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

Evidence of a metabolic memory to early-life dietary restriction in male C57BL/6 mice

<p>Background: Dietary restriction (DR) extends lifespan and induces beneficial metabolic effects in many animals. What is far less clear is whether animals retain a metabolic memory to previous DR exposure, that is, can early-life DR preserve beneficial metabolic effects later in life even after the resumption of ad libitum (AL) feeding. We examined a range of metabolic parameters (body mass, body composition (lean and fat mass), glucose tolerance, fed blood glucose, fasting plasma insulin and insulin-like growth factor 1 (IGF-1), insulin sensitivity) in male C57BL/6 mice dietary switched from DR to AL (DR-AL) at 11 months of age (mid life). The converse switch (AL-DR) was also undertaken at this time. We then compared metabolic parameters of the switched mice to one another and to age-matched mice maintained exclusively on an AL or DR diet from early life (3 months of age) at 1 month, 6 months or 10 months post switch.</p> <p>Results: Male mice dietary switched from AL-DR in mid life adopted the metabolic phenotype of mice exposed to DR from early life, so by the 10-month timepoint the AL-DR mice overlapped significantly with the DR mice in terms of their metabolic phenotype. Those animals switched from DR-AL in mid life showed clear evidence of a glycemic memory, with significantly improved glucose tolerance relative to mice maintained exclusively on AL feeding from early life. This difference in glucose tolerance was still apparent 10 months after the dietary switch, despite body mass, fasting insulin levels and insulin sensitivity all being similar to AL mice at this time.</p> <p>Conclusions: Male C57BL/6 mice retain a long-term glycemic memory of early-life DR, in that glucose tolerance is enhanced in mice switched from DR-AL in mid life, relative to AL mice, even 10 months following the dietary switch. These data therefore indicate that the phenotypic benefits of DR are not completely dissipated following a return to AL feeding. The challenge now is to understand the molecular mechanisms underlying these effects, the time course of these effects and whether similar interventions can confer comparable benefits in humans.</p&gt

Compensatory ingestion upon dietary restriction in Drosophila melanogaster

Dietary restriction extends the lifespan of numerous, evolutionarily diverse species. In D. melanogaster, a prominent model for research on the interaction between nutrition and longevity, dietary restriction is typically based on medium dilution, with possible compensatory ingestion commonly being neglected. Possible problems with this approach are revealed by using a method for direct monitoring of D. melanogaster feeding behavior. This demonstrates that dietary restriction elicits robust compensatory changes in food consumption. As a result, the effect of medium dilution is overestimated and, in certain cases, even fully compensated for. Our results strongly indicate that feeding behavior and nutritional composition act concertedly to determine fly lifespan. Feeding behavior thus emerges as a central element in D. melanogaster aging

Overfeeding Reduces Insulin Sensitivity and Increases Oxidative Stress, without Altering Markers of Mitochondrial Content and Function in Humans

BACKGROUND: Mitochondrial dysfunction and increased oxidative stress are associated with obesity and type 2 diabetes. High fat feeding induces insulin resistance and increases skeletal muscle oxidative stress in rodents, but there is controversy as to whether skeletal muscle mitochondrial biogenesis and function is altered. METHODOLOGY AND PRINCIPAL FINDINGS: Forty (37±2 y) non-obese (25.6±0.6 kg/m2) sedentary men (n = 20) and women (n = 20) were overfed (+1040±100 kcal/day, 46±1% of energy from fat) for 28 days. Hyperinsulinemic-euglycemic clamps were performed at baseline and day 28 of overfeeding and skeletal muscle biopsies taken at baseline, day 3 and day 28 of overfeeding in a sub cohort of 26 individuals (13 men and 13 women) that consented to having all 3 biopsies performed. Weight increased on average in the whole cohort by 0.6±0.1 and 2.7±0.3 kg at days 3 and 28, respectively (P<0.0001, without a significant difference in the response between men and women (P = 0.4). Glucose infusion rate during the hyperinsulinemic-euglycemic clamp decreased from 54.8±2.8 at baseline to 50.3±2.5 mmol/min/kg FFM at day 28 of overfeeding (P = 0.03) without a significant difference between men and women (P = 0.4). Skeletal muscle protein carbonyls and urinary F2-isoprostanes increased with overfeeding (P,<.05). Protein levels of muscle peroxisome proliferator-activated receptor gamma coactivator-1a (PGC1a) and subunits from complex I, II and V of the electron transport chain were increased at day 3 (all P<0.05) and returned to basal levels at day 28. No changes were detected in muscle citrate synthase activity or ex vivo CO2 production at either time point. CONCLUSIONS: Peripheral insulin resistance was induced by overfeeding, without reducing any of the markers of mitochondrial content that were examined. Oxidative stress was however increased, and may have contributed to the reduction in insulin sensitivity observed.Dorit Samocha-Bonet, Lesley V. Campbell, Trevor A. Mori, Kevin D. Croft, Jerry R. Greenfield, Nigel Turner and Leonie K. Heilbron

Metabolic and Behavioral Compensations in Response to Caloric Restriction: Implications for the Maintenance of Weight Loss

BackgroundMetabolic and behavioral adaptations to caloric restriction (CR) in free-living conditions have not yet been objectively measured.Methodology and principal findingsForty-eight (36.8+/-1.0 y), overweight (BMI 27.8+/-0.7 kg/m(2)) participants were randomized to four groups for 6-months;Controlenergy intake at 100% of energy requirements; CR: 25% calorie restriction; CR+EX: 12.5% CR plus 12.5% increase in energy expenditure by structured exercise; LCD: low calorie diet (890 kcal/d) until 15% weight reduction followed by weight maintenance. Body composition (DXA) and total daily energy expenditure (TDEE) over 14-days by doubly labeled water (DLW) and activity related energy activity (AREE) were measured after 3 (M3) and 6 (M6) months of intervention. Weight changes at M6 were -1.0+/-1.1% (CONTROL), -10.4+/-0.9% (CR), -10.0+/-0.8% (CR+EX) and -13.9+/-0.8% (LCD). At M3, absolute TDEE was significantly reduced in CR (-454+/-76 kcal/d) and LCD (-633+/-66 kcal/d) but not in CR+EX or controls. At M6 the reduction in TDEE remained lower than baseline in CR (-316+/-118 kcal/d) and LCD (-389+/-124 kcal/d) but reached significance only when CR and LCD were combined (-351+/-83 kcal/d). In response to caloric restriction (CR/LCD combined), TDEE adjusted for body composition, was significantly lower by -431+/-51 and -240+/-83 kcal/d at M3 and M6, respectively, indicating a metabolic adaptation. Likewise, physical activity (TDEE adjusted for sleeping metabolic rate) was significantly reduced from baseline at both time points. For control and CR+EX, adjusted TDEE (body composition or sleeping metabolic rate) was not changed at either M3 or M6.ConclusionsFor the first time we show that in free-living conditions, CR results in a metabolic adaptation and a behavioral adaptation with decreased physical activity levels. These data also suggest potential mechanisms by which CR causes large inter-individual variability in the rates of weight loss and how exercise may influence weight loss and weight loss maintenance.Trial registrationClinicalTrials.gov NCT00099151.Leanne M. Redman, Leonie K. Heilbronn, Corby K. Martin, Lilian de Jonge, Donald A. Williamson, James P. Delany, Eric Ravussin, for the Pennington CALERIE tea

A family history of type 2 diabetes increases risk factors associated with overfeeding

Aims/hypothesis: The purpose of the study was to test prospectively whether healthy individuals with a family history of type 2 diabetes are more susceptible to adverse metabolic effects during experimental overfeeding. Methods: We studied the effects of 3 and 28 days of overfeeding by 5,200 kJ/day in 41 sedentary individuals with and without a family history of type 2 diabetes (FH+ and FH− respectively). Measures included body weight, fat distribution (computed tomography) and insulin sensitivity (hyperinsulinaemic–euglycaemic clamp). Results: Body weight was increased compared with baseline at 3 and 28 days in both groups (p<0.001), FH+ individuals having gained significantly more weight than FH− individuals at 28 days (3.4±1.6 vs 2.2±1.4 kg, p<0.05). Fasting serum insulin and C-peptide were increased at 3 and 28 days compared with baseline in both groups, with greater increases in FH+ than in FH− for insulin at +3 and +28 days (p<0.01) and C-peptide at +28 days (p<0.05). Fasting glucose also increased at both time points, but without a significant group effect (p=0.1). Peripheral insulin sensitivity decreased in the whole cohort at +28 days (54.8±17.7 to 50.3±15.6 μmol min−1 [kg fat-free mass]−1, p=0.03), and insulin sensitivity by HOMA-IR decreased at both time points (p<0.001) and to a greater extent in FH+ than in FH− (p=0.008). Liver fat, subcutaneous and visceral fat increased similarly in the two groups (p<0.001). Conclusions: Overfeeding induced weight and fat gain, insulin resistance and hepatic fat deposition in healthy individuals. However, individuals with a family history of type 2 diabetes gained more weight and greater insulin resistance by HOMA-IR. The results of this study suggest that healthy individuals with a family history of type 2 diabetes are predisposed to adverse effects of overfeeding.D. Samocha-Bonet, L.V. Campbell, A. Viardot, J. Freund, C.S. Tam, J.R. Greenfield and L.K. Heilbron

Caloric Restriction Alters the Metabolic Response to a Mixed-Meal: Results from a Randomized, Controlled Trial

OBJECTIVES: To determine if caloric restriction (CR) would cause changes in plasma metabolic intermediates in response to a mixed meal, suggestive of changes in the capacity to adapt fuel oxidation to fuel availability or metabolic flexibility, and to determine how any such changes relate to insulin sensitivity (S(I)). METHODS: Forty-six volunteers were randomized to a weight maintenance diet (Control), 25% CR, or 12.5% CR plus 12.5% energy deficit from structured aerobic exercise (CR+EX), or a liquid calorie diet (890 kcal/d until 15% reduction in body weight)for six months. Fasting and postprandial plasma samples were obtained at baseline, three, and six months. A targeted mass spectrometry-based platform was used to measure concentrations of individual free fatty acids (FFA), amino acids (AA), and acylcarnitines (AC). S(I) was measured with an intravenous glucose tolerance test. RESULTS: Over three and six months, there were significantly larger differences in fasting-to-postprandial (FPP) concentrations of medium and long chain AC (byproducts of FA oxidation) in the CR relative to Control and a tendency for the same in CR+EX (CR-3 month P = 0.02; CR-6 month P = 0.002; CR+EX-3 month P = 0.09; CR+EX-6 month P = 0.08). After three months of CR, there was a trend towards a larger difference in FPP FFA concentrations (P = 0.07; CR-3 month P = 0.08). Time-varying differences in FPP concentrations of AC and AA were independently related to time-varying S(I) (P<0.05 for both). CONCLUSIONS: Based on changes in intermediates of FA oxidation following a food challenge, CR imparted improvements in metabolic flexibility that correlated with improvements in S(I). TRIAL REGISTRATION: ClinicalTrials.gov NCT00099151

A mitochondrial mutator plasmid that causes senescence under dietary restricted conditions

BACKGROUND: Calorie or dietary restriction extends life span in a wide range of organisms including the filamentous fungus Podospora anserina. Under dietary restricted conditions, P. anserina isolates are several-fold longer lived. This is however not the case in isolates that carry one of the pAL2-1 homologous mitochondrial plasmids. RESULTS: We show that the pAL2-1 homologues act as 'insertional mutators' of the mitochondrial genome, which may explain their negative effect on life span extension. Sequencing revealed at least fourteen unique plasmid integration sites, of which twelve were located within the mitochondrial genome and two within copies of the plasmid itself. The plasmids were able to integrate in their entirety, via a non-homologous mode of recombination. Some of the integrated plasmid copies were truncated, which probably resulted from secondary, post-integrative, recombination processes. Integration sites were predominantly located within and surrounding the region containing the mitochondrial rDNA loci. CONCLUSION: We propose a model for the mechanism of integration, based on innate modes of mtDNA recombination, and discuss its possible link with the plasmid's negative effect on dietary restriction mediated life span extension