Calorie-restricted Mice Research Articles

Coordinated multitissue transcriptional and plasma metabonomic profiles following acute caloric restriction in mice

Caloric restriction (CR) increases healthy life span in a range of organisms. The underlying mechanisms are not understood but appear to include changes in gene expression, protein function, and metabolism. Recent studies demonstrate that acute CR alters mortality rates within days in flies. Multitissue transcriptional changes and concomitant metabolic responses to acute CR have not been described. We generated whole genome RNA transcript profiles in liver, skeletal muscle, colon, and hypothalamus and simultaneously measured plasma metabolites using proton nuclear magnetic resonance in mice subjected to acute CR. Liver and muscle showed increased gene expressions associated with fatty acid metabolism and a reduction in those involved in hepatic lipid biosynthesis. Glucogenic amino acids increased in plasma, and gene expression for hepatic gluconeogenesis was enhanced. Increased expression of genes for hormone-mediated signaling and decreased expression of genes involved in protein binding and development occurred in hypothalamus. Cell proliferation genes were decreased and cellular transport genes increased in colon. Acute CR captured many, but not all, hepatic transcriptional changes of long-term CR. Our findings demonstrate a clear transcriptional response across multiple tissues during acute CR, with congruent plasma metabolite changes. Liver and muscle switched gene expression away from energetically expensive biosynthetic processes toward energy conservation and utilization processes, including fatty acid metabolism and gluconeogenesis. Both muscle and colon switched gene expression away from cellular proliferation. Mice undergoing acute CR rapidly adopt many transcriptional and metabolic changes of long-term CR, suggesting that the beneficial effects of CR may require only a short-term reduction in caloric intake.

Calorie Restriction in Mice: Effects on Body Composition, Daily Activity, Metabolic Rate, Mitochondrial Reactive Oxygen Species Production, and Membrane Fatty Acid Composition

Different levels of calorie restriction (CR) (125, 85, 50, or 40 kcal/wk for 1, 3, and 6 months) were examined in mice by using the paradigm of Weindruch and colleagues. Lean and total body mass increased on 125 and 85 kcal/wk, but there was negligible growth on low-energy intake. There was no CR-induced reduction in either daily activity or mass-specific metabolic rate. There was no CR-effect on in vitro reactive oxygen species production by liver or muscle mitochondria at 3 months, but after 6 months the effect was significantly reduced in liver mitochondria from 40 kcal/wk mice compared to 125 kcal/wk mice. Changes in the fatty acid composition of phospholipids from liver, kidneys, heart, brain, and skeletal muscle were observed following 1 month of CR.

Do long-lived mutant and calorie-restricted mice share common anti-aging mechanisms?—a pathological point of view

Rodent models are an invaluable resource for studying the mechanism of mammalian aging. In recent years, the availability of transgenic and knockout mouse models has facilitated the study of potential mechanisms of aging. Since 1996, aging studies with several long-lived mutant mice have been conducted. Studies with the long-lived mutant mice, Ames and Snell dwarf, and growth hormone receptor/binding protein knockout mice, are currently providing important clues regarding the role of the growth hormone/insulin like growth factor-1 axis in the aging process. Interestingly, these studies demonstrate that these long-lived mutant mice have physiological characteristics that are similar to the effects of calorie restriction, which has been the most effective experimental manipulation capable of extending lifespan in various species. However, a question remains to be answered: do these long-lived mutant and calorie-restricted mice extend their lifespan through a common underlying mechanism?

Adipocytokines and Lipid Levels in Ames Dwarf and Calorie-Restricted Mice

Ames dwarf mice are long-lived and insulin sensitive, and have a normal or reduced percentage of body fat. Calorie restriction (CR) is known to improve insulin sensitivity and reduce body fat. The purpose of this study was to evaluate the mechanism of improved insulin sensitivity in the Ames dwarfs and the effects of CR on adipose signaling and metabolism in normal and dwarf mice. Enhanced insulin sensitivity in dwarf mice may be partly due to increased release of adiponectin and the reduced release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Altered levels of adipocytokines might be consequent to the decreased lipid synthesis, plasma triglycerides, and free fatty acid levels. In normal mice, CR improves insulin sensitivity by affecting the release of adipocytokines, and decreasing circulating fatty acid and triglycerides concentrations as well as liver triglyceride accumulation. However, CR may reduce rather than enhance some of the insulin effects in the highly insulin-sensitive dwarf mice.

Gene Expression in Lung: Early Response to Ad Libitum Refeeding after Calorie Restriction in Adult Mice

Our goal is to identify and manipulate for therapeutic purposes gene expression in lung that is determinative of alveolar regeneration. One obstacle toward this goal is identification of the time of onset of gene expression determinative of alveolar regeneration following an experimental manipulation that induces alveolar regeneration. To identify the onset of signaling at the RNA level that initiates alveolar regeneration, we reasoned to regenerate alveoli requires, for each alveolus, the conversion of a flat component of gas-exchange surface comprised of epithelium, extracellular matrix, cells within the matrix, and endothelial cells, into a fold (septum). The biological processes involved in forming a septum include cell replication, angiogenesis, cell motion and guidance, and formation of extracellular matrix. Using microarray and RT-PCR analysis of gene expression in lung, we found evidence at the RNA-level, of signaling for cell replication and extracellular matrix formation within 3 h of ad libitum refeeding following calorie restriction in mice, a manipulation that induces regeneration of alveoli lost during calorie restriction. We conclude that the first three hours of ad libitum refeeding following calorie restriction is the period in which genes determinative of alveoli regeneration in this model are expressed. Supported by HL20366, HL37666, and HL73558.

Adaptive Stress Response in Segmental Progeria Resembles Long-Lived Dwarfism and Calorie Restriction in Mice

How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPDG602D/R722W/XPA−/−) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80 −/− mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage.

Increase in Activity During Calorie Restriction Requires Sirt1

Sir2 (silent information regulator 2) is a nicotinamide adenine dinucleotide-dependent deacetylase required for longevity due to calorie restriction in yeast and Drosophila. In mammals, calorie restriction induces a complex pattern of physiological and behavioral changes. Here we report that the mammalian Sir2 ortholog, Sirt1, is required for the induction of a phenotype by calorie restriction in mice.

Housing Density Does Not Influence the Longevity Effect of Calorie Restriction

This study examined the effect of housing density on the longevity-extending and disease-delaying actions of calorie restriction (CR). Singly or multiply housed (four per cage) mice were either fed ad libitum (AL) or were on CR beginning at 2 months. All CR mice were fed 40% less food than were multiply housed AL mice. CR increased median longevity by 19%, and housing density had no effect on this increase. CR also reduced neoplastic lesions in both housing groups, but lymphoma, the most common neoplasm, was reduced more in singly than in multiply housed mice. Singly housed AL mice ate 40% more food than did multiply housed AL mice, but weighed the same and lived as long as multiply housed AL mice. These results indicate that CR can extend life span as effectively in multiply as in singly housed mice, even though housing density can differentially affect the cancer-reducing effect of CR.

Early hypothalamic response to age-dependent gene expression by calorie restriction

Molecular events linking the initial detection of calorie restriction (CR) to changes in gene expression throughout the organism that ultimately retard aging in CR animals are unknown. This study measured changes in gene expression induced by CR and by aging in the hypothalamus, which likely plays a central role in the initial perception of and response to CR. Hypothalamic expression profiling was done in young (4–6 months) ad libitum fed (AL), young CR (2.5–4.5 months of CR), and old (26–28 months) AL male C57BL/6 mice. CR altered the expression of 137 genes and aging altered 1222. Only 8 age-related genes were oppositely regulated by CR. To test whether reduced plasma glucose is a signal in altering hypothalamic gene expression, we examined GLUT4 transgenic mice (C57BL/6 background; 4–6 months), which have reduced plasma glucose similar to that of CR mice. Twenty-seven genes differed between transgenic and non-transgenic mice; nine of these were only altered by CR. The decreased plasma glucose had a limited role in CR mediated hypothalamic gene expression.

Mouse liver plasma membrane redox system activity is altered by aging and modulated by calorie restriction

Caloric restriction (CR) is known as the only non-genetic method proven to slow the rate of aging and extend lifespan in animals. Free radicals production emerges from normal metabolic activity and generates the accumulation of oxidized macromolecules, one of the main characteristics of aging. Due to its central role in cell bioenergetics, a great interest has been paid to CR-induced modifications in mitochondria, where CR has been suggested to decrease reactive oxygen species production. The plasma membrane contains a trans-membrane redox system (PMRS) that provides electrons to recycle lipophilic antioxidants, such as α-tocopherol and coenzyme Q (CoQ), and to modulate cytosolic redox homeostasis. In the present study, we have investigated age differences in the PMRS in mouse liver and their modulation by CR. Aging induced a decrease in the ratio of CoQ10/CoQ9 and α-tocopherol in liver PM from AL-fed mice that was attenuated by CR. CoQ-dependent NAD(P)H dehydrogenases highly increased in CR old mice liver PMs. On the other hand, the CoQ-independent NADH-FCN reductase activity increased in AL-fed animals; whereas, in mice under CR this activity did not change during aging. Our results suggest that liver PMRS activity changes during aging and that CR modulates these changes. By this mechanism CR maintains a higher antioxidant capacity in liver PM of old animals by increasing the activity of CoQ-dependent reductases. Also, the putative role of PMRS in the modulation of redox homeostasis of cytosol is implicated.

Mitochondrial DNA as a Cancer Biomarker

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.

SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and Caenorhabditis elegans. Because adipose tissue is essential in energy homeostasis and also plays a role in life span determination, we decided to investigate the function of sirtuin members in fat. We report here that murine SIRT3 is expressed in brown adipose tissue and is localized on the mitochondria inner membrane. Caloric restriction activates SIRT3 expression in both white and brown adipose. Additionally, cold exposure up-regulates SIRT3 expression in brown fat, whereas elevated climate temperature reduces the expression. Enforced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of the uncoupling protein PGC-1alpha, UCP1, and a series of mitochondria-related genes. Both ADP-ribosyltransferase and deacetylase activities of SIRT3 are required for this action. Furthermore, the SIRT3 deacetylase mutant exhibits a dominant negative effect by inhibiting UCP1 expression. This inhibitive effect can be abolished by the coexpression of PGC-1alpha, indicating a major role of PGC-1alpha in the SIRT3 action. In addition, SIRT3 stimulates CREB phosphorylation, which reportedly activates PGC-1alpha promoter directly. Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species production while increasing cellular respiration. Finally, SIRT3, along with genes related to mitochondrial function, is down-regulated in the brown adipose tissue of several genetically obese mice. In summary, our results demonstrate that SIRT3 activates mitochondria functions and plays an important role in adaptive thermogenesis in brown adipose.

Serine utilization in mouse liver: Influence of caloric restriction and aging

The influence of caloric restriction (CR) on the activities of hepatic serine metabolizing enzymes in young (3 months) and old (30 months) mice was studied. Serine dehydratase (SDH) activity increased markedly with age in both diet groups and in old mice was higher in the CR group. No effects of CR were observed in the young. Serine:pyruvate transaminase (SPT) and glycerate kinase activities were unaffected by age and diet. However, glycerate dehydrogenase activity was decreased in old CR mice but not in young CR. The results of this study show that long-term CR influenced serine utilization only in the pathway catalyzed by SDH. This suggests that in mouse liver this pathway is critical for serine utilization in gluconeogenesis, while the SPT pathway plays a minor role. The increase in SDH activity with long-term CR is consistent with sustained increase in gluconeogenesis.

The effects of intentional weight loss as a latent variable problem

Although obesity is associated with increased mortality rate and short-term weight loss improves risk factors for mortality, it has not been convincingly shown that weight loss among obese people results in reduced mortality rate. When considering the human literature, it has been pointed out that weight loss is often a sign of illness and that investigators therefore need to separate intentional from unintentional weight loss. It has generally been assumed that among people who state that they do not intend to lose weight, weight change subsequently observed is unintentional. Among such people, weight loss has been consistently associated with increased mortality rate. Complementarily, it has generally been assumed that among people who state that they do intend to lose weight, weight change subsequently observed is intentional. In these people who are intending to lose weight, some studies show apparent benefits of weight loss, some are neutral, and some show deleterious effects. The overall conclusion that some reviewers have drawn from this literature is that intentional weight loss (IWL) is at best not beneficial and may even be harmful with respect to mortality rate. We believe that this conclusion is drawn by inappropriately conflating weight loss (or more generally weight change) among people intending to lose weight with IWL (or change). Herein, under certain assumptions, we: (1) show that the association between mortality rate and weight loss among people intending to lose weight and between mortality rate and IWL are two different things; (2) show that the association between IWL and mortality rate is an inherently unobservable entity; (3) derive a method for estimating the plausible range of true effect of IWL on mortality rate if one is willing to make a number of restrictive, but perhaps reasonable assumptions; and (4) illustrate the method by application to a data set involving middle-age onset calorie restriction in mice.

Adaptive stress response in segmental progeria resembles long-lived dwarfism and calorie restriction in mice

Adaptive stress response in segmental progeria resembles long-lived dwarfism and calorie restriction in mice

Caloric restriction transiently improves motor performance but hastens clinical onset of disease in the Cu/Zn‐superoxide dismutase mutant G93A mouse

Caloric restriction (CR) prolongs lifespan in insects, rodents, and nonhuman primates, a process attributed to a reduction in oxidative stress. Transgenic mice that overexpress the mutant human Cu/Zn-superoxide dismutase (SOD1) gene (G93A mice) are an animal model of amyotrophic lateral sclerosis showing progressively lower motor neuron weakness and increased oxidative stress. We investigated the effect of CR on motor performance, clinical onset, disease progression, and lifespan in G93A mice. Starting at 40 days of age, 14 separately caged G93A mice were randomly divided into two groups: ad libitum (AL; n = 6) and calorie-restricted (CR; n = 8) with a diet equal to 60% of AL. The CR mice (mean +/- SEM: 14.0 +/- 0.7 g) weighed 31% less than the AL mice (20.3 +/- 1.0 g) (P = 0.0002). From 74 to 93 days of age, the CR mice performed better on the rotarod than the AL mice: fall time, P = 0.039; fall speed, P = 0.009. The CR mice had a faster rate of reaching clinical onset than the AL mice (hazard ratio = 4.3, P = 0.0006). The CR and AL mice reached clinical onset of disease at age 99 +/- 1 and 110 +/- 2 days, respectively (P = 0.0003), with no significant difference in disease progression. The CR mice tended to reach endpoint sooner than the AL mice (age-specific death: 125 +/- 3 vs. 133 +/- 3 days, respectively, P = 0.09). We conclude that CR diet transiently improves motor performance but hastens clinical onset of disease in G93A mice. These results suggest that CR diet is not a protective strategy for patients with amyotrophic lateral sclerosis (ALS) and hence is contraindicated.

Fructose metabolizing enzymes from mouse liver: influence of age and caloric restriction

The influence of caloric restriction (CR) on the activities of liver fructose metabolizing enzymes and metabolite levels were studied in young (3 months) and old (30 months) mice. Fructokinase activity was increased (P<0.05) in both young and old CR mice when compared to controls while triokinase activity was increased (P<0.05) only in old CR versus control mice. Aldolase was not altered by CR in either old or young mice. No age-related differences in activities were observed in controls although a trend towards an increase was observed for triokinase, while significant age-related increases were observed for fructokinase and triokinase, but not aldolase, in CR mice. Both young and old mice on CR showed significant decreases in fructose and fructose-1-phosphate, however, no age-related changes in metabolite levels were observed for either control or CR mice. A fructose-1-phosphate kinase activity was also measured and found to be unchanged in both young and old mice on CR, but the activity was significantly lower in the old mice compared with young. We show here that the enzymes involved in fructose metabolism are influenced by CR and that this could contribute to alterations in gluconeogenesis and glycolysis observed with CR.

Behavioral and physiologic responses to caloric restriction in mice

The purpose of the review is to highlight the influences of ambient temperature ( T a) and caloric restriction (CR) on metabolism, cardiovascular function and behavior in mice. Standard vivarium ambient temperatures ( T a≅23 °C) are a mild cold stress for mice requiring elevated metabolic rate and food intake. Increasing T a into the zone of thermoneutrality (TMN≅29–33 °C) markedly reduces food intake, metabolic rate, heart rate (HR) and blood pressure in mice. Mice are members of a diverse, yet unique group of homeothermic animals that respond to thermal and energetic challenges by allowing body temperature ( T b) to fall to less than 31 °C, a condition known as torpor. In mice housed at standard T a, torpor is induced by a single night of fasting or a few days of CR. The mechanisms responsible for initiating torpor are related to reduced caloric availability, but do not require leptin. Mice housed at TMN and subjected to CR exhibit physiologic reductions in metabolic rate and HR, but do not appear to enter torpor. Finally, mice exhibit differential locomotor activity responses during CR that depends on T a. At standard T a, mice display increased light-phase home-cage activity with CR. This response is virtually eliminated when CR is performed at TMN. We suggest that researchers using mice to investigate energy homeostasis and cardiovascular physiology carefully consider the influence of T a on physiology and behavior.

Hypotension and bradycardia during caloric restriction in mice are independent of salt balance and do not require ANP receptor.

We hypothesized that caloric restriction (CR)-induced hypotension would correlate with increased sodium excretion through an atrial natriuretic peptide (ANP)-dependent mechanism. To test this hypothesis, the cardiovascular parameters of c57/Bl mice were measured with radiotelemetry while urine was collected. The 23-h mean blood pressure (BP) dropped from 108.6 +/- 1.8 to 92.7 +/- 2.4 mmHg, and 23-h heart rate dropped from 624 +/- 5 to 426 +/- 13 beats/min over 7 days of CR at 29 degrees C. Contrary to our hypothesis, urine sodium excretion decreased by 55% by day 7 of CR. Consistent with decreased sodium excretion was the drop in plasma ANP (from 82.4 +/- 4.3 to 68.0 +/- 5.8 pg/ml). To explore the possibility that CR lowers BP through an ANP receptor-dependent mechanism that is independent of its effect on sodium retention, we measured the cardiovascular parameters of mice deficient in the ANP receptor (NPR1(-/-)) or the ANP clearance receptor (NPR3(-/-)). Mean BP fell from 117.1 +/- 3.9 to 108.0 +/- 4.7 mmHg in the NPR1(-/-) mice and from 87.0 +/- 2.4 to 78.4 +/- 1.7 mmHg in the NPR3(-/-) mice during CR. These data indicate that the hypotension induced by CR does not depend on increased sodium excretion. Rather, it appears that the mouse responds to the low BP induced by CR with an increase in sodium reabsorption. Furthermore, circulating ANP levels and data from NPR1(-/-) and NPR3(-/-) mice suggest that the ANP pathway may not be involved in the cardiovascular response to CR.

Krebs cycle enzymes from livers of old mice are differentially regulated by caloric restriction

Krebs cycle enzyme activities and levels of five metabolites were determined from livers of old mice (30 months) maintained either on control or on long-term caloric restriction (CR) diets (28 months). In CR mice, the cycle was divided into two major blocks, the first containing citrate synthase, aconitase and NAD-dependent isocitrate dehydrogenase which showed decreased activities, while the second block, containing the remaining enzymes, displayed increased activity (except for fumarase, which was unchanged). CR also resulted in decreased levels of citrate, glutamate and α-ketoglutarate, increased levels of malate, and unchanged levels of aspartate. The α-ketoglutarate/glutamate and malate/α-ketoglutarate ratios were higher in CR, in parallel with previously reported increases with CR in pyruvate carboxylase activity and glucagon levels, respectively. The results indicate that long-term CR induces a differential regulation of Krebs cycle in old mice and this regulation may be the result of changes in gene expression levels, as well as a complex interplay between enzymes, hormones and other effectors. Truncation of Krebs cycle by CR may be an important adaptation to utilize available substrates for the gluconeogenesis necessary to sustain glycolytic tissues, such as brain.