Serum glucose, glucose tolerance, corticosterone and free fatty acids

ELSEVIER SCIENCE IRELAND

Mechanisms of Ageing and Development 73 (1994) 209-221

Serum glucose, glucose tolerance, corticosterone and free fatty acids during aging in energy restricted mice Steven B. Harris a, Mark W. Gunion b, Mark J. Rosenthal c R o y L. W a l f o r d a aDepartment of Pathology, Center for the Health Sciences, University of California at Los Angeles, Los Angeles, CA 90024, USA bSepulveda Veterans Administration Hospital, B36, Building 5, 16111 Plummet, Sepulveda, CA 91343, USA "Research Service 151, Sepulveda Veterans Administration Hospital, 16111 Plummer, Sepulveda, CA 91343, USA

(Received 23 March; accepted 26 July 1993)

Abstract

Energy restriction, the only method known to increase maximum life span in laboratory animals, was used as a tool to test hypotheses regarding possible mechanisms of aging. Serum glucose and corticosterone (CS) concentrations in mice of a long-lived hybrid mouse strain, aged 7, 17, and 29 months, and on 50%, 80%, and 100% of ad libitum intake, were measured. Serum glucose and CS concentrations were also measured in response to intraperitoneal (i.p.) glucose challenge in mice at ages 7 and 29 months. Serum glucose and CS concentrations were also measured at several time points over 36 h, to assess their diurnal variation. There were no differences in single fasting glucose concentrations in 7- and 29omonth-old mice at the same degree of energy restriction, but energy restriction decreased glucose concentrations. Serum CS concentrations were generally increased restricted animals with respect to fully fed ones. Average serum glucose concentrations were found to be significantly decreased by dietary restriction. Glucose tolerance curves were unchanged by age in ad libitum fed or 50% restricted animals, but in 80% ad libitum groups, older animals showed evidence of decreased glucose tolerance with respect to young animals. For each age, peak serum glucose concentrations after i.p. glucose loading varied with degree of energy restriction, with more severely restricted animals showing less glucose tolerance. Average serum CS concentrations were elevated at 7 months by restriction, especially at night and long after feeding, but we found no differences with age or diet in average CS concentrations. Our serum glucose results sup* Corresponding author. 0047-6374/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0047-6374(94)01391-K

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port the hypothesisthat nonenzymaticglycation is mechanisticallyinvolved in normal aging. Our serum CS results do not support the hypothesis that CS contributes significantlyto the pathophysiology of normal aging in mice. Key words:

Aging; Energy restriction; Dietary restriction; Glucose; Corticosterone

1. Introduction

Dietary restriction, and specifically energy restriction, has proven the only effective method of extending maximum life span in laboratory animals, and in delaying onset of many age-related pathologies and pathologic changes [1,2]. The mechanism(s) whereby energy restriction exerts these global effects is unknown but the overall findings suggest that dietary restriction may modify underlying process(es) in part responsible for aging [3]. Recently, two hypotheses concerning a proposed endocrinological basis of aging have been advanced which are amenable to investigation with chronic energy restriction models. Sapolski et al. [41 have noted that aged rodents hypersecrete corticosterone (CS) in response to stress, and also that glucocorticoids such as CS may damage brain centers involved in suppressing glucocorticoid release, thereby perhaps potentiating a cycle of damage. A number of common features-of glucocorticoid excess and aging have led these investigators to propose that a senescent cascade of glucocorticoid hypersecretion causes some of the changes of aging. Several hormonal control systems in the brain appear to be possibly susceptible to runaway feedback from hormone damage, and a neurohumoral hysteresis theory of aging has also been proposed, involving estrogen, glucose, and CS [5]. A hypothesis of Cerami [6] suggests that glucose is an important toxin in the body, and that long term damage to DNA and protein from nonenzymatic glycation may be responsible for some of the aspects of aging. In this theory, the rate of such damaging glycation would be directly dependent on concentrations of glucose in body fluids. In order to test some of the endocrine-based hypotheses about aging, we examined how certain metabolic and endocrine factors in rodents responded to a lifetime of chronic energy restriction. The present study measured basal serum concentrations of glucose and CS, as well as their response to glucose challenge, in severely restricted, mildly restricted, and ad libitum fed young and old mice. 2. Methods 2.1. M i c e

The long-lived C3BIORF l hybrid strain of mouse has been used in previous dietary restriction studies in this laboratory [8,9]. These mice are bred from C57BL10.R111 and C3H.Sw/Sn. lines obtained originally from Jackson Laboratories, Bar Harbor, Maine. Female hybrid progeny were weaned at 21-28 days of age, individually caged in plastic cages on wood chip bedding, and assigned to one of three dietary regimens. Three age cohorts were studied, mice 7-, 17- and 29-month-

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old at the beginning of the study. Mice in each of three age cohorts, born within a 6-week period, were maintained under conventional (non-barrier) conditions, with temperature (20-24°C), humidity (50-60%) and 12 h. (06:00-18:00 h) lighting constant throughout the study. Survival curves for these mice under these conditions have been shown to be well rectangularized [8,9]. To monitor for infections, sentinel mice were kept in the same room as experimental mice, and sentinel serum samples were screened every 6 months for antibody titers against 11 common pathogens. Positive titers were not found during this study. 2.2. Diets C3H.SW/Sn dams were fed Purina Laboratory Chow TM (Ralston Purina, St. Louis, MO) during gestation and nursing of the hybrid study animals. Based on previous life-extending dietary restriction studies in our laboratory [8,9], mice at weaning were assigned to one of three diet groups. (1) The fully fed (ad libitum fed) group ate diet C (specified in Ref. 9), a 20% casein-purified diet. These mice were fed 10.0, 10.0 and 15.0 g of diet C given Monday, Wednesday, and Friday, respectively. This-represented a total feeding of 126 kcal/week (526 kJ/week), but food was not completely consumed, and on inspection some food was always present in the cages of these animals. These mice were obese by inspection after about 6 months of age. (2) The mildly restricted group was restricted to approximately 80% of the ad libitum intake, which prevented obesity. They received 86 kcal/week (360 kJ/week), given as 3.5 g daily on Monday through Thursday, and a 10.0-g feeding on Friday. (3) The severely restricted group ate restricted diet R, a 35% casein, vitamin and mineral enriched purified diet [9]. They received 49 kcal/week, given as 3.5 g Monday and Wednesday, and 7.0 g on Friday). The compositions of diets C and R were chosen so that mildly and severely energy restricted groups ate very nearly the same weekly amounts of protein, fat, vitamins, and minerals, with restricted animals differing only in carbohydrate (energy), and fiber intake [9]. Fully fed animals ate greater quantities of all food components. All feedings were done in the morning at 08:00 h, and no food was routinely visible in the cages of either mildly or severely restricted animals within a few hours after feeding. 2.3. Study 1: Baseline serum glucose and corticosterone, 24 h after feeding Nine groups of eight animals were studied, consisting of cohorts of three different ages (7, 17, and 29 months) from each of the three lifetime diet groups (Fully Fed, Mildly Restricted, and Severely Restricted). Blood was collected from 10:00 to 12:00 h, 26-28 h from last feeding in all animals. To minimize animal stress and its possible attendant CS and glucose increases, blood was collected in the room housing the animals, but in an adjacent room area. Each animal was not disturbed until the time of collection, then in turn each cage was removed from its place and the animal rapidly transferred directly from cage to an airtight jar containing anaesthetic agent (enflurane, USP, Anaquest/BOC Health Care, Madison, WI). The animal was removed from anaesthesia immediately after losing the righting reflex (20-30 s), and bled from the retro-orbital venous plexus by means of a 339-/zl capacity heparinized capillary tube (350 mm x 1.08 mm, Monoject/Sherwood Medical, St. Louis, MO). Total time from removal of an animal cage from the rack to completion of collection

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of blood was typically less than 60 s. To avoid disturbing still-to-be-bled animals, animals which had been bled were not returned to the racks until completion of the experiment. Previous pilot studies demonstrated no significant differences in serum CS and glucose concentrations between suddenly decapitated animals and animals anesthetized in this manner (data not shown), nor were significant differences seen in serum concentrations of glucose or CS in animals bled at the beginning of the series for each day, and those bled at the end. Animals were weighed after the drawing of blood. Collected blood was pipetted immediately into plastic microcentrifuge tubes on ice, allowed to clot for several hours, then microcentrifuged for 5 min and the serum removed for storage at -79°C until assay. For each type of serum assay done in this study and those below (glucose, insulin, CS, free fatty acid), all samples from each study were assayed together in a single assay run, in duplicate, and the assay repeated for any sample in which duplicate values failed to agree within 10%.

2.4. Study 2: Glucose tolerance testing and CS stress response For this study 7- to 8- and 29- to 30-month-old animals were used from-the Fully Fed, Mildly Restricted, and Severely Restricted groups. Each of the resulting six groups consisted of a pool of 12-20 animals, from which animals were selected sequentially to be bled on a rotating basis, as often as once a week. Animals-were bled (as described above) from 10:00 to 12:00 h, all groups having been fed the previous morning. Animals were briefly removed from their cages, weighed, and either bled immediately (for the zero time measurement) or else given a glucose load of 1.5 mg glucose per gram body weight by. i.p. injection of a 150 g/1 glucose solution. After injection, animals were returned to their cages for a specified time (30, 60, or 120 rain) before being bled. Thus, the glucose tolerance curve measurement at each time point represents the mean of serum concentration values for six to eight animals which had each been bled only once on a given day. To minimize daily and procedural sources of variance, one animal to represent each of the four time points (0, 30, 60, or 120 min) was selected from each of the six age/diet groups on each testing day, for a total of 24 animals and samples taken each day. In addition, an injection and bleeding schedule was chosen to thoroughly mix animals from the six groups and four test time points on each testing day. For Study 2, collected blood was pipetted immediately into small plastic microcentrifuge tubes holding 5 ~tl aproteinin 1 mg/ml (Sigma, St. Louis, MO) vortexed, and allowed to clot on ice. After all blood had been collected, serum was collected as previously described and stored until assay. 2.5. Study 3." Diurnal variation of glucose, corticosterone and jhee fatty acids This study followed Study 2 by approximately a month, and these measurements were conducted on the same pools of young (8-month) and old (30-month) fully fed, mildly restricted, and severely restricted animals used in study 2. Animals were fed normally on their diets, and blood and serum was collected as in Study 2, from six animals in each of the six groups, for concentrations of glucose, CS, and free fatty acids, at 6-h intervals for 36 h, beginning after a feeding. As in Study 2, all animals in each pool were bled several times during this study, but animals were selected in sequence and each animal was bled not more frequently than once per week.

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2. 6. Assays Serum glucose was measured with an enzymatic (hexokinase) assay and free fatty acids using a commercial reagent kit (Wako, Osaka, Japan). Serum corticosterone (ICN Biomedicals, Costa Mesa, CA) and insulin (Novo, Denmark) were measured using commercial R I A kits. 2. 7. Data analysis Data are presented as means 4- S.E.M. The data were analyzed using multiple regression. Comparisons between groups of animals were made using a t-test or (for post hoc comparisons) Duncan's test. 3. R e s u l t s

3.1. Animal weights At all ages, body weights differed significantly between group's eating differing amounts of food (Table 1, Fig. 1). Body weight was stable throughout life in the Severely Restricted mice, but rose slowly with aging in the Mildly Restricted group. The Fully Fed group showed significant weight gain between 7 and 17 months, but none thereafter. 3.2. Study 1: Mid-morning serum glucose and CS, 26 h after feeding Within each intake group, age did not have an effect on significant effect on fasting mid-morning serum glucose concentrations, save that severely restricted animals showed a significant rise in serum glucose at 17 months, while mildly restricted animals showed a significant fall in fasting glucose at the same age. Mice in the fully fed group showed no significant fasting glucose change with aging (Table 2, Fig. 1). Dietary restriction depressed average fasting glucose; concentrations in all three age groups. Average glucose concentrations for fasting Fully Fed, Mildly Restricted, and Severely Restricted groups were 113, 92, and 68 mg/dl respectively. Diet (energy intake) also had a significant effect on glucose concentrations at each age, except between the two restricted groups at 17 months, and between the mildly restricted and fully fed groups at 29 months.

Table 1 Body weights of mice of different ages under varying degrees of dietary restriction since weaning Feeding

50% Ad libitum Mildly restricted Ad libitum fed

Age 7 Months

17 Months

29 Months

21 ± 0.5a 28 -4- 0.9b 36 q- 1.6¢

21 ± 0.6a 32 ± 0.8 b 51 .4- 1.6c

23 .4- 1.1 a 39 .4- 1.5b 48 .4- 2.6¢

Estimated as 100-110 kcal/week. Each group represents eight mice. Values are mean weights in ± S.E.M. Means in each column not sharing a common superscript are significantly different (P < 0.05). grams

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T h e r e w a s a s i g n i f i c a n t d r o p in m i d - m o r n i n g f a s t i n g C S c o n c e n t r a t i o n s b e t w e e n 7- a n d 2 9 - m o n t h m i c e in t h e r e s t r i c t e d g r o u p s , b u t n o s u c h a g e - r e l a t e d c h a n g e f o r t h e fully f e d a n i m a l s , w h i c h h a d m u c h l o w e r C S c o n c e n t r a t i o n s . All g r o u p s s h o w e d a d e c l i n e in m i d - m o r n i n g C S b e t w e e n 17 a n d 29 m o n t h s ( T a b l e 3, Fig. 1).

Table 2 Serum glucose levels of mice of different ages under varying degrees of dietary restriction since weaning Feeding


Age 7 Months

17 Months

29 Months

58 ± 6 a 94 4- 4 b 123 ± II c

84 ± 3 a 74 + 4 b I l l 4- 8 c

63 ± 3 a 107 ± 5 b 105 ± 8 b

Estimated as 100-110 kcal/week. Each group represents eight mice. Values are mean glucose concentrations in milligrams per deciliter ± S.E.M. Means in each column not sharing a common superscript are significantly different (P < 0.05).

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Table 3 Midmorning serum corticosterone levels of mice of different ages under varying degrees of dietary restriction since weaning

Feeding


Age 7 Months

17 Months

29 Months

398 ± 35a 130 ± 23b 72 ± 15c

365 ± 45a 365 ± 48a 108 ± 28b

210 ± 35a 57 ± 11b 42 ± 6b

Estimated as 100-! I0 kcal/week. Each group representseight mice. Valuesare mean serumcorticosterone levels in nanograms/ml ± S.E.M. Means in each column not sharing a common superscript are significantly different (P < 0.05).

On average, dietary restriction increased mid-morning fasting CS concentrations substantially. Mean CS serum concentrations (across all ages) for the fully fed, mildly restricted, and severely restricted groups were 74, 184 and 324 ng/ml respectively. Severely restricted animals of each age showed a significantly greater CS than fully fed animals, and also a significantly greater CS than mildly restricted animals at 7 and 29 months. 3.3. Study 2: Glucose tolerance test and stress CS secretion test

The results of the glucose tolerance tests and CS measurements during the test (a measure of stress CS secretion) are shown in Fig. 2. Dietary effects proved to be more important than aging effects. Fully fed and young mildly restricted animals did not show a significant rise in glucose from baseline during the test, although a trend is evident. Old mildly restricted animals and both groups of severely restricted animals showed a significant (P < 0.05) glucose rise at 30 and 60 min, but their glucose concentration values had returned to baseline by 120 min. Neither severely restricted nor fully fed animals showed a difference between age groups in glucose tolerance, but in the mildly restricted groups, older animals showed a significantly larger glucose rise (less glucose tolerance) than younger animals. No effect of glucose injection was seen with our insulin assay (Fig. 2), although in general fully fed animals had significantly higher insulin concentrations throughout than the two restricted groups. No clear-cut effect of glucose injection on free fatty acid (FFA) concentrations was seen in our assay for most groups of animals (Fig. 2). Young severely restricted animals initially had significantly higher F F A concentrations than old animals, and these were suppressed significantly by glucose. Corticosterone (CS) secretion in response to the glucose injection is shown in Fig. 2. Mildly restricted animals did not respond significantly, while only old animals responded among the severely restricted group (P < 0.05 at 60 min). Both young and old animals responded significantly (P < 0.05) in the fully fed groups, with the older animals having a significantly higher CS concentration than younger ones at 60 min.

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