Calorie-restricted Animals Research Articles

Calorie Restriction Reduces Oxidative Stress by SIRT3-Mediated SOD2 Activation

A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases.

Caloric restriction delays aging-induced cellular phenotypes in rhesus monkey skeletal muscle

Sarcopenia is the age-related loss of skeletal muscle mass and function and is characterized by a reduction in muscle mass and fiber cross-sectional area, alterations in muscle fiber type and mitochondrial functional changes. In rhesus monkeys, calorie restriction (CR) without malnutrition improves survival and delays the onset of age-associated diseases and disorders including sarcopenia. We present a longitudinal study on the impact of CR on early stage sarcopenia in the upper leg of monkeys from ~ 16 years to ~ 22 years of age. Using dual-energy X-ray absorptiometry we show that CR delayed the development of maximum muscle mass and, unlike Control animals, muscle mass of the upper leg was preserved in CR animals during early phase sarcopenia. Histochemical analyses of vastus lateralis muscle biopsies revealed that CR opposed age-related changes in the proportion of Type II muscle fibers and fiber cross-sectional area. In contrast the number of muscle fibers with mitochondrial electron transport system enzyme abnormalities (ETS ab) was not significantly affected by CR. Laser capture microdissection of ETS ab fibers and subsequent PCR analysis of the mitochondrial DNA revealed large deletion mutations in fibers with abnormal mitochondrial enzyme activities. CR did not prevent stochastic mitochondrial deletion mutations in muscle fibers but CR may have contributed to the maintenance of affected fibers.

Effects of Moderate Calorie Restriction on Testosterone Production and Semen Characteristics in Young Rhesus Macaques (Macaca mulatta)1

We have previously reported a modest influence of moderate calorie restriction (CR) on testicular gene expression in young adult rhesus macaques (Macaca mulatta); however, it is unclear if these modifications correspond to subsequent changes in testicular function or sperm physiology. This study extends our earlier findings to examine potential physiological differences due to this differential gene expression. Animals were subjected to 30% CR (CR, n = 5) or were fed a standard control diet (CON, n = 5) starting during their peripubertal period. Circulating testosterone (T) levels were measured across a 24-h period after 7 yr of dietary treatment and were found to be similar in CR and CON males; however, maintenance of daily minimum T levels was significantly higher in the CR animals. Semen collection was performed on the same cohort of animals three times per male (CR, n = 4; CON, n = 4) after 8 yr of treatment, and samples were assessed by a variety of measures. Parameters, including semen quality and sperm cell viability and function, showed less variability in semen samples taken from CR males, but overall testicular function and sperm quality were comparable regardless of diet. There is mounting evidence that CR may promote health and longevity in a wide range of organisms, including nonhuman primates. Importantly, our data suggest that moderate CR has no obvious lasting detrimental effect on testicular function and sperm parameters in young adult primates and may in fact help maintain higher levels of circulating T.

A Calorie-Restricted Diet Decreases Brain Iron Accumulation and Preserves Motor Performance in Old Rhesus Monkeys

Caloric restriction (CR) reduces the pathological effects of aging and extends the lifespan in many species, including nonhuman primates, although the effect on the brain is less well characterized. We used two common indicators of aging, motor performance speed and brain iron deposition measured in vivo using MRI, to determine the potential effect of CR on elderly rhesus macaques eating restricted (n = 24; 13 males, 11 females) and standard diets (n = 17; 8 males, 9 females). Both the CR and control monkeys showed age-related increases in iron concentrations in globus pallidus (GP) and substantia nigra (SN), although the CR group had significantly less iron deposition in the GP, SN, red nucleus, and temporal cortex. A diet x age interaction revealed that CR modified age-related brain changes, evidenced as attenuation in the rate of iron accumulation in basal ganglia and parietal, temporal, and perirhinal cortex. Additionally, control monkeys had significantly slower fine motor performance on the Movement Assessment Panel, which was negatively correlated with iron accumulation in left SN and parietal lobe, although CR animals did not show this relationship. Our observations suggest that the CR-induced benefit of reduced iron deposition and preserved motor function may indicate neural protection similar to effects described previously in aging rodent and primate species.

Caloric restriction or resveratrol supplementation and ageing in a non-human primate: first-year outcome of the RESTRIKAL study in Microcebus murinus

A life-long follow-up of physiological and behavioural functions was initiated in 38-month-old mouse lemurs (Microcebus murinus) to test whether caloric restriction (CR) or a potential mimetic compound, resveratrol (RSV), can delay the ageing process and the onset of age-related diseases. Based on their potential survival of 12 years, mouse lemurs were assigned to three different groups: a control (CTL) group fed ad libitum, a CR group fed 70% of the CTL caloric intake and a RSV group (200 mg/kg.day–1) fed ad libitum. Since this prosimian primate exhibits a marked annual rhythm in body mass gain during winter, animals were tested throughout the year to assess body composition, daily energy expenditure (DEE), resting metabolic rate (RMR), physical activity and hormonal levels. After 1 year, all mouse lemurs seemed in good health. CR animals showed a significantly decreased body mass compared with the other groups during long day period only. CR or RSV treatments did not affect body composition. CR induced a decrease in DEE without changes in RMR, whereas RSV induced a concomitant increase in DEE and RMR without any obvious modification of locomotor activity in both groups. Hormonal levels remained similar in each group. In summary, after 1 year of treatment CR and RSV induced differential metabolic responses but animals successfully acclimated to their imposed diets. The RESTRIKAL study can now be safely undertaken on a long-term basis to determine whether age-associated alterations in mouse lemurs are delayed with CR and if RSV can mimic these effects.

Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates

Calorie restriction (CR) increases longevity and retards the development of many chronic diseases, but the underlying metabolic signals are poorly understood. Increased fatty acid (FA) oxidation and reduced FA synthesis have been hypothesized to be important metabolic adaptations to CR. However at steady state, FA oxidation must match FA intake plus synthesis; FA intake is low, not high, during CR. It is therefore not clear how FA dynamics are altered during CR. Accordingly, we measured food intake patterns, whole‐body fuel selection, endogenous FA synthesis and gene expression in mice on CR. Within two days of starting CR, a shift occurred to a cyclic pattern of whole‐body FA metabolism, with an initial phase of elevated endogenous FA synthesis (respiratory exchange ratio [RER]>1.10, lasting 4–6 hours after food provision), followed by a prolonged phase of FA oxidation (RER=0.70, lasting 18–20 hours). CR mice oxidized four times as much fat per day as ad libitum fed (AL) controls (367 vs 97 mg/d) despite reduced fat intake. This increase in FA oxidation was balanced by a 3‐fold increase in adipose tissue FA synthesis. We conclude that CR induces a surprising metabolic pattern characterized by periods of elevated FA synthesis alternating with periods of FA oxidation disproportionate to dietary FA intake. This pattern may have implications for understanding the reduced oxidative damage and disease risk in CR animals.Grant Funding Source: NIH

EAT LESS TO BURN MORE FAT

Over 70 years ago, a team of scientists discovered that a reduction of daily caloric intake can prolong lifespan in rats. Since then, a moderate caloric restriction has been associated with increased longevity in a variety of organisms ranging from yeast to mice. Animals subjected to caloric restriction typically exhibit reduced occurrence of age-related disorders such as cardiovascular diseases, diabetes and oxidative damage, resulting in an overall longer life expectancy. Although the exact mechanisms promoting these health benefits remain elusive, it has been argued that caloric restriction could reduce oxidative stress in cells, therefore preventing excessive oxidative damage. Furthermore, when carbohydrates are oxidized in the mitochondria they tend to produce more oxidative stress than lipids, so it has been further hypothesized that an increased reliance on fatty acids vs carbohydrates could mediate the health benefits of caloric restriction. However, the nutrient composition of the caloric restriction diet is similar to that of a regular diet; that is, the two diets have the same relative lipid content. Therefore, this hypothesis cannot hold unless calorie-restricted animals are synthesizing more lipids than their satiated counterparts. In this study, Matthew Bruss and his colleagues from the University of California at Berkeley and the Children's Hospital in Oakland (USA) sought to test this hypothesis by examining fuel selection patterns in calorie-restricted mice.The team reduced the caloric intake of mice by 30% and measured their oxygen consumption and CO2 production to examine their fuel preferences over several weeks. The authors initially discovered that the calorie-restricted mice wolfed down their entire daily food ration within an hour of the food arriving and essentially fasted for the next 23 h. Monitoring the fuel sources that the animals used while fasting, the team found that during the first 6 h after being fed, the mice used carbohydrates for their energetic needs and synthesized their own fatty acids. During the rest of the day, the rodents relied almost exclusively on lipids as metabolic fuels, burning four times more lipids than if they were on a normal diet, and three times more than their actual daily fat intake.As these results strongly suggested that calorie-restricted mice synthesized more lipids, Bruss and his colleagues further examined changes in the animals' lipid production. The team confirmed that calorie-restricted mice exhibited elevated rates of lipid synthesis and retention. Furthermore, they found that shortly after their meal the mice started producing and storing the majority of these lipids in their adipose tissues.Using classical physiological techniques, Bruss and colleagues elegantly demonstrated unique metabolic adjustments in calorie-restricted mice. The team clearly showed that the calorie-restricted mice switched to a predominant reliance on their own newly synthesized fatty acids as oxidative fuels and there was a pattern to the timing of this shift, which should most definitely be kept in mind for future experimental designs. The next logical step is probably to ask the question: is burning more fat the key to living a long, healthy, calorie-restricted life?

Sexual dimorphism in the lasting effects of moderate caloric restriction during gestation on energy homeostasis in rats is related with fetal programming of insulin and leptin resistance

AimWe aimed to characterize the lasting effect of moderate caloric restriction during early pregnancy on offspring energy homeostasis, by focusing on the effects on food intake and body weight as well as on the insulin and leptin systems.MethodsMale and female offspring of 20% caloric restricted dams (from 1 to 12 days of pregnancy) (CR) and from control dams were studied. These animals were fed after weaning with a normal-fat (NF) diet until the age of 4 months, and then moved to a high-fat (HF) diet. Blood parameters were measured under fed and 14-h fasting conditions at different ages (2, 4 and 5 months). Food preferences were also assessed in adult animals.ResultsAccumulated caloric intake from weaning to the age of 5 months was higher in CR animals compared with their controls, and this resulted in higher body weight in adulthood in males, but not in females. Both male and female CR animals already showed higher insulin levels at the age of 2 months, under fed conditions, and higher HOMA-IR from the age of 4 months, compared with their controls. CR male animals, but not females, displayed higher preference for fat-rich food than their controls in adulthood and higher circulating leptin levels when they were under HF diet.ConclusionIt is suggested that hyperinsulinemia may play a role in the etiology of hyperphagia in the offspring of caloric restricted animals during gestation, with different outcomes on body weight depending on the gender, which could be associated with different programming effects on later leptin resistance.

Inhibition of Thymic Adipogenesis by Caloric Restriction Is Coupled with Reduction in Age-Related Thymic Involution

Aging of thymus is characterized by reduction in naive T cell output together with progressive replacement of lymphostromal thymic zones with adipocytes. Determining how calorie restriction (CR), a prolongevity metabolic intervention, regulates thymic aging may allow identification of relevant mechanisms to prevent immunosenescence. Using a mouse model of chronic CR, we found that a reduction in age-related thymic adipogenic mechanism is coupled with maintenance of thymic function. The CR increased cellular density in the thymic cortex and medulla and preserved the epithelial signatures. Interestingly, CR prevented the age-related increase in epithelial-mesenchymal transition (EMT) regulators, FoxC2, and fibroblast-specific protein-1 (FSP-1), together with reduction in lipid-laden thymic fibroblasts. Additionally, CR specifically blocked the age-related elevation of thymic proadipogenic master regulator, peroxisome proliferator activated receptor gamma (PPARgamma), and its upstream activator xanthine-oxidoreductase (XOR). Furthermore, we found that specific inhibition of PPARgamma in thymic stromal cells prevented their adipogenic transformation in an XOR-dependent mechanism. Activation of PPARgamma-driven adipogenesis in OP9-DL1 stromal cells compromised their ability to support T cell development. Conversely, CR-induced reduction in EMT and thymic adipogenesis were coupled with elevated thymic output. Compared with 26-mo-old ad libitum fed mice, the T cells derived from age-matched CR animals displayed greater proliferation and higher IL-2 expression. Furthermore, CR prevented the deterioration of the peripheral TCR repertoire diversity in older animals. Collectively, our findings demonstrate that reducing proadipogenic signaling in thymus via CR may promote thymopoiesis during aging.

Anti-oxidative and anti-inflammatory vasoprotective effects of caloric restriction in aging: Role of circulating factors and SIRT1

Endothelial dysfunction, oxidative stress and inflammation are associated with vascular aging and promote the development of cardiovascular disease. Caloric restriction (CR) mitigates conditions associated with aging, but its effects on vascular dysfunction during aging remain poorly defined. To determine whether CR exerts vasoprotective effects in aging, aortas of ad libitum (AL) fed young and aged and CR-aged F344 rats were compared. Aging in AL-rats was associated with impaired acetylcholine-induced relaxation, vascular oxidative stress and increased NF-κB activity. Lifelong CR significantly improved endothelial function, attenuated vascular ROS production, inhibited NF-κB activity and down-regulated inflammatory genes. To elucidate the role of circulating factors in mediation of the vasoprotective effects of CR, we determined whether sera obtained from CR animals can confer anti-oxidant and anti-inflammatory effects in cultured coronary arterial endothelial cells (CAECs), mimicking the effects of CR. In CAECs cultured in the presence of AL serum TNFα elicited oxidative stress, NF-κB activation and inflammatory gene expression. By contrast, treatment of CAECs with CR serum attenuated TNFα-induced ROS generation and prevented NF-κB activation and induction of inflammatory genes. siRNA knockdown of SIRT1 mitigated the anti-oxidant and anti-inflammatory effects of CR serum. CR exerts anti-oxidant and anti-inflammatory vascular effects, which are likely mediated by circulating factors, in part, via a SIRT1-dependent pathway.

Cystathionine γ‐lyase and cystathionine β‐synthase expression and activity change with age and caloric restriction

Hydrogen sulfide (H2S), a gas signaling molecule with actions on the cardiovascular system, is endogenously produced by cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS). We have previously shown an increase in H2S signaling efficacy with age and caloric restriction (CR) in rat aorta and hypothesized that CSE and CBS expression would change similarly. Aorta and liver tissues were collected from rats of ages 8 to 38 months maintained on CR or ad libitum (AL) diets. Protein and RNA were extracted for western blotting and real time PCR, respectively. H2S production was determined in tissue homogenates. While age did not affect H2S production or RNA expression, diet did: aorta and liver H2S production and CSE RNA expression were higher in CR than AL animals for all ages (t‐test, p=0.041). Age and diet both affected protein expression (2‐way ANOVA, p=0.0056): in aorta, both enzymes increased with age, with expression highest in AL animals at older ages; in liver, CSE increased with age, with expression highest in CR animals at older ages, whereas CBS decreased with age with no diet affect. These data show that for aorta, enzyme expression increases with age while RNA expression does not, and CR animals with lower CSE and CBS produce more H2S than AL animals. However, this may not be true of non‐vascular tissues, such as liver. This suggests a decrease in enzyme function with age that may be attenuated by CR.

Insulin Sensitivity as a Key Mediator of Growth Hormone Actions on Longevity

Reduced insulin sensitivity and glucose intolerance have been long suspected of having important involvement in aging. Here we report that in studies of calorie restriction (CR) effects in mutant (Prop1(df) and growth hormone receptor knockout [GHRKO]) and normal mice, insulin sensitivity was strongly associated with longevity. Of particular interest was enhancement of the already increased insulin sensitivity in CR df/df mice in which longevity was also further extended and the lack of changes in insulin sensitivity in calorically restricted GHRKO mice in which there was no further increase in average life span. We suggest that enhanced insulin sensitivity, in conjunction with reduced insulin levels, may represent an important (although almost certainly not exclusive) mechanism of increased longevity in hypopituitary, growth hormone (GH)-resistant, and calorie-restricted animals. We also report that the effects of GH treatment on insulin sensitivity may be limited to the period of GH administration.

Improvements in body fat distribution and circulating adiponectin by alternate-day fasting versus calorie restriction

Calorie restriction (CR) and alternate-day fasting (ADF) beneficially affect several aspects of adipose tissue physiology, but direct comparisons between regimens have yet to be performed. The present study evaluated the effects of ADF versus CR on body fat distribution and circulating adiponectin levels and examined the kinetic mechanisms that underlie changes in fat distribution. Thirty female C57BL/6J mice were randomized to one of five groups for 4 weeks: (a) CR-25% (25% energy restriction daily), (b) ADF-75% (75% restriction on fast day), (c) ADF-85% (85% restriction on fast day), (d) ADF-100% (100% restriction on fast day) and (e) control (ad libitum fed). Body weights of the CR mice were lower than that of the ADF and control groups posttreatment. After 4 weeks of diet, the proportion of visceral fat decreased ( P<.001) and the proportion of subcutaneous fat increased ( P<.001) similarly in ADF and CR animals. Adiponectin increased ( P<.05) by 62–86% in the ADF groups and by 69% in the CR group. Triglyceride (TG) synthesis and de novo lipogenesis were augmented ( P<.05) in the subcutaneous fat pad of ADF and CR animals, relative to control. No differences in net lipolysis were observed, resulting in greater TG accumulation in the subcutaneous fat pad, with a shift in the ratio of TG between depots. These findings indicate that ADF (both modified and true) produces similar beneficial modulations in body fat distribution and adiponectin levels as daily CR.

Control of anesthesia-induced hypothermia by ambient temperature regulation in rats

(1) The goal of this study was to control anesthesia-induced hypothermia in rats, which was addressed through four experimental steps. (2) First, identify the effects of isoflurane anesthetic on Sprague-Dawley rats at an ambient temperature of 23 °C. (3) Second, determine an ambient temperature that is thermo-neutral and counteracts anesthesia hypothermia. (4) Third, test the calculated thermo-neutral point. (5) Fourth, identify differences in colonic temperature response to different body weights and to calorie-restricted animals. (6) Our findings showed there were differences in core temperature response and skin adipose deposition between 350 and 450 g rats. (7) Also there was a greater hypothermic trend in rats that were calorie restricted to 350 g vs. ad libitum fed 350 g rats.

In Vitro Cellular Adaptations of Indicators of Longevity in Response to Treatment with Serum Collected from Humans on Calorie Restricted Diets

Calorie restriction (CR) produces several health benefits and increases lifespan in many species. Studies suggest that alternate-day fasting (ADF) and exercise can also provide these benefits. Whether CR results in lifespan extension in humans is not known and a direct investigation is not feasible. However, phenotypes observed in CR animals when compared to ad libitum fed (AL) animals, including increased stress resistance and changes in protein expression, can be simulated in cells cultured with media supplemented with blood serum from CR and AL animals. Two pilot studies were undertaken to examine the effects of ADF and CR on indicators of health and longevity in humans. In this study, we used sera collected from those studies to culture human hepatoma cells and assessed the effects on growth, stress resistance and gene expression. Cells cultured in serum collected at the end of the dieting period were compared to cells cultured in serum collected at baseline (before the dieting period). Cells cultured in serum from ADF participants, showed a 20% increase in Sirt1 protein which correlated with reduced triglyceride levels. ADF serum also induced a 9% decrease in proliferation and a 25% increase in heat resistance. Cells cultured in serum from CR participants induced an increase in Sirt1 protein levels by 17% and a 30% increase in PGC-1α mRNA levels. This first in vitro study utilizing human serum to examine effects on markers of health and longevity in cultured cells resulted in increased stress resistance and an up-regulation of genes proposed to be indicators of increased longevity. The use of this in vitro technique may be helpful for predicting the potential of CR, ADF and other dietary manipulations to affect markers of longevity in humans.

The Effects of a Calorie‐Reduced Diet on Periodontal Inflammation and Disease in a Non‐Human Primate Model

Low-calorie diets are commonplace for reducing body weight. However, no information is available on the effects of a reduced-calorie diet on periodontal inflammation and disease. The purpose of this study was to evaluate the clinical effects of a long-term calorie-restriction (CR) diet on periodontitis in an animal model of periodontitis. Periodontitis was induced in 55 young, healthy, adult rhesus monkeys (Macaca mulatta) by tying 2.0 silk ligatures at the gingival margins of maxillary premolar/molar teeth. Animals on a CR diet (30% CR; N = 23) were compared to ad libitum diet controls (N = 32). Clinical measures, including the plaque index (PI), probing depth (PD), clinical attachment level (CAL), modified gingival index (GI), and bleeding on probing (BOP) were recorded at baseline and 1, 2, and 3 months after ligature placement. Significant effects of CR were observed on the development of inflammation and the progression of periodontal destruction in this model. Compared to controls, CR resulted in a significant reduction in ligature-induced GI (P <0.0001), BOP (P <0.0015), PD (P <0.0016), and CAL (P <0.0038). Periodontal destruction, as measured by CAL, progressed significantly more slowly in the CR animals than in the controls (P <0.001). These clinical findings are consistent with available evidence that CR has anti-inflammatory effects. Moreover, these experimental findings are the first observations, to the best of our knowledge, that CR dampens the inflammatory response and reduces active periodontal breakdown associated with an acute microbial challenge.

Enhanced activation of a “nutrient‐sensing” pathway with age contributes to insulin resistance

Calorie restriction improves life span whereas nutrient excess leads to obesity and unfavorable metabolic consequences, supporting the role for a cellular "nutrient sensor" in aging. Hexosamine biosynthetic pathway (HBP) is a candidate nutrient-sensing pathway. We hypothesized that altered nutrient sensing (by HBP) with age may provide a link among aging, nutrient flux, and insulin resistance. Using a hyperinsulinemic clamp in young rats, we show that experimental activation of HBP, through the systemic infusion of glucosamine, induced severe insulin resistance (36% decline in peripheral insulin action; P<0.05), increased adipose tissue gene expression of fat-derived peptides (PAI-1 by 4-fold, angiotensinogen 3-fold, leptin 2-fold, resistin 4-fold, and adiponectin 4-fold; P<0.01 compared with young saline-infused), and enhanced glycosylation of transcription factors, thus mimicking a physiological and biological phenotype of aging. We further demonstrate a greater activation of nutrient-sensing HBP with age in both old ad libitum-fed and calorie-restricted rats. Interestingly, old calorie-restricted animals rapidly develop insulin resistance when exposed to glucosamine, despite their "young" phenotype. These results suggest that altered nutrient sensing by HBP with age may be the link among nutrients, insulin resistance, and age-related diabetes.

Self-Modeling Curve Resolution Recovery of Temporal Metabolite Signal Modulation in NMR Spectroscopic Data Sets: Application to a Life-Long Caloric Restriction Study in Dogs

A novel model-free statistical approach (self modeling curve resolution, SMCR) has been applied to recover biochemical information from complex overlapping signals in (1)H NMR spectra of blood serum in a long-term study of caloric restriction (CR) in the dog (n = 24 control fed (CF) and n = 24 CR animals). A new statistical spectroscopic construct, the spectrotype, is proposed which is a spectroscopic subset description or component of a metabolic phenotype. Characterization of the (1)H NMR profiles according to their evolutionary contribution of each spectrotype gives clues to the kinetics of the macro-biochemical response profiles and the identity of the underlying biochemical constituents, governing the evolutionary global response to an intervention. This information can be used to monitor and predict the end point of the biological process and to identify the mechanisms responsible for those changes. Here a SMCR strategy together with a pattern recognition method, principal component analysis (PCA) was used to resolve sets of spectrotypes, without a priori information. From the (1)H NMR evolutionary response profiles, two spectrotypes were identified and resolved; spectrotype 1 dominated by lipids featuring contributions from phosphatidylcholine, lipoprotein lipid fatty acyl groups from triglycerides, phospholipids, and cholesteryl esters plus total cholesterol (i.e., both esterified and unesterified); spectrotype 2 comprising glucose signals and a poorly resolved envelope of albumin and N-acetylated glycoprotein resonances. The relative contributions of these spectrotypes in each sample were calculated. For both caloric restricted (CR) and control fed (CF) dogs between ages 1 and 9 years, the contribution of spectrotype 2 > spectrotype 1, whereas for dogs aged between 9 and 12 years spectrotype 1 > spectrotype 2. Therefore, SMCR analysis pinpointed ages where nutrition and aging metabolic changes became significant within serum samples as well as providing the individual longitudinal contribution profiles associated with each spectrotype, which could potentially be used as part of a strategy to monitor and predict longevity and morbidity in populations. Hence SMCR is a useful addition to the chemometric "toolbox" for metabolic analysis and should have diverse applications within other biomedical conditions characterized by subtle time-dependent changes.

Abstract 4188: Dietary energy balance impacts spontaneous pancreatic lesions in the K5.COX-2 transgenic model of pancreatic cancer

Abstract Pancreatic cancer is the fourth leading cause of cancer death in both men and women in the United States. Obesity, which has increased dramatically over the past 40 years, has emerged as an important risk factor for pancreatic cancer, although the mechanisms underlying the obesity-pancreatic cancer association have not been identified. Calorie restriction (CR), a dietary strategy for inducing negative energy balance and preventing or reversing obesity, has significant anti-cancer effects in several species, for a variety of tumor types, and for both spontaneous and chemically-induced tumors. However, the effects of dietary energy balance modulation on pancreatic tumor formation have not been well studied. We hypothesized that spontaneous tumor development in the K5.COX-2 transgenic mouse model of pancreatitis-driven pancreatic cancer would be reduced in lean mice, relative to overweight or diet-induced obese (DIO) mice. In order to test this hypothesis, we placed 6-8 week old K5.COX-2 transgenic mice on one of three diets for 14 weeks (n=12/group): lean (30% CR), overweight (AIN-76A diet fed ad libitum), or a high calorie/high fat DIO regimen. K5.COX-2 transgenic mice develop spontaneous pancreatic lesions as early as 4 weeks with 100% becoming moribund within 6-8 months. Based on histologic analysis of the percentage of pancreas that is comprised of enlarged and dilated ductal lesions, the CR animals were significantly protected from spontaneous pancreatic lesion formation (7.5%±1.44) as compared to the overweight (45%±10.5) and DIO (57.5%±9.46) groups. Furthermore, CR mice had smaller lesions at time of harvest with none exhibiting signs of moribundity. Conversely, the DIO group generally had the largest lesions, although there was no statistical difference between the overweight and DIO groups. In addition, several mice in the overweight and DIO groups had to be sacrificed early due to extensive abdominal distention, morbidity from enlarged cystic lesions, and other signs of distress. Further investigation is needed to discern how CR exerts its effects. Preliminary analyses suggest that IGF-1 may be playing a significant role. Moreover, pro-inflammatory cytokines were shown to be significantly reduced in sera of CR-animals as compared to the other two groups. In conclusion, using a model of pancreatitis-induced pancreatic tumorigenesis, our findings indicate that dietary energy balance modulation can alter the development of pancreatic cancer. This could have direct implications for the prevention and control of pancreatic tumors due to chronic inflammation and/or obesity.

Enzymes of glycerol and glyceraldehyde metabolism in mouse liver: effects of caloric restriction and age on activities

The influence of caloric restriction on hepatic glyceraldehyde- and glycerol-metabolizing enzyme activities of young and old mice were studied. Glycerol kinase and cytoplasmic glycerol-3-phosphate dehydrogenase activities were increased in both young and old CR (calorie-restricted) mice when compared with controls, whereas triokinase increased only in old CR mice. Aldehyde dehydrogenase and aldehyde reductase activities in both young and old CR mice were unchanged by caloric restriction. Mitochondrial glycerol-3-phosphate dehydrogenase showed a trend towards an increased activity in old CR mice, whereas a trend towards a decreased activity in alcohol dehydrogenase was observed in both young and old CR mice. Serum glycerol levels decreased in young and old CR mice. Therefore increases in glycerol kinase and glycerol-3-phosphate dehydrogenase were associated with a decrease in fasting blood glycerol levels in CR animals. A prominent role for triokinase in glyceraldehyde metabolism with CR was also observed. The results indicate that long-term caloric restriction induces sustained increases in the capacity for gluconeogenesis from glycerol.