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Plasma Glucose and the Action of Calorie Restriction on Aging

Departments of ¹ Physiology, ² Cellular & Structural Biology, ³ Pathology, ⁴ Pharmacology, and⁵ the Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio.
⁶ Research Service, South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio.
⁷ Pfizer Research Laboratories, Groton, Connecticut.
⁸ Department of Pathology, Case Western Reserve University, Cleveland, Ohio.

Address correspondence to Roger McCarter, PhD, The Pennsylvania State University, Center for Developmental and Health Genetics, Gardner House, University Park, PA 16802. E-mail: rjm28{at}psu.edu

	Abstract

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We tested the hypothesis that retardation of aging by caloric restriction is due in part to decreased levels of plasma glucose over the life span. Male C57BL/6 mice expressing a human GLUT4 minigene (transgenic [TG] mice) and their nontransgenic littermates (NTG mice) were maintained under specific pathogen-free conditions. Mice were fed ad libitum (A mice) or 40% less than ad libitum (R mice) from age 6 weeks. Over the life span there were three different levels of plasma glucose, with NTGA mice having the highest daily levels, TGR mice the lowest daily values, and TGA and NTGR mice having similar levels intermediate between these values. Despite differences in plasma glucose, the differences measured in longevity (50% and 10% survival), physiology and tissue pathology were associated with diet rather than with levels of plasma glucose. We conclude that decreased plasma glucose over the life span is not an important factor in the action of calorie restriction on aging processes.

The purpose of this study was to test the hypothesis that decreased plasma glucose is an important factor in the retardation of aging by CR. The test was made possible by the availability of transgenic (TG) mice that express a human GLUT4 minigene, leading to the overexpression of GLUT4 protein in heart, skeletal muscle, and adipose tissue and that exhibit lower levels of plasma glucose (6). Our preliminary studies demonstrated that these TG mice eat the same amount of food on a daily basis as their nontransgenic (NTG) littermates do (7). Also, we found that diurnal levels of plasma glucose of TG mice were the same as those of NTG mice fed a restricted diet (40% less food than mice fed ad libitum). In addition, we found that restriction of the food intake of TG mice further lowered plasma glucose to levels significantly less than those of restricted wild-type mice and/or TG mice fed ad libitum. Thus, using fully fed (TGA) or restricted (TGR) mice and their NTG littermates, four different groups of mice were available having three different levels of plasma glucose, as follows: NTGA > TGA = NTGR > TGR. To test the hypothesis, we measured characteristics known to be modulated by aging and by CR, (i.e., survival, physiological parameters, and tissue pathology) in four groups of mice housed under specific pathogen-free (SPF) conditions: NTG and TG mice fed ad libitum (NTGA and TGA mice) and NTG and TG mice fed 40% less than ad libitum from 6 weeks of age (NTGR and TGR mice). The results demonstrate no effect of reduced plasma glucose over the life span on aging and survival of these mice.

	METHODS

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Animals
TG mice were those developed in a C57BL/6 background by Jeffrey Pessin at the University of Iowa (8,9). These mice express a human GLUT 4 minigene (11.5 kb) in a tissue-specific manner using the endogenous promoter. This genetic manipulation results in a 2- to 4-fold increase in the GLUT4 insulin-regulatable glucose transporter protein present in cells of skeletal muscle, cardiac muscle, and adipose tissue, with no detectable change in levels of GLUT4 present in other tissues, including the brain (8). Several studies using these mice have demonstrated that young TG mice have plasma glucose levels about 15%–20% lower than their NTG littermates (e.g., 6,10). Mice were bred and housed under SPF conditions in the barrier facility of the Audie L. Murphy Memorial Veterans Hospital (San Antonio, TX). All procedures for handling mice were approved by the Institutional Animal Care Committee of the University of Texas Health Sciences Center at San Antonio and by the Committee for Animal Studies of the Audie L. Murphy Memorial Veterans Hospital. Male TG mice and their NTG littermates (the progeny of heterozygous TG males and C57BL/6 females) were fed ad libitum a diet of Teklad Mouse chow LM-485 (20% protein, 6% fat, 69% carbohydrate, and 5% insoluble fiber; Harlan Teklad, Madison, WI). Water was provided ad libitum throughout life. This was acidified (pH 2.7) and filtered by reverse osmosis. At 6 weeks of age, animals were divided into four groups and housed four per cage: NTGA and TGA mice were fed ad libitum, with food consumption monitored at monthly intervals. NTGR and TGR mice were fed, at 3:00 PM, 60% of the food consumed by ad libitum-fed mice (i.e., a 40% restriction of food intake). Our previous studies in mice and in rats have demonstrated that multiple (rather than single) housing does not affect the life-extending effects of CR and that mineral supplementation is not necessary under these conditions (11,12). Eighty mice in each of the four groups were designated for survival studies, and an additional 30 mice in each group were used in longitudinal studies of physical activity, total daily energy expenditure, and body temperature.

Cross-sectional studies were carried out using 15 mice in each of the four groups at ages 6, 12, and 28 months. These studies were more invasive than those listed above, involving the collection of blood samples over a 24-hour period and tail-tendon samples. Selected mice of this study were necropsied at the designated ages for identification of tissue pathology. Only those mice whose tissues exhibited minimum autolysis were studied for the presence and severity of tissue pathology.

Cross-Sectional Measures
Plasma metabolites.-- Blood samples (200 µL) were collected by orbital sinus puncture to ensure that samples were obtained within 60 seconds of the entry of personnel into the animal facility. Previous experience showed that mice are easily startled and that reproducible measurements are possible only if plasma samples are rapidly obtained before hormonal values are altered by activation of stress responses. Samples were obtained from the same animal at 2-week intervals at four different times of the day; i.e., at 8:00 AM, 2:00 PM, 5:00 PM, and 12:00 AM. Average daily values were obtained by integrating measurements over the 24-hour time period. (Plasma corticosterone samples were obtained at 8:00 AM and 5:00 PM, at similar 2-week intervals). Blood samples were collected in capillary tubes, immediately spun down in microcentrifuge tubes to remove cells, and then frozen in liquid nitrogen for later analysis.

Plasma glucose was assayed by the glucose oxidase method using the Sigma Trinder Kit 315 (St. Louis, MO). Plasma insulin was determined using the Linco Insulin RIA kit (Linco, now part of Millipore, St.Charles, MO) and plasma corticosterone values were measured using the RSL 125I Corticosterone Kit for mice (ICN, Carson, CA). Plasma leptin was determined by enzyme-linked immunosorbent assay (ELISA) using the ADI Mouse Leptin Kit (#100 LEM; Alpha Diagnostic International [ADI], San Antonio, TX). All samples were run in triplicate.

Tendon breaking time.-- Tail-tendon breaking time was used as a measure of protein cross-linking. Increased breaking time is related to the degree of collagen cross-linking and is directly related to resistance to denaturation by urea. Tendons were dissected from the tails of 15 mice in each of the four groups at ages 6 and 12 months, washed in buffered saline solution, immediately frozen on dry ice, and shipped to Dr. Monnier's laboratory at Case Western Reserve University. Assays followed routine procedures of this laboratory (13) (i.e., weights of 2.7 g were attached to the tendons using 4.0 silk sutures, and this system was immersed in a 7 M urea solution [pH 7.5 at 40°C]). The time taken for rupture of the tendon to occur under this constant load was recorded.

Longitudinal Measures
Body weight.-- Body weight was measured correct to ± 0.1 g using a Sartorius Precision balance (Terre Haute, IN) every 2 weeks until age 24 weeks and then every month thereafter.

Physical activity.-- Spontaneous movement of mice in their cages was measured as in our several previous studies (14). In brief, the mouse cage was placed in an optical activity monitor (Digiscan; Omnitech Electronics, Columbus, OH). In the monitor, the cage is traversed by a series of interlocking infrared light beans. Movement of the animal around the cage interrupts these beams. Beam breaks are interpreted by supplied software to generate a continuous record of distance moved by the animal over a 24-hour period. Mice were placed, singly housed, in the monitor for a 72-hour period. This permitted a 24-hour acclimation period, and recordings over the following 48 hours were then averaged to provide a record of activity every 10 minutes over a 24-hour period.

Daily energy expenditure.-- During the 72-hour measurement of physical activity, air was drawn through the cage (500 mL/min in a 2-L cage). Analysis of the composition of the gas entering and leaving the cage permitted calculation of total daily energy expenditure using indirect calorimetry. The system used was that employed in several of our previous studies of metabolic rates of rats and mice (15,16). Oxygen and carbon dioxide gas contents were determined by precision instruments (Ametek SA3; CDA, AEI Company, Pittsburgh, PA) every second together with continuous recordings of humidity, pressure, and temperature, with regulation of airflow by mass-air controllers. The system was calibrated on a weekly basis using laboratory standard gases. As with physical activity measures, we used a 24-hour period of acclimation for each mouse to its cage, then records were obtained over the following 48 hours. Measurements obtained every second were averaged on an hourly basis to provide an average 24-hour measure of oxygen consumption, carbon dioxide production, and respiratory quotient (RQ, ratio of CO₂ production to O₂ consumption). The equations of Consolazio and colleagues (17) were used to determine daily energy expenditure.

Body temperature.-- The measures of activity and metabolic rate necessarily used singly-housed mice. In contrast, body temperature was measured continuously over a 48-hour period with mice in their usual home cages. This was achieved by implanting temperature transmitters in the thoracic cavity of a single mouse in each of a cage of four mice. The implantation involved a small abdominal incision, with the transmitters (Vital View System; Mini Mitter, Bend, OR) all implanted in the same location in the peritoneal cavity. The transmitters are passive, i.e., have no need of battery replacement, and so were left in mice over the entire life span. These studies revealed no differences in survival, physical activity, or metabolic rate associated with the presence of the transmitter. Fifteen mice in each group received an implant at age 4 months. Recovery from the surgery was rapid (1 week) and complete. Measurements of daily temperature were then obtained at ages 6, 12, and 28 months by placing the home cage on an energizer platform and collecting data continuously over the 48-hour period. Before implantation, all transmitters were calibrated to ± 0.05°C using a precision mercury-in-glass thermometer.

Pathology
Necropsy and histology.-- After mice were necropsied for gross pathological lesions, the following organs and tissues were excised and preserved in 10% buffered formalin: brain, pituitary gland, heart, lung, trachea, thymus, aorta, esophagus, stomach, small intestine, colon, liver, pancreas, spleen, kidneys, urinary bladder, reproductive system (prostate, testes, epididymis, and seminal vesicles), thyroid gland, adrenal glands, parathyroid glands, psoas muscle, knee joint, sternum, and vertebrae. Any other tissue with gross lesions was also excised. The fixed tissues were processed conventionally, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin–eosin. Although autolysis of varying severity occurred, it did not prevent the histopathological evaluation of lesions, with the exception of three mice. Diagnosis of each histopathological change was made with histological classifications in aging mice described by Bronson and Lipman (18).

Pathological assessment.-- A list of pathological lesions was constructed for each mouse that included both neoplastic and non-neoplastic diseases. Based on these histopathological data, the tumor burden, disease burden, and severity of each lesion in each mouse were assessed. The disease burden was calculated as the sum of the histopathological changes in a mouse as described by Bronson and Lipman (18). The severity of neoplastic and nephrologic lesions was assessed with the grading system previously described (19,20).

The percentage of tumor-bearing mice and overall and age-specific incidence of disease were calculated for each experimental group. The percentage of tumor-bearing mice was calculated as the percentage of mice that had one or more neoplastic lesions. For this assessment, all neoplastic lesions were counted regardless of the severity of tumors, i.e., both incidental (not severe enough to be the cause of death) and fatal (severe enough to be the cause of death) tumors were counted.

Grading of lesions.-- The severity of neoplastic lesions and glomerulonephritis were determined using grading systems (19,20). Severity of these lesions was determined because their high prevalence allowed a finer dissection of the effect of the treatments on the extent of neoplastic lesions and kidney pathology.

Glomerulonephritis was graded in order of increasing severity: Grade 0 = no lesions; Grade 1 = minimal change in glomeruli (minimal glomerulosclerosis); Grade 2 = Grade 1 with a few (< 10) casts in renal tubules; Grade 3 = Grade 1 with > 10 casts in renal tubules; and Grade 4 = Grade 3 with interstitial fibrosis.

Grading of neoplastic lesions was based on a modification of previously reported criteria (19,20): Grade 1 = primary site only; Grade 2 = primary site and intra-organ or one other organ metastasis; Grade 3 = metastasis to two to three organs; and Grade 4 = metastasis to more than four organs or Grade 3 + additional pathology, e.g., pleural effusion, ascites, and subcutaneous edema. Hydrothorax, ascites, and subcutaneous edema were the common complications associated with advanced neoplastic diseases.

Probable cause of death.-- The probable cause of death in each mouse was determined by the severity of diseases found by necropsy, and was assessed independently by two pathologists. For neoplastic diseases, cases that had Grades 3 and/or 4 lesions were categorized as death by neoplastic lesions. For non-neoplastic diseases, cases that had a severe lesion, e.g., Grade 4, associated with other histopathological changes (pleural effusion, ascites, congestion and edema in lung) were categorized by death by non-neoplastic lesion. In > 90% of the cases, there was agreement by the two pathologists. In cases in which there was not agreement or in which no disease was considered severe enough, cause of death was categorized as unknown.

Data Analysis
Survival curves were estimated using product limit estimates, and curves were compared using the log-rank test (21). The median and 10th percentile survival times were compared using the quantile test (22). Average 24-hour values of plasma metabolites (glucose, insulin, leptin) were computed from the area under the curve using the trapezoidal rule. These values were similar to the average of values measured at 8:00 AM, 2:00 PM, 5:00 PM, and 12:00 AM (correlation = 0.995). Average values of 24-hour physical activity and body temperature were obtained, respectively, by summing each mouse's average hourly activity over 24 hours and by averaging hourly body temperature over 24 hours. Average values were analyzed using analysis of variance (ANOVA). Comparisons among mean values were Bonferroni-adjusted. Residual analysis was used to confirm that the assumptions underlying the ANOVA were satisfied (23).

Because plasma corticosterone was measured at only two times of the day (8:00 AM and 5:00 PM), we did not compute 24-hour averages. The logarithms of the concentrations measured at 8:00 AM and 5:00 PM were analyzed separately using ANOVA. Comparisons among means were Bonferroni-adjusted. Means and standard errors were computed from untransformed data.

Total number of pathologies present in mice at necropsy was analyzed using ANOVA. A square root transformation was used to better satisfy the assumptions underlying the ANOVA. Comparisons among means were Bonferroni-adjusted. The presence of tumors, neoplasm, glomerulonephritis, acidophilic macrophage polymorphonucleocytes, and lymphocyte infiltration was analyzed using the chi-square test. If expected frequencies were small, exact tests were used (24). Tumors were observed only in old mice. In these, the number per animal was analyzed using ANOVA. A square root transformation was used to better satisfy the assumptions underlying the ANOVA. Significance of differences between mean values was assumed at p <.05.

	RESULTS

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Body Weight
Variation of body weight with age, diet, and genetic status is shown in Figure 1. The data demonstrate the absence of an effect of overexpression of GLUT4 protein on body weight over the life span. Rather, mice fed 40% less food than ad libitum, regardless of GLUT4 status, exhibited body weights about 35% less than mice fed ad libitum. Food consumed by mice fed ad libitum was similarly not affected by GLUT4 status over the life span (Figure 2).

View larger version (11K): [in this window] [in a new window]   Figure 1. Average body weight versus age for mice in each of the four groups. Results are mean ± standard error of the mean (SEM) with n = 77–80 mice per group. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum

View larger version (19K): [in this window] [in a new window]   Figure 2. Food consumed per mouse per week versus age. Mice were housed four per cage, and consumption was measured for each of 20 cages per group. Results are mean ± standard error of the mean (SEM), n = 20 mice per group. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum

View larger version (21K): [in this window] [in a new window]   Figure 3. Average 24-hour plasma glucose levels of the four groups of mice versus age. Results are mean ± standard error of the mean (SEM), n = 12 mice per cage. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. *Significantly less than NTGA value (p <.05). **Significantly less than all other values (p <.05)

View larger version (20K): [in this window] [in a new window]   Figure 4. Average 24-hour plasma insulin concentrations versus age for each of the four groups of mice. Values are mean ± standard error of the mean (SEM), n = 9–12 mice per group. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. *Significantly less than NTGA mice (p <.05). **Significantly less than all other groups of mice at given age (p <.05)

The data in Figure 5 show the variation of average daily plasma leptin with age, diet, and genotype. NTGA mice exhibited significantly higher values of plasma leptin than all other groups over the life span. TGA mice, although having body weights similar to those of NTGA mice, exhibited lower levels of plasma leptin and had significantly higher daily leptin levels than those of restricted mice. In general, over the life span, average daily leptin levels were in the order NTGA > TGA > NTGR TGR.

View larger version (16K): [in this window] [in a new window]   Figure 5. Average 24-hour plasma leptin concentration versus age of each of the four groups of mice. Values are mean ± standard error of the mean (SEM), n = 12 mice per group. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. *Significantly less than NTGA mice (p <.05). Significantly less than TGA mice (p <.05)

In mice aged 4–6 months, plasma corticosterone levels in the morning (8:00 AM) were significantly elevated in restricted versus ad libitum-fed mice, with NTGR mice exhibiting higher values of this hormone than TGR mice (Figure 6). There was no significant effect of plasma glucose level on corticosterone concentration in ad libitum-fed mice (i.e., values in NTGA mice not significantly different from those in TGA mice). Restricted mice exhibited a significant decline of plasma corticosterone from 4–6 to 12–14 months of age, with no significant effect of age thereafter (data not shown). At 5:00 PM, no significant differences were found for this hormone between NTGA, TGA, and NTGR mice. However, for TGR mice, plasma corticosterone levels were significantly lower than those of other groups of mice at ages 12–14 and 28–30 months (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 6. Mean plasma corticosterone concentration at 8:00 AM and 5:00 PM for mice of the four groups aged 4–6 months. Values are mean ± standard error of the mean (SEM), n = 3–12 mice per group. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. *Significantly less than value at 8:00 AM for NTGR mice (p <.05). Significantly less than value at 5:00 PM (p <.05)

View larger version (19K): [in this window] [in a new window]   Figure 7. Average distance traveled in spontaneous activity per mouse over a 24-hour period, for each of the four groups as a function of age. Values are mean ± standard error of the mean (SEM), n = 12 mice per group. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. *Significantly less than NTGR value at given age (p <.05). Significantly greater than NTGA, TGA values at 28–30 months of age (p <.05). Significantly less than TGA value at 4–6 months of age (p <.05)

View larger version (14K): [in this window] [in a new window]   Figure 8. Diurnal variation of body temperature in the four groups of mice aged 4–6 months. Values are mean ± standard error of the mean (SEM), n = 12. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. Temperatures of mice eating the restricted diet were significantly lower than those of mice feeding ad libitum, between the hours of 7:00 PM and 1:00 PM the following day (p <.05)

Average 24-hour body temperatures at ages 4–6, 12–14, and 28–30 months are shown in Figure 9. There was remarkable consistency of temperature for a given dietary group at ages 4–6 and 12–14 months. The results demonstrate a significantly lower body temperature in restricted versus ad libitum-fed mice at all ages. There was an age-related increase in temperature of restricted mice and an increase in temperature of mice fed ad libitum at 28–30 months of age, with temperatures of TGA mice being higher than those of all other mice. In the case of NTG mice, the difference in temperature associated with diet was diminished but not eliminated with age.

View larger version (20K): [in this window] [in a new window]   Figure 9. Mean 24-hour body temperatures of mice as a function of age and diet. Values are mean ± standard error of the mean (SEM), n = 12. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. *Significantly less than mean values of mice fed ad libitum at given age (p <.05). Significantly less than mean temperature of TGA mice aged 28–30 months (p <.05)

View larger version (14K): [in this window] [in a new window]   Figure 10. Diurnal pattern of spontaneous cage activity and body temperature for nontransgenic mice fed 40% less than ad libitum (NTGR) aged 4–6 months. Values are mean ± standard error of the mean (SEM), n = 12

View larger version (20K): [in this window] [in a new window]   Figure 11. Mass-specific 24-hour metabolic rate of mice as a function of age. Values are mean ± standard error of the mean (SEM), n = 12. NTGA, nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. *Mean value significantly less than those of mice fed the restricted diet at age 4–6 months (p <.05)

View larger version (14K): [in this window] [in a new window]   Figure 12. Diurnal variation of respiratory quotient (RQ; ratio of carbon dioxide production to oxygen consumption) for mice aged 4–6 months. Values are mean ± standard error of the mean (SEM), n = 12. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum. Values of RQ for NTGR mice were significantly less than those of NTGA mice from 12:00 AM to 2:00 PM; greater than those of NTGA mice from 4:00 PM to 9:00 PM

View larger version (14K): [in this window] [in a new window]   Figure 13. Tendon breaking time for mice aged 12–14 months. Values are mean ± standard deviation, n = 10. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum. *Significantly different values than mice fed ad libitum (p <.05)

View larger version (15K): [in this window] [in a new window]   Figure 14. Total number of pathologies present at various ages in tissues of mice. Values are mean ± standard error of the mean (SEM), n = 4–10. *Significantly lower values recorded in mice fed the restricted diet versus mice fed ad libitum at age 12–14 months

View larger version (14K): [in this window] [in a new window]   Figure 15. Percentage of mice surviving versus age in each of the four groups of mice, n = 77–80. NTGA = nontransgenic mice fed ad libitum; NTGR = nontransgenic mice fed 40% less than ad libitum; TGA = transgenic mice fed ad libitum; TGR = transgenic mice fed 40% less than ad libitum

	DISCUSSION

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Plasma glucose is a promising agent for involvement in mechanisms of aging. This promise arises from: (i) its status as a closely regulated and essential tissue nutrient; (ii) the recognition that long-term exposure of macromolecules to reducing sugars leads to nonenzymatic glycation reactions (AGE), which irreversibly alter chemical properties of these molecules; and (iii) the clinical finding that diabetics having elevated levels of plasma glucose exhibit characteristic features of accelerated aging. These facts led Cerami (2) to propose the Glycation Hypothesis of Aging. Consistent with this proposal, evidence has accumulated demonstrating an inverse relationship between rates of formation of AGE and species-specific life span (3). There is also evidence that formation of AGE promotes oxidative stress and, conversely, that increased oxidative stress promotes formation of AGE. Such positive feedback cycles would promote the accelerated tissue degeneration typical of aging (34).

As elevated plasma glucose appears to accelerate degenerative processes, it is possible that reduced levels of plasma glucose of CR animals might constitute part of the anti-aging action of CR. A test of this hypothesis was made possible by the creation of the TG GLUT4 mouse by Pessin and colleagues (8). Several studies using young (2-month-old) mice have demonstrated that fasted TG GLUT4 mice exhibit plasma glucose values about 20% less than those of fasted NTG littermates (6,8,10). One of these studies also reported no difference in levels of plasma insulin in the conscious and/or anesthetized state between TG and NTG mice (10). Other researchers, using hemizygotic variants of these mice having higher levels of GLUT4 protein expression, found significantly lower levels of both plasma glucose and insulin in young, fasted mice (35,36). However, for our purposes a significant lowering of both plasma glucose and insulin would not be useful because CR also results in decreased plasma insulin (4) and insulin has been identified as yet another potential factor in mechanisms of aging (33). Our preliminary studies indicated no difference in body weight, food consumption, and plasma insulin values in young (3 month) as well as old (24 month) male TG and NTG mice (7). Lifelong studies were therefore initiated to measure characteristics known to be influenced by CR in aging male mice. It should be noted that the intent of our studies was different from those of prior studies: Issues addressed by the earlier work relate to glucose homeostasis and its regulation by GLUT4 proteins (6,8,10,35,36). In our studies, the focus was on the average daily value of plasma metabolites in vivo and their variation over the life span. We obtained samples of blood at four different times throughout the 24-hour cycle in conscious mice at different ages. Previous measurements of plasma metabolites of GLUT4 mice were obtained mostly in fasted, anesthetized young animals at a single age.

The data demonstrate no difference in body weight over the life span of TG and NTG mice, fed either ad libitum or 40% less than ad libitum (Figure 1), respectively. Similarly, no differences were found in food consumed by TG and NTG mice when fed ad libitum, and CR mice consumed all food provided to them each day (Figure 2). The average daily values of plasma glucose over the life span (Figure 3) demonstrate that significant differences (associated with diet and genetic status) between groups of mice persisted over the life span. Over the life span of all mice there were three different levels of plasma glucose, with NTGA mice having consistently highest daily values, TGR mice lowest daily values, and TGA and NTGR mice having similar levels intermediate between these values. If decreased plasma glucose of CR mice is an important factor in the anti-aging action, these three different levels should have resulted in three different patterns of longevity, functional decline, and tissue pathology. Figure 15 shows that survival of the four different groups of mice was related to nutritional status and not related to levels of plasma glucose. The possible confounding effect of low plasma insulin on this conclusion is addressed by the results of Figure 4. Average 24-hour measurements of insulin were not significantly different between NTGA and TGA mice over the life span. Also, TGR mice exhibited levels of plasma insulin significantly lower than those of NTGR mice, but had survival characteristics similar to those of NTGR mice.

The effect of CR on elevating plasma corticosterone levels (8:00 AM) was observed in NTGR mice of all ages and in TGR mice at age 4–6 months. It seems that corticosterone levels were depressed in TGR mice at 12–14 and 28–30 months of age, in association with the extremely low values of plasma insulin in these mice. These low plasma insulin and corticosterone values did not affect the ability of CR to extend longevity in TGR mice (Figure 15). However, it should be noted that the effect of CR on increasing levels of spontaneous physical activity was blunted in TGR mice, suggesting the possible interplay of plasma glucose, insulin, corticosterone, and physical activity in these mice. In contrast, previously reported effects of CR on survival, body temperature, tendon breaking times, plasma leptin levels, and mass-adjusted metabolic rate were found for both NTGR and TGR mice. This result suggests little effect of variable plasma insulin and corticosterone levels in these mice on responses to CR. The absence of effect of plasma insulin and corticosterone on these properties is of interest in view of current discussion regarding the possible effects of insulin and corticosterone signaling on aging across phylogenetic lines (37). Our results (Figures 7, 14, and 15) indicate that variable plasma insulin and corticosterone levels over the life span did not affect usual measures of aging (survival, function, and tissue pathology).

No effects of variable plasma glucose on tissue pathology and on tendon breaking time were found with age. CR suppressed the age-related increase in tendon breaking time (Figure 13); this effect was not related to levels of plasma glucose. This result suggests that metabolic changes induced by CR are more powerful than the relatively weak glycosylating effects of plasma glucose [i.e., defense mechanisms such as the macrophage system may be upregulated by CR in reducing the presence of damaged AGE-related proteins (38)]. The expected effect of CR in suppressing the age-related accumulation of tissue pathology was found for mice aged 12–14 months but not at age 28–30 months. It should be noted however that, consistent with the SPF status of mice and excellent husbandry practices, levels of observed pathology were low throughout the life span for all groups.

Summary
Ad libitum feeding and restriction of food intake of TG and NTG mice resulted in three different levels of daily plasma glucose. The differences were sustained throughout the life span. Despite these differences, survival, function, and tissue pathology were characterized by levels of food intake and not by levels of plasma glucose. We conclude that aging of these mice was determined by nutritional status rather than by lifelong levels of plasma glucose, i.e., that decreased plasma glucose is not an important factor in the action of CR on aging of these mice.

	Acknowledgments

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This work was supported by National Institutes of Health Grant PO1 AG-14674.

We thank Ms. Vivian Diaz and Ms. Erica Castillo for their excellent technical assistance and Ms. Carol Brytczuk for her assistance with the manuscript.

	Footnotes

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Decision Editor: Huber R. Warner, PhD

Received February 2, 2007

Accepted May 18, 2007

	References

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