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Nutrition and Aging

Improved Glucose Tolerance with Lifetime Diet Restriction Favorably Affects Disease and Survival in Dogs

Nestlé Purina PetCare Research, St. Louis, MO 63164 and ^* Department of Mathematics, Washington University, St. Louis, MO 63130

¹To whom correspondence should be addressed. E-mail: Brian.Larson{at}rdmo.nestle.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Labrador retrievers (42 of original 48) were used to assess the effects of lifetime diet restriction on glucose tolerance at ages 9–12 y. Restricted-fed (RF) dogs were fed 75% of the same diet consumed by control-fed (CF) pair-mates. An intravenous glucose tolerance test was done annually (maximal stimulation, nonsteady-state). Diet treatment, age, and interactions were fixed effects. Statistical procedures used included mixed-model, repeated-measures ANOVA; least-squares means; Tukey’s multiple comparison; paired t tests; and Spearman rank correlations. Glucose k-value and half-life, and insulin sensitivity (total, and 9, 10, 11 y, and per lean mass) were higher (P < 0.05) in RF than in CF dogs. Late-phase insulin release [area under the curve (AUC) 30–120 min] was less (P < 0.05) in RF than in CR dogs. Early-phase insulin release (AUC 0–5 min), y 12 insulin sensitivity and insulinogenic index did not differ between RF and CF dogs. Insulin peak, and total AUC increased (P < 0.05) with age, whereas the glucose k-value and glucose half-life were not affected by age. Insulin sensitivity was negatively, and insulin AUC 30–120 min, peak and glucose were positively correlated with body weight, body condition score, fat mass, percentage of fat and abdominal fat/total tissue. Higher insulinogenic indices tended (P = 0.053) to be associated with greater median survival and dogs with higher insulin sensitivity were at lower (P < 0.05) risk of dying or receiving chronic disease treatment. Time to first osteoarthritis treatment or death was greater with lower basal glucose and higher insulin sensitivity (P < 0.05), but diet restriction explained most of this relationship’s variation. Glucose disposal efficiency and insulin response were associated with increased quality and length of life in diet-restricted dogs.

KEY WORDS: • diet restriction • glucose tolerance • insulin sensitivity • chronic disease • dogs

Diet restriction has been shown to extend the lifespan of many species, ranging from single cell organisms to insects, rodents and dogs (1,2). Masoro and colleagues (3) proposed that the reduced energy effect of diet restriction modulated the aging process in rats, and that energy use characteristics were altered, rather than rate of energy use. Subsequently, it was shown that total daily energy expenditure [total body or lean body mass (LBM),¹ basis] was lower in monkeys that were energy restricted rather than having unrestricted access to food (4). Weindruch et al. (5), in a study of aging effects on gene expression, found that lifetime energy restriction of mice prevented or delayed many characteristic age-related cellular energy deficits and shifted energy production toward alternative pathways. Another important and related consequence of diet or energy restriction is less accumulation of body fat compared with control-fed (CF) rodents (6,7) and nonhuman primates (4,8,9).

Diet restriction in mammals has consistently resulted in a reduction in circulating glucose and insulin compared with those that had unrestricted access or CF counterparts (2,3,8,10–13). Diet-restricted mammals have been observed to be more insulin sensitive (8,11,14). Reduced insulin sensitivity and pancreatic ß-cell responsiveness are regarded as early indicators of defects in glucose regulation (15) and possibly predictive of subsequent deleterious effects.

Hyperglycemia and hyperinsulinemia have been associated with cellular damage and chronic disease (16–19). Masoro (20) proposed that lifetime reduced levels of glucose and insulin may explain in part the delayed senescence observed in studies of diet restriction. The positive relationship of diet restriction with longevity is reflected in rodents by delayed incidence and severity of chronic diseases endemic to the particular strains tested (21–23). Early indications were that diet restriction may be responsible for reduced chronic disease in nonhuman primates (24). Subsequently, Bodkin and colleagues (25) showed that rhesus monkeys with unrestricted access to food had a 2.6-fold greater risk of death than dietary-restricted monkeys, and hyperinsulinemia led to a 3.7-fold increased risk of death.

After an initial 2-y evaluation of diet restriction effects on orthopedic development (26), this study was extended as follows: 1) to determine the effects of lifetime diet restriction, 2) to evaluate biomarkers of aging and 3) to discover impediments to successful aging. The summary results of this lifetime study of dogs were published recently (2). This report elaborates the glucose tolerance component of that study.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Study conduct.

Labrador retrievers (n = 48) from seven litters were used in the study, which consisted of a paired-feeding design. Animal care procedures were approved by the Ralston Purina Animal Care and Use Committee. Dogs were paired at 6 wk of age by gender and body weight (BW) within litter, and randomly assigned to one of two dietary treatment groups. Each treatment group [CF and restricted-fed (RF)] was offered the same diet; only the quantity offered differed between treatments. Beginning at 8 wk of age, one dog in each pair consumed the diet ad libitum and the other dog in each pair was fed 75% of the amount of food that its pair-mate had consumed the previous day.

When dogs were 3.25 y old, two adjustments were made to the feeding protocol. All dogs were switched from a growth formula diet (27% protein) to an adult formula diet (21% protein; Table 1). In addition, the amount of food was reduced and held constant to prevent insidious development of obesity in dogs that had unrestricted access to food. The amount fed to the 24 dogs that previously had had unrestricted access was calculated by estimating the ideal body weight for each dog on the basis of skeletal size in reference to other dogs of the same breed. These dogs were then fed 0.26 kJ of metabolizable energy (ME) per gram of estimated ideal body weight (maintenance requirement for large breed dogs; R. D. Kealy, unpublished data). This group of dogs was designated as CF. The remaining 24 dogs were each fed 25% less than the amount fed to respective pair-mates the previous day. This group of dogs was designated as RF. Details of the experimental design and procedures have been described (2,26–28).

Data collection.

Dogs were weighed weekly as puppies, periodically as adolescents, and weekly as adults. Beginning at 6 y of age, body condition was evaluated annually to assess degree of leanness or obesity, and a body condition score (BCS) ranging from 1 (emaciated) to 9 (severely obese) was assigned (BCS chart, Ralston Purina Company, St. Louis. MO). Beginning at 6 y of age, amounts of body lean, fat and bone mass were estimated annually, using dual-energy X-ray absorptiometry (DEXA; Lunar, Madison, WI). Abdominal fat was estimated by defining that particular region of interest within the DEXA database. Indirect blood pressure estimates and electrocardiogram parameters were collected (BAS Vetronics, West Lafayette, IN). Ultrasound heart scans were used to evaluate cardiac function (ALOKA 1100, ALOKA, Tokyo, Japan). Beginning at 8 y of age, osteoarthritis (OA) scores were assigned using methodology previously described (27,28).

Glucose tolerance test.

Intravenous glucose tolerance tests (IVGTT) were performed annually from 9 to 12 y of age. Food-deprived dogs were maximally stimulated with an infusion of a 500 g/L solution of D-glucose (2 g/kg body); venous blood samples were collected before and 5, 30, 45, 60 and 120 min after glucose administration. Serum glucose was determined spectrophotometrically (Ciba-Corning Express Plus 550, Walpole, MA) using a hexokinase reagent procedure (Polestar Labs, Escondido, CA) and plasma insulin was estimated by RIA (Insulin RIA, Diagnostic Systems Laboratory, Webster, TX) (Michigan State University, Animal Health Diagnostic Laboratory, E. Lansing, MI).

Glucose tolerance test calculations.

Time factors were calculated and reported in two ways: 1) the absolute amount of time required to reach baseline for insulin and 2) the half-life for glucose disappearance from serum (t_1/2, time required for the glucose concentration to decrease by half). Linear regression analysis of a semilogarithmic plot of glucose concentration vs. time was used to calculate the t_1/2, which was estimated to be between 5 and 45 min after glucose infusion. The k-value (29), also known as fractional disappearance or turnover, or clearance coefficient or rate, was calculated from the formula: k = (0.693/t_1/2) x 100 (where 0.693 = ln 2) and expressed as the percentage decrease between 5 and 45 min (%/min). The rate of insulin decline is reported as absolute reduction in concentration per time. Glucose and insulin peaks were recorded and insulin and glucose were calculated. The highest estimates of insulin and glucose were considered peak values and the increments of insulin and glucose concentrations above their respective food-deprived baselines were considered I and G, respectively. The areas under the curve (AUC) for insulin were determined from 0 to 5 min (early-phase insulin release), 30 to 120 min (late-phase insulin release) and for the total 120 min collection. Early insulin response to glucose infusion was also calculated as the insulinogenic index, I/G (30). Insulin sensitivity was calculated using the "simple method" formula according to Galvin et al. (31), in which insulin sensitivity = [(-10⁶)(least-squares slope of log₁₀ (glucose) vs. time from 5 min to 45 min)]/[(insulin AUC from 0 to 45 min) – (area under insulin curve from 0 to 45 min that would have occurred had the insulin level maintained its baseline value)]. Although this calculation of insulin sensitivity was derived from data collected under nonsteady-state conditions, it has been determined (31) that there is a high correlation (r = 0.85) between the euglycemic clamp (steady-state) and the simple method (nonsteady-state) insulin sensitivity results among a diverse group of glucose tolerance subjects (human and canine). Data from one dog (CF) with diabetes mellitus were deleted from the analyses, but data from three treated hypothyroid dogs were retained.

Statistical analyses.

Longitudinal variables were analyzed with a mixed-model, repeated-measures ANOVA approach applied consistently to all variables (32). Dietary treatment, age and their interaction were considered fixed effects of interest. Paired t tests for insulin sensitivity analyses were conducted separately within each year in paired-dog data analysis. Gender and its interactions with other fixed effects were not significant. Random effects accounted for variation among litters, pairs within litters and dog within pair. F-tests were used to evaluate significance of fixed effects of dietary treatment, age and their interaction. Means of each diet treatment at each year of age were estimated with least-squares means. Differences between dietary treatments or between year of age for a variable were assessed by applying Tukey’s multiple comparison procedure to the least-squares means and their standard errors. The Spearman rank correlation coefficient was used to measure tendencies of variables to have a monotonic relationship (33). Analyses of relationships of biological markers to survival were conducted using Cox proportional hazards regression models (34,35). Cox’s model was used to explain differences in survival due to varying levels of IVGTT covariates, in terms of their effect on the hazard function. As the hazard increases, survival rates decrease more rapidly. Survival was modeled in three ways: 1) time to death, 2) time to death or first treatment for all chronic disease or 3) time to death or first treatment, specifically for OA.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Energy intake during IVGTT testing at 9–12 y of age, when expressed on a BW basis, was similar in control and RF dogs (0.223 ± 0.006 vs. 0.233 ± 0.005 kJ/g BW, respectively). However, when energy intake was expressed on a LBM basis, CF dogs required 15% more (P < 0.05) energy to maintain lean tissue than RF dogs (0.323 ± 0.006 vs. 0.274 ± 0.004 kJ/g LBM, respectively).

Glucose and insulin.

When maximally challenged with glucose infusion, RF dogs had 22% lower (P < 0.05) peak insulin, and the resulting insulin tended (P = 0.052) to be lower (19% reduction) in RF dogs (Table 2). Additionally, basal insulin increased (P < 0.05) with age. Peak and glucose levels were not affected by age, whereas peak and insulin increased with age (P < 0.05).

Insulin indices.

The insulinogenic index did not differ between CF and RF dogs. However, insulin sensitivity was increased (P < 0.05) by 58% for the entire 4-y IVGTT period in RF compared with CF dogs. Insulin sensitivity, when expressed on a LBM basis, was 147% greater (P < 0.05) for RF than CF dogs. In a paired analysis, RF dogs had higher (P < 0.05) insulin sensitivity than their CF pair-mates at ages 9, 10 and 11 y (Table 3). By age 12 y, insulin sensitivity did not differ between RF and CF dogs in a paired analysis. In the CF group, any 2 y insulin sensitivity values were similar.

Correlation coefficients were calculated to quantify relationships between IVGTT measurements and selected morphometric or biochemical variables. Basal serum glucose was not highly correlated with any of the selected variables (Table 4). Peak and glucose were moderately correlated with variables related to body composition, especially those relating to body fat content. Glucose tolerance, reflected by the k-value, was not highly correlated with selected variables. Basal, peak and insulin were not highly correlated with most of the selected variables; however, there was a moderate correlation with the variables and the 30–120 min insulin AUC (Table 5). Insulin decline rate and time to baseline were not remarkably correlated with any selected variable. Insulin sensitivity was highly correlated with insulin AUC 30–120 min. Moderate insulin sensitivity correlations were found with peak and insulin, abdominal fat per total body tissue, total fat mass, BW and BCS. Low correlations existed between insulin sensitivity and lean mass, basal glucose, food or energy intake, basal insulin, k-value or insulinogenic index. Bile acids, serum triglycerides, blood pressure and OA scores were not remarkably correlated with IVGTT variables (correlations not shown).

Basal glucose and insulin tended (P = 0.065 and 0.096, respectively) to be positively related to survival time until death. Most variation in these relationships was explained by the diet restriction treatment; therefore, these relationships did not contribute information. The insulinogenic index also tended (P = 0.053) to be a positively associated predictor of survival. Interestingly, this early insulin response index added more information and had a strong tendency (P = 0.057) to predict survival beyond diet restriction treatments.

"Survival time to death or first chronic disease treatment."

High insulin sensitivity was positively associated with low hazard of dying from or needing treatment for chronic disease (P < 0.05). This relationship tended (P = 0.055) to be significant even when accounting for diet restriction, thereby supplying important additional information to understanding hazards to survival from chronic disease.

"Survival time to death or first OA treatment."

Basal glucose and insulin sensitivity were associated with survival time to death due to OA or first OA treatment (P < 0.05). The relationship for basal glucose was negative and for insulin sensitivity, positive. However, diet restriction treatment explained most of this relationship. The insulinogenic index was more difficult to interpret. Insulin index tended (P = 0.079) to show an interaction with diet restriction treatments. This interactive association became significant (P < 0.05) when accounting for diet restriction. Higher indices appeared to reduce risks in OA hazard for CF dogs but may have been a hazard for RF dogs.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
There were no clinical signs of malnutrition at any point during this lifetime trial. Serum indicators of protein status, such as total protein, albumin and urea nitrogen, showed no treatment effects that would suggest protein deficiency or potential direct effects upon eventual treatment differences (data not shown). Based on mean daily food intake, the calculated reductions of carbohydrates, fat and protein for RF dogs were 52, 12 and 22 g/d, respectively. The proportions of energy reduction consisted of 50% from carbohydrates and 25% each from protein and fat.

RF dogs consumed 22.5% less diet energy from the start of the trial through 12 y of age (96.9 ± 2.6 kJ vs. 75.0 ± 1.9 kJ of ME/d for CF dogs and RF dogs, respectively) (2). Interestingly, food intake (synonymous with diet intake) was not highly correlated with IVGTT variables. This lack of high correlation may reflect the numerous digestive, absorptive and metabolic steps between food consumption and subsequent cellular responses that are subject to complex regulatory influences. Typical of animals reaching a food restriction steady state, RF and CF dogs had similar energy intakes on a BW basis for 9–12 y. However, the CF dogs required 15% more energy to maintain lean mass (energy intake/LBM basis). Perhaps related metabolically, 3,5,3'-triiodothyronine was 16% higher in CF dogs than in their restricted pair-mates during the trial (2)

The IVGTT was used to assess effective and efficient use and/or storage of glucose. Physiologically, glucose tolerance involves a complex interaction among pancreatic ß-cell insulin secretion, actions of insulin to increase glucose disappearance and decrease endogenous glucose production (insulin sensitivity), liver clearance rate of insulin, and the ability of glucose itself (independent of insulin) to increase glucose uptake and suppress the endogenous glucose production (glucose efficiency) (15). In this study, diet-restricted dogs demonstrated marked advantages in glucose tolerance and related IVGTT variables over their CF counterparts.

Diet-restricted dogs in this study had 7 and 32% lower basal circulating glucose and insulin concentrations (5.19 vs. 5.59 mmol/L and 48.4 vs. 70.8 pmol/L for RF and CF, respectively) (2). Similar basal glucose and insulin differences were reported in studies with diet-restricted rats and monkeys (3,6,8–13,36). It was reported previously that these RF dogs’ basal glucose decreased more with age than that of CF dogs (2). This result is unlike that noted for any other mammal studied under diet restriction conditions in which basal glucose generally increases with age. At the same time, basal insulin increased, creating unique insulin-glucose relationships in aging dogs.

We showed reduced glucose k-value, half-life and AUC for RF compared with CF dogs. Kealy et al. (2) noted that peak and glucose, and time to glucose baseline were 15, 16 and 55% lower in these RF dogs compared with CF dogs. Diet restriction improved glucose tolerance in monkeys and rats (8,10,11,37–39), rat glucose homeostasis and insulin-induced suppression of hepatic glucose production (40). Ortmeyer and co-workers (13,41,42) showed that in diet-restricted monkeys, intermediate glucose metabolism (glycogen synthase regulated by insulin) differed from controls. This same group of researchers recently demonstrated that energy restriction and the avoidance of obesity markedly reduced glucose disposal defects through atypical protein kinase C activation in nonhuman primate skeletal muscle cells (43). This lack of adipose-induced inhibition of atypical protein kinase C combined with activation of the insulin receptor results in increased glucose transporter migration to the cell surface, allowing greater glucose uptake and insulin sensitivity.

Insulin sensitivity is the ability of insulin to augment glucose’s self-regulation by suppressing hepatic glucose production and increasing glucose utilization (44). Whether evaluating these data by year (9 through 12) or in total, the magnitude of the effect of diet restriction on canine insulin sensitivity was evident. Limited studies in primates indicate similar improvements of insulin sensitivity with diet restriction (11,39). Insulin sensitivity, however, is only part of the glucose tolerance response. A decrease in insulin sensitivity will cause a compensatory insulin secretion (hyperinsulinemia) by pancreatic ß-cells due to their increased glucose sensitivity to maintain glucose tolerance (15). When that compensation is compromised, glucose efficiency is reduced and that reduction may be pathogenic to the point that diabetes develops. One dog from the CF group in this study developed diabetes. CF dogs had increased basal, peak, and total AUC insulin combined with increased insulin rate of decline and time to reach basal levels.

With the exception of the one CF dog with diabetes, these IVGTT data may indicate that pancreatic ß-cells of the remaining dogs in this study were not compromised in their ability to secrete insulin in response to circulating glucose. However, insulin secretion of CF dogs appeared to overcompensate during late-phase insulin release (insulin AUC, 30–120 min) in an unsuccessful attempt to maintain glucose tolerance. The additional insult of increased late-phase insulin release correlated well with fat mass.

Exploring the relationship of body fat content to survival in diet restriction research began years ago when it was unexpectedly discovered that only in diet-restricted rats did body fat content correlate with longevity (5). A threshold effect has been suggested by correlations of body fat with insulin-stimulated glucose uptake, in monkeys with <22% body fat (45), and in humans with <28% body fat (46). Recently, it was proposed that when a threshold of age-associated fat mass is reached, no further reduction in insulin insensitivity occurs (40). These results may demonstrate saturation, and that body fat is an active endocrine organ (47). In our study, insulin sensitivity and body fat mass were highly correlated. At a threshold somewhat > 5000 g of body fat, insulin sensitivity remained at a consistently low level. During the time period of IVGTT (9 through 12 y of age) the mean weight of dogs in this trial was 28.2 kg (24.1 kg and 33.2 kg for RF and CF, respectively). Therefore, a threshold of 20% body fat may exist at which dogs with less body fat would likely be more insulin sensitive.

Excessive accumulation of abdominal fat in nonhuman primates (48) and humans (47,49) has been associated with hyperinsulinemia and impaired glucose tolerance. However in this study, abdominal fat per total tissue and total fat mass were similarly correlated with IVGTT variables, suggesting that fat depot may not be important to outcome in dogs.

Kealy and co-workers (2) reported that diet restriction increased the median life span of these dogs by 1.8 y (11.2 vs. 13.0 y, CF and RF dogs, respectively). Although early-phase insulin response to glucose (insulinogenic index) did not differ between RF and CF dogs, this study associated the insulinogenic index with hazards to survival.

Dogs face increasing challenges from chronic disease as they age. The age to which 50% of these dogs survived without requiring medication for any chronic disease was increased by 19% (9.4 y for the CF group vs. 11.2 y for the RF group) (2). Insulin resistance or conversely, the role of insulin sensitivity in the etiology of disease is not novel. A recent review elaborates insulin resistance relationships to hyperinsulinemia, diabetes, obesity, hypertension, dyslipidemia, cardiovascular disease and cancer (50). Our research indicates that improving insulin sensitivity through diet restriction (or perhaps by other nutritional methods) correlates with reduced hazard for chronic disease

The most prevalent chronic disease noted in this study was OA, including both "first treated" and total diagnoses. Previously, we reported that initial radiographic evidence for OA of hip joints appeared at an older age and was less severe in RF dogs (27). Additionally, RF dogs survived longer without requiring first medication for OA (mean age of 10.3 y for CF vs. 13.3 y for RF dogs) (2). Reduced basal glucose levels and higher insulin sensitivity, both achieved as a result of diet restriction, were associated with reduced hazard to survival due to OA. Although specific mechanisms were not identified by this research, the osteoarthritic consequences of unrestricted food access and the effect on glucose tolerance appear to be important for the dog.

In summary, among glucose tolerance variables, early-phase insulin response-to-glucose (insulinogenic index) and insulin sensitivity were the strongest predictors of hazard to survival and chronic disease, respectively. Although the implications are circumstantial, optimum glucose tolerance and insulin response appear to be integrally involved in the health and longevity of dogs. Long-term control of food intake to minimize circulating glucose and enhance strategic insulin response will improve the quality and increase the quantity of the dog’s life. Imposing strict dietary control, thereby controlling body fat levels early in the dog’s life, can be an effective method of achieving these goals. Finally, the IVGTT measurements and several critical calculations using those data may become effective clinical diagnostic tools for monitoring the health of dogs.

	FOOTNOTES

 
² Abbreviations used: AUC, area under the curve; BCS, body condition score; BW, body weight; DEXA, dual-energy X-ray absorptiometry; IVGTT, intravenous glucose tolerance test; k-value, fractional glucose disappearance; LBM, lean body mass; OA, osteoarthritis.

Manuscript received 11 March 2003. Initial review completed 4 May 2003. Revision accepted 18 June 2003.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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