a mechanism involving elevated fatty acyl coAs and subsequent release of inhibition by hexokinase (67,68). Since the hexokinase to glucokinase activity ratio may reflect sensitivity of the ß cell to prevailing glucose (69), DR may result in reduced in ß-cell sensitivity and thus regulate fasting insulin and glucose levels by lowering fatty acyl coA levels within ß cells, secondary to reduced abdominal fat mass and reduced portal vein fatty acids. Continual exposure to elevated fatty acids, however, leads to a reduction in ß-cell secretion (64,70,71), which is inadequate to maintain glucose tolerance. Furthermore, fatty acids have also been implicated in the up-regulation of UCP2 within ß cells, which leads to secretory dysfunction (60), suggesting that a related mechanism by which DR may improve secretory function may be through a reduction in ß-cell UCP2 levels. Short-term food restriction in rodents has been shown to reduce UCP2 levels in several tissues (72).

Changes in circulating levels of insulin and glucose have been observed shortly after initiation of DR in rats (73), monkeys (74), and humans (75,76), and before changes in body composition can be detected. This suggests that DR may affect these hormones and substrates independent of a loss of fat mass. Also, DR is known to improve glucose regulation in both lean rhesus monkeys (6) and genetically obese rats (77). Considering the emerging role of adipose tissue as an endocrine organ, it is possible that early DR-induced alterations in levels of cytokines such as TNF-{alpha} (tumor necrosis factor-{alpha}) or IL-6 (interleukin-6) or other secretory products such as free fatty acids, leptin, resistin (78–81), or some other still unidentified signal(s) may be pivotal to the mechanism by which DR exerts its effects on glucose homeostasis and longevity (82).

Although relatively low insulin secretion can be indicative of dysfunctional secretory process or capacity, e.g., in elderly subjects (15) or in glucose-tolerant adult offspring of individuals with type 2 diabetes (83), this is clearly not the situation with the highly insulin-sensitive R monkeys. In addition to the lower requirement for insulin in the periphery because of elevated insulin sensitivity, another possible explanation for lower secretion in the face of a similar glucose stimulus may relate to organ size. Apart from its recognized effects on body composition in general (31,84–86), restriction of energy intake, whether in starvation or nonmalnutritional DR, is known to reduce the size of most organs, especially those of the digestive tract (87). A smaller ß-cell mass could explain a lower insulin response, yet we did not detect a difference in X0, a CPMM-estimated parameter that represents the amount of C-peptide available for release in the first phase. Ultrasound imaging of the organs of individuals with anorexia nervosa and other food-deprivation disorders has shown that pancreatic size is strongly associated with energy intake (88). Insulin (C-peptide) secretion is lower in these individuals as well. Their reduction in energy intake is also typically accompanied by inadequate micronutrient intake, unlike our R monkeys whose diet was supplemented with vitamins and minerals. It is possible that DR offsets the loss in pancreatic size with enhanced capacity of the ß cells that remain, resulting in an amount of C-peptide and insulin available for release equivalent to that of C monkeys. If the capacity of each ß cell were not enhanced in this way, a reduced ß cell content due to lower organ size would seem to suggest the cells would have to compensate by becoming more sensitive to glucose. DR has been shown previously to preserve ß-cell function in prediabetic rats (77).

In summary, we estimated body composition, ß-cell secretion, and peripheral insulin sensitivity in middle-aged rhesus monkeys 10 years into a longitudinal study of the effects of aging and moderate DR. R monkeys weighed less and had less total and abdominal body fat compared with ad libitum-fed C, a contrast that we found to be critical to the interpretation of differences between groups in several variables. R monkeys also exhibited lower basal ß-cell sensitivity and plasma C-peptide, which explained their consistently lower average fasting insulin. However, in the dynamic phases of secretion, while mean ß-cell sensitivity did not differ between C and R groups, certain C monkeys clearly demonstrated greater ß-cell sensitivity, which perhaps, along with reduced hepatic extraction, explained their greater peripheral insulin levels. These hyperinsulinemic monkeys also exhibited greater serum triglycerides and lower peripheral insulin sensitivity than either R or other more normoinsulinemic C monkeys, reminiscent of the insulin-resistant, obese, and dyslipidemic profile characterizing "syndrome X." DR lowered body fat, enhanced insulin sensitivity, and either preserved or reduced ß-cell sensitivity to glucose compared with ad libitum feeding. Jointly or individually, these characteristics effectively reduced the amount of insulin to which peripheral tissues of these animals were exposed, and likely contributed to reduced disease risk. A mediating effect of body fat was also demonstrated on the relationship between DR and plasma insulin or basal ß-cell sensitivity to glucose, highlighting a contribution of a reduced body fat content and/or a more favorable fat distribution to the effects that we would otherwise attribute to caloric restriction per se.


   Acknowledgments
 
We are grateful for the hard work of the Wisconsin Primate Research Center (WPRC) animal care, technical, and veterinary staff, and also for the many helpful suggestions provided by Drs. Gianna Toffolo and Claudio Cobelli of Padua University in running the models. This study was presented as a poster at the American Diabetes Association's 62nd Annual Scientific Sessions in San Francisco, June 2002, and an abstract only was published in Diabetes [Diabetes. 2002;51(Suppl. 2):1490]. This work was supported by grant PO1 AG 11915 from the National Institutes of Health. The WPRC is funded by NIH P51 RR00167. This is WPRC publication number 42-022.

Address correspondence to Joseph W. Kemnitz, Wisconsin Primate Research Center, 1220 Capitol Court, Madison, WI 53715. E-mail: kemnitz{at}primate.wisc.edu


   Footnotes
 
James R. Smith,, PhD, Decision Editor

Received January 28, 2003

Accepted February 25, 2003


   References
Top
 Abstract
 Research Design and Methods
 Results
 Discussion
 References
 

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