Jackie S. Duncan

Energy expenditure of calorically restricted rats is higher than predicted from their altered body composition

Debate exists over the impact of caloric restriction (CR) on the level of energy expenditure. At the whole animal level, CR decreases metabolic rates but in parallel body mass also declines. The question arises whether the reduction in metabolism is greater, smaller or not different from the expectation based on body mass change alone. Answers to this question depend on how metabolic rate is normalized and it has recently been suggested that this issue can only be resolved through detailed morphological investigation. Added to this issue is the problem of how appropriate the resting energy expenditure is to characterize metabolic events relating to aging phenomena. We measured the daily energy demands of young and old rats under ad libitum (AD) food intake or 40% CR, using the doubly labeled water (DLW) method and made detailed morphological examination of individuals, including 21 different body components. Whole body energy demands of CR rats were lower than AD rats, but the extent of this difference was much less than expected from the degree of caloric restriction, consistent with other studies using the DLW method on CR animals. Using multiple regression and multivariate data reduction methods we built two empirical predictive models of the association between daily energy demands and body composition using the ad lib animals. We then predicted the expected energy expenditures of the CR animals based on their altered morphology and compared these predictions to the observed daily energy demands. Independent of how we constructed the prediction, young and old rats under CR expended 30 and 50% more energy, respectively, than the prediction from their altered body composition. This effect is consistent with recent intra-specific observations of positive associations between energy metabolism and lifespan and theoretical ideas about mechanisms underpinning the relationship between oxygen consumption and reactive oxygen species production in mitochondria.

Life-long vitamin C supplementation in combination with cold exposure does not affect oxidative damage or lifespan in mice, but decreases expression of antioxidant protection genes

Oxidative stress is suggested to be central to the ageing process, with endogenous antioxidant defence and repair mechanisms in place to minimize damage. Theoretically, supplementation with exogenous antioxidants might support the endogenous antioxidant system, thereby reducing oxidative damage, ageing-related functional decline and prolonging life- and health-span. Yet supplementation trials with antioxidants in animal models have had minimal success. Human epidemiological data are similarly unimpressive, leading some to question whether vitamin C, for example, might have pro-oxidant properties in vivo. We supplemented cold exposed (7+/-2 degrees C) female C57BL/6 mice over their lifespan with vitamin C (ascorbyl-2-polyphosphate), widely advocated and self administered to reduce oxidative stress, retard ageing and increase healthy lifespan. No effect on mean or maximum lifespan following vitamin C treatment or any significant impact on body mass, or on parameters of energy metabolism was observed. Moreover, no differences in hepatocyte and lymphocyte DNA oxidative damage or hepatic lipid peroxidation was seen between supplemented and control mice. Using a DNA macroarray specific for oxidative stress-related genes, we found that after 18 months of supplementation, mice exhibited a significantly reduced expression of several genes in the liver linked to free-radical scavenging, including Mn-superoxide dismutase. We confirmed these effects by Northern blotting and found additional down-regulation of glutathione peroxidase (not present on macroarray) in the vitamin C treated group. We suggest that high dietary doses of vitamin C are ineffective at prolonging lifespan in mice because any positive benefits derived as an antioxidant are offset by compensatory reductions in endogenous protection mechanisms, leading to no net reduction in accumulated oxidative damage.

Hypothalamic energy balance gene responses in the Sprague-Dawley rat to supplementation of high-energy diet with liquid ensure and subsequent transfer to chow.

Energy dense, high fat, high sugar, foods and beverages in our diet are a major contributor to the escalating global obesity problem. Here, we examine the physiological and neuroendocrine effects of feeding rats a solid high‐energy (HE) diet with or without a liquid supplement (Ensure) and the consequence of subsequently transferring animals back to chow (C). Outbred Sprague‐Dawley rats were fed C until 49–56 days of age, and then transferred a HE diet for 3 weeks before allocation to one of two weight‐matched groups. Over the next 10 weeks, one group remained on HE diet, whereas the other had access to the liquid diet, chocolate Ensure (EN), in addition to HE diet (HE + EN). Half the rats from each group were then killed, and the remainder were returned to C for 3 weeks. Supplementation of the HE diet with EN accelerated weight gain and increased daily energy intake, adipose tissue mass, and circulating leptin levels. Transferring animals back to C caused a decrease in bodyweight in the HE + EN group, whereas HE animals were weight stable. Both groups also exhibited voluntary hypophagia, although the magnitude and duration of this response was greater in HE + EN animals. The only effect of Ensure on the hypothalamic genes studied was on tyrosine kinase B expression in the ventromedial hypothalamic nucleus (VMH), which was increased in rats given the supplement. Withdrawal of the obesogenic diets decreased gene expression for cocaine‐and‐amphetamine regulated transcript (CART) and dynorphin (DYN) in the arcuate nucleus (ARC), and DYN and brain‐derived neurotrophic factor (BDNF) in the VMH, whereas neuropeptide Y (NPY) gene expression in the ARC was increased. These changes were independent of previous dietary history. EN supplementation generates distinct physiological responses, yet has a minimal effect on hypothalamic neuropeptide or receptor gene expression, possibly due to the development of leptin resistance. Withdrawal of obesogenic diets induces changes in the gene expression consistent with NPY, CART and BDNF attempting to oppose weight gain on either HE or HE + EN.

Additional anthropometric measures may improve the predictability of basal metabolic rate in adult subjects

Background:The most commonly used predictive equation for basal metabolic rate (BMR) is the Schofield equation, which only uses information on body weight, age and sex to derive the prediction. However, because body composition is a key influencing factor, there will be error in calculating an individuals basal requirements based on this prediction.Objective:To investigate whether adding additional anthropometric measures to the standard measures can enhance the predictability of BMR and to cross-validate this within a separate subgroup.Design:Cross-sectional study of 150 Caucasian adults from Scotland, with a body mass index range of 16.7–49.3 kg/m2. All subjects underwent measurement of BMR, body composition, and 148 also had basic skinfold and circumference measures taken. The resultant equation was tested in a subgroup of 39 obese males.Results:The average difference between the predicted (Schofield equation) and measured BMR was 502 kJ/day. There was a slight systematic bias in this error, with the Schofield equation underestimating the lowest values. The average discrepancy between predicted and actual BMR was reduced to 452 kJ/day, with the addition of fat mass, fat-free mass, an overall 10% improvement on the Schofield equation (P=0.054). Using an equation derived from principal components analysis of anthropometry measurements similarly decreased the difference to 458 kJ/day (P=0.039). Testing the equation in a separate group indicated a 33% improvement in predictability of BMR, compared to the Schofield equation.Conclusions:In the absence of detailed information on body composition, utilizing anthropometric data provides a useful alternative methodology to improve the predictability of BMR beyond that achieved from the standard Schofield prediction equation. This should be confirmed in more individuals, both within the obese and normal weight category.

Lifelongα-tocopherol supplementation increases the median life span of C57BL/6 mice in the cold but has only minor effects on oxidative damage

The effects of dietary antioxidant supplementation on oxidative stress and life span are confused. We maintained C57BL/6 mice at 7 +/- 2 degrees C and supplemented their diet with alpha-tocopherol from 4 months of age. Supplementation significantly increased (p = 0.042) median life span by 15% (785 days, n = 44) relative to unsupplemented controls (682 days, n = 43) and also increased maximum life span (oldest 10%, p = 0.028). No sex or sex by treatment interaction effects were observed on life span, with treatment having no effect on resting or daily metabolic rate. Lymphocyte and hepatocyte oxidative DNA damage and hepatic lipid peroxidation were unaffected by supplementation, but hepatic oxidative DNA damage increased with age. Using a cDNA macroarray, genes associated with xenobiotic metabolism were significantly upregulated in the livers of female mice at 6 months of age (2 months supplementation). At 22 months of age (18 months supplementation) this response had largely abated, but various genes linked to the p21 signaling pathway were upregulated at this time. We suggest that alpha-tocopherol may initially be metabolized as a xenobiotic, potentially explaining why previous studies observe a life span extension generally when lifelong supplementation is initiated early in life. The absence of any significant effect on oxidative damage suggests that the life span extension observed was not mediated via any antioxidant properties of alpha-tocopherol. We propose that the life span extension observed following alpha-tocopherol supplementation may be mediated via upregulation of cytochrome p450 genes after 2 months of supplementation and/or upregulation of p21 signaling genes after 18 months of supplementation. However, these signaling pathways now require further investigation to establish their exact role in life span extension following alpha-tocopherol supplementation.

Altered Expression of SOCS3 in the Hypothalamic Arcuate Nucleus during Seasonal Body Mass Changes in the Field Vole, Microtus agrestis

We have previously shown that cold‐acclimated (8 °C) male field voles (Microtus agrestis) transferred from short day (SD, 8 h light) to long day (LD, 16 h light) photoperiod exhibit an increase in body mass lasting 4 weeks, after which they stabilise at a new plateau approximately 7.5 g (24.8%) higher than animals maintained in SD. By infusing voles with exogenous leptin, we have also demonstrated that SD voles respond to the hormone by reducing body mass and food intake, whereas LD animals increasing body mass are resistant to leptin treatment. In the present study, we investigated whether seasonal changes in body mass could be linked to modulation of the leptin signal by suppressor of cytokine signalling‐3 (SOCS3). We used in situ hybridisation to examine hypothalamic arcuate nucleus (ARC) expression of SOCS3, neuropeptide Y (NPY), agouti‐related peptide (AgRP), pro‐opiomelanocortin (POMC) and cocaine‐ and amphetamine‐regulated transcript (CART) genes in 90 voles exposed to either SD or LD for up to 11 weeks. LD voles increasing body mass had significantly higher levels of SOCS3 mRNA than SD or LD voles with a stable body mass. There were no associated changes in expression of NPY, AgRP, POMC and CART genes. These results suggest that voles that regulate body mass at either the lower (SD) or upper (LD) plateau remain sensitive to leptin action, whereas SOCS3‐mediated leptin resistance is a short‐term mechanism that enables animals to move between the stable body mass plateaus. Our data provide evidence that expression of SOCS3 in the ARC is involved in the modulation of the strength of the leptin signal to facilitate seasonal cycles in body mass and adiposity.

Plasma leptin levels are related to body composition, sex, insulin levels and the A55V polymorphism of the UCP2 gene

Objective:Circulating leptin levels show a high degree of individual variability even after the main effect of body fatness is accounted for. We therefore wanted to determine the roles of variation in body composition, age, sex and polymorphisms of the UCP2 gene and promoter region on levels of circulating leptin.Subjects:One hundred and fifty Caucasian subjects, which represented a cross-section of the population from NE, Scotland, were recruited.Measurements:Body composition was measured using dual X-ray absorptiometry. Fasted circulating leptin, insulin, T3 and T4 levels were measured, and all individuals were genotyped for the UCP2 polymorphisms A55V, –866G>A and exon-8 ins/del.Results:The results indicate that circulating leptin was significantly related to sex and principle component (PC) scores representing overall adipose tissue mass and a second representing the contrast of central to peripheral bone mineral content. Residual leptin was associated with the A55V polymorphism (P< 0.001) explaining 11.3% of the residual variance. There was a marginal effect associated with exon-8 ins/del (P=0.045) explaining 4.4% of the residual variance in leptin. Loge transformed circulating fasting insulin was related to PC scores representing general adiposity and sex. Residual Loge insulin was associated with the A55V and exon-8 ins/del polymorphisms explaining 5.7% (P=0.015) and 5% (P=0.026) of the residual variation, respectively. The –866G>A polymorphism was not significantly associated with residual leptin or insulin. Leptin and insulin were significantly (P=0.007) correlated. Statistically removing the effect of insulin on leptin still showed association between leptin and A55V (P=0.002). Removing the effect of leptin on insulin, the A55V polymorphism was no longer significant (P=0.120). After accounting for the correlation between insulin and leptin, the exon-8 ins/del was no longer significant for residual leptin (P=0.119) or Loge insulin (P=0.252).Conclusion:These data suggest that the A55V polymorphism directly affected the levels of leptin but not via an effect on insulin.

Introduction of a high-energy diet acutely up-regulates hypothalamic cocaine and amphetamine-regulated transcript, Mc4R and brown adipose tissue uncoupling protein-1 gene expression in male Sprague-Dawley rats.

Obesity is an escalating problem in Western societies. Susceptibility to weight gain within an obesogenic environment is variable. It remains unclear how the range of weight gain responses are generated. It is possible that an individuals immediate and/or sustained appetite for apparently palatable foods, or metabolic adaptations to a new diet could be important. The present study therefore examined the short‐ to medium‐term effects of a high‐energy (HE) diet on bodyweight, food intake, and energy balance‐related signalling systems. Sprague‐Dawley rats were fed either chow or an HE diet for 12 h, 24 h, 48 h or 14 days. Blood hormones and metabolites were assayed, and expression of uncoupling protein‐1 (UCP‐1) and hypothalamic energy‐balance related genes were determined by Northern blotting or in situ hybridisation, respectively. Short‐term exposure (12 h, 24 h, 48 h) to the HE diet had no effect on grams of food consumed, but caloric intake was increased. Exposure to HE diet for 14 days (medium term) established a bodyweight differential of 7.7 g, and animals exhibited a transient increase in caloric intake of 5 days duration. Terminal levels of leptin, insulin, glucose and non‐esterified fatty acids (NEFAs) were all increased in HE‐fed animals. UCP‐1 mRNA was elevated in interscapular brown adipose tissue from HE‐fed rats only at 12 h. Cocaine and amphetamine‐regulated transcript (CART) and Mc4R gene expression in the hypothalamus were increased after 12 h and 24 h on an HE diet, respectively. The rats appear to passively over‐consume calories as a result of consuming a similar weight of a more energy dense food. This evokes physiological responses, which adjust caloric intake over several days. Circulating NEFA and insulin concentrations, UCP‐1, Mc4R and CART gene expression are increased as an immediate consequence of consuming HE diet, and may be involved in countering hypercaloric intake. Circulating leptin is increased in the HE‐fed animals after 48 h, reflecting their increasing adiposity.

Calorie-restricted mice that gorge show less ability to compensate for reduced energy intake.

Caloric restriction in mice can trigger gorging behaviour, which is characterized by periods of excessive food ingestion in a short time. Animals that gorge are thought to have a reduced metabolism compared to those that nibble their food over a longer period and might therefore be more able to compensate for reduced energy intake. We examined whether mice that gorged showed less weigh loss during restriction. We placed female mice (n=60) on a restriction of 75% of their ad libitum food intake (FI) for 22 days. FI and body mass (BM) were measured at 1, 2 and 24 h after food provision. Ten controls remained feeding ad lib and we selected the 10 strongest gorgers and 10 strongest non-gorgers for comparison. Mice had BM, FI, resting metabolic rate (RMR), body composition, body temperature, daily energy expenditure (DEE) and circulating levels of the regulatory hormones leptin and ghrelin measured. Gorgers had a significantly lower BM at the end of restriction than non-gorgers or controls, indicating that they were less able to compensate for the reduced energy. Both groups of restricted mice had reduced RMR, however reduced activity was only used as an energy saving mechanism in non-gorgers. Gorging mice had a significantly lower level of circulating leptin than controls and non-gorgers but no differences in ghrelin levels. Gorging mice were, in fact, less able to compensate for reduced energy intake, as they reduced RMR by a similar extent as non-gorgers, but did not reduce activity compared to non-gorgers on the same restriction level. The reduced leptin levels may drive the gorging behaviour.