Real time in vivo methods are needed to better understand the interplay between diet and the gastrointestinal microbiota. Therefore, a rodent indirect calorimetry system was equipped with hydrogen (H2) and methane (CH4) sensors. H2 production was readily detected in C57BL/6J mice and followed a circadian rhythm. H2 production was increased within 12 hours after first exposure to a lowly-digestible starch diet (LDD) compared to a highly-digestible starch diet (HDD). Marked differences were observed in the faecal microbiota of animals fed the LDD and HDD diets. H2 was identified as a key variable explaining the variation in microbial communities, with specific taxa (including Bacteroides and Parasutterella) correlating with H2 production upon LDD-feeding. CH4 production was undetectable which was in line with absence of CH4 producers in the gut. We conclude that real-time in vivo monitoring of gases provides a non-invasive time-resolved system to explore the interplay between nutrition and gut microbes in a mouse model, and demonstrates potential for translation to other animal models and human studies.
Nutrition in early life has permanent consequences affecting development and later-life health. In this study we investigated the potential of monosaccharides in the post-weaning diet to program adult metabolic health. Female and male C57BL/6JRccHsd mice were time-mated and fed a low-fat diet ad libitum. Litters were culled to six pups per nest and randomly assigned to foster dams. Mice were weaned onto one of three experimental diets containing 32 energy% as glucose (GLU), fructose (FRU) or an equimolar mixture of glucose and galactose (GAL). At six weeks of age, all mice received the same high-fat diet (HFD). Food intake, body weight, and body composition were measured biweekly. Whole-body metabolism was analysed towards the end of the post-weaning diet and during the last stretch of the high-fat feeding period in GLU and FRU mice. Additionally, an oral glucose tolerance test and a fasting-refeeding challenge as an indicator of metabolic flexibility were performed. At the end of the post-weaning intervention period, no significant differences were found in body weight or lean mass across all dietary groups, and in 24-hour energy expenditure and substrate oxidation between GLU and FRU. At week 15, cumulative food intake, body weight, and fat mass were significantly lower in GAL females compared to GLU females. Glucose tolerance in all groups, and energy expenditure, fuel utilization, and metabolic flexibility between GLU and FRU did not reveal programmed differences in metabolic phenotype. None of the parameters studied indicated long-lasting effects in males. This study provides evidence of the potential of galactose in the post-weaning diet to program a reduction in food intake resulting in lower fat mass during adulthood. Our findings highlight the relevance of carbohydrates as a dietary intervention target for future clinical studies in the field of metabolic programming. Acknowledgements : Financially supported by NWO-Applied and Technical Sciences grant 13509
Starches of low digestibility are associated with improved glucose metabolism. We hypothesise that a lowly digestible-starch diet (LDD) versus a highly digestible-starch diet (HDD) improves the capacity to oxidise starch, and that this is sex-dependent. Mice were fed a LDD or a HDD for 3 weeks directly after weaning. Body weight (BW), body composition (BC), and digestible energy intake (dEI) were determined weekly. At the end of the intervention period, whole-body energy expenditure (EE), respiratory exchange ratio (RER), hydrogen production, and the oxidation of an oral 13C-labelled starch bolus were measured by extended indirect calorimetry. Pancreatic amylase activity and total 13C hepatic enrichment were determined in females immediately before and 4 h after administration of the starch bolus. For both sexes, BW, BC, and basal EE and RER were not affected by the type of starch, but dEI and hydrogen production were increased by the LDD. Only in females, total carbohydrate oxidation and starch-derived glucose oxidation in response to the starch bolus were higher in LDD versus HDD mice; this was not accompanied by differences in amylase activity or hepatic partitioning of the 13C label. These results show that starch digestibility impacts glucose metabolism differently in females versus males.
Objective: To assess the lipid quality focusing on trans fatty acids (TFA) content of standardized milk formulas marketed in Mexico for infants aged from 0 to 36 months. Material and Methods: A total of 27 infant formulas from eight different leading brands were analyzed. Nine of them belonged to stage 1 (age 12 months). Acquired products were treated by duplicate for extraction of total lipid content with the modified Folch method before their expiration date. Fatty acids were esterified in an alkaline medium followed by an acid-catalyzed esterification. Analysis was performed on a gas chromatograph (5890 Series II; Hewlett-Packard, USA) with a flame ionization detector. Results: Thirty-four fatty acids (C8 to C22) were identified. Most products complied with ESPHAGAN compositional requirements. Only one product exceeded the suggested limit (>3%) for TFA. Long chain polyunsaturated fatty acids (LC-PUFAs) content was consistently meager (≈78%), with low amounts of arachidonic (Conclusion: Most milk formulas complied with ESPHAGAN global recommendations. The content of TFA and LC-PUFAs was scarce in the majority of samples.
Abstract In nature Siberian hamsters utilize the decrement in day length following the summer solstice to implement physiological adaptations in anticipation of the forthcoming winter, but also exploit an intrinsic interval timer to initiate physiological recrudescence following the winter solstice. However, information is lacking on the temporal dynamics in natural photoperiod of photoperiodically regulated genes and their relationship to physiological adaptations. To address this, male Siberian hamsters born and maintained outdoors were sampled every month over the course of one year. As key elements of the response to photoperiod, thyroid hormone signalling components were assessed in the hypothalamus. From maximum around the summer solstice (late-June), Dio2 expression rapidly declined in advance of physiological adaptations. This was followed by a rapid increase in Mct8 expression (T3/T4 transport), peaking early-September before gradually declining to minimum expression by the following June. Di o3 showed a transient peak of expression beginning late-August. A recrudescence of testes and body mass occurred from mid-February, but Dio2 expression remained low until late-April of the following year, converging with the time of year when responsiveness to short-day length is re-established. Other photoperiodically regulated genes show temporal regulation, but of note is a transient peak in Gpr 50 around late-July.
The maternal polyinosinic:polycytidylic acid (poly(I:C)) animal model is frequently used to study how maternal immune activation may impact neuro development in the offspring. Here, we present the first systematic review and meta-analysis on the effects of maternal poly(I:C) injection on immune mediators in the offspring and provide an openly accessible systematic map of the data including methodological characteristics. Pubmed and EMBASE were searched for relevant publications, yielding 45 unique papers that met inclusion criteria. We extracted data on immune outcomes and methodological characteristics, and assessed the risk of bias. The descriptive summary showed that most studies reported an absence of effect, with an equal number of studies reporting an increase or decrease in the immune mediator being studied. Meta-analysis showed increased IL-6 concentrations in the offspring of poly(I:C) exposed mothers. This effect appeared larger prenatally than post-weaning. Furthermore, poly(I:C) administration during mid-gestation was associated with higher IL-6 concentrations in the offspring. Maternal poly(I:C) induced changes in IL-1β, Il-10 and TNF-α concentrations were small and could not be associated with age of offspring, gestational period or sampling location. Finally, quality of reporting of potential measures to minimize bias was low, which stresses the importance of adherence to publication guidelines. Since neurodevelopmental disorders in humans tend to be associated with lifelong changes in cytokine concentrations, the absence of these effects as identified in this systematic review may suggest that combining the model with other etiological factors in future studies may provide further insight in the mechanisms through which maternal immune activation affects neurodevelopment.
Metabolic programming can occur not only in the perinatal period, but also post-weaning. This study aims to assess whether fructose, in comparison to glucose, in the post-weaning diet programs body weight, adiposity, glucose tolerance, metabolic flexibility, and health at adult age.Three-week-old male and female C57BL6/JRccHsd mice are given an intervention diet with 32 energy percent (en%) glucose or fructose for only 3 weeks. Next, all animals are switched to the same 40 en% high fat diet for 9 weeks. Neither body weight nor adiposity differs significantly between the animals fed with glucose or fructose diets at any point during the study in both sexes. Glucose tolerance in adulthood is not affected by the post-weaning diet, nor are activity, energy expenditure, and metabolic flexibility, as measured by indirect calorimetry. At the end of the study, only in females fasting serum insulin levels and HOMA-IR index are lower in post-weaning fructose versus glucose diet (p = 0.02), without differences in pancreatic β-cell mass.Our present findings indicate no adverse programming of body weight, adiposity, glucose tolerance, and metabolic flexibility by dietary (solid) fructose in comparison to glucose in the post-weaning diet in mice.
Indirect calorimetry (InCa) is an essential tool for human and animal studies of energy metabolism. InCa measures the metabolic gases consumed (oxygen, O2) and produced (carbon dioxide, CO2) by the organism to calculate energy expenditure (EE). The ratio of CO2 produced to O2 consumed, or the respiratory exchange ratio (RER), indicates substrate utilisation (carbohydrate, fat, and protein) at the whole body level. Significant improvements in commercial InCa systems for rodents have increased its accuracy and resolution, yet there have been few attempts to extend the technique by measuring other physiological gases. These gases can be of microbial origin, like hydrogen (H2) and methane (CH4), which result from fermentation by the gut microbiome. Furthermore, conventional InCa cannot distinguish between the oxidation of exogenous (e.g. dietary) and endogenous (stored in the body) substrates. Stable isotopic tracers, like 13C-enriched nutrients, make this distinction possible by analysis of the ratio of 13CO2/12CO2 in expired air. These gases (H2, CH4, 13CO2, 12CO2) are rarely measured in mouse studies and, when they are, they need expensive equipment and considerable time and effort. Dietary starches can be lowly or highly digestible. Lowly digestible starches generally produce lower glycaemic responses, provide fermentative substrates for the intestinal microbiota, and are thought to prevent excessive adiposity and favour metabolic health. There are some indications that starch digestibility has different metabolic effects in females and males. The evidence for this is very limited and, where both sexes have been studied, little attention has been paid to other aspects of carbohydrate metabolism, besides oral glucose tolerance tests and static tests for glycaemia. Moreover, despite the potential of starches to impact metabolism, there are no evidence-based recommendations for starch intake for young children. This is important because early life nutrition has the power to condition the metabolic function of the organism in the long term, a phenomenon known as metabolic programming. It is not known whether starch digestibility can program the organism metabolically, neither whether programming by starches can occur in the post-weaning period, a period where the organism faces a dramatic change in dietary macronutrient composition and density. This thesis aimed to 1) develop an show the added value of an extended mouse metabolic phenotyping tool based on InCa for the real-time study of microbiota activity and the oxidation of exogenous vs endogenous substrates; and 2) to apply this tool to study the direct and metabolic programming effects of starches consumed during the early post-weaning period. In Chapter 2, we aimed to examine whether we would be able to study microbiota activity non-invasively, continuously and in real time by extending a commercial InCa system (eInCa) with H2 and CH4 sensors (eInCa). Hydrogen production was circadian and depended on food intake and starch digestibility, as tested in mice fed a lowly (LDD) or a highly digestible-starch diet (HDD). Hydrogen production explained ~20% of the variation in faecal bacterial composition, and correlated with specific bacterial genera known to produce H2 in vitro. No CH4 production could be measured, and this was consistent with the absence of faecal methanogenic archaea. We concluded that eInCa is a useful tool to study diet-microbiota-host interactions in real time. We then incorporated 13CO2 and 12CO2 sensors into the same eInCa system and aimed to demonstrate their added value in Chapter 3. The system detected to differences in 13CO2 enrichment based on the natural 13C enrichment of the diet and daily food intake patterns. By combining 13CO2 enrichment and conventional InCa data, we were able to quantify the oxidation rates of 13C glucose or 13C palmitate ingested by lean and obese mice, separately from total (exogenous + endogenous) glucose and fat oxidation rates. The oxidation of ingested palmitate was negatively correlated to the animal’s fat mas and positively correlated to metabolic flexibility. We concluded that enabling 13CO2 enrichment analysis in eInCa makes it a powerful tool for the quantification of specific substrate oxidation in physiological and pathophysiological conditions . In Chapter 4, we aimed to test whether the capacity to oxidise the starch molecule is influenced by prior exposure to an LDD or an HDD, and if this effect is similar for both females and males. The oxidation of a highly digestible starch bolus was higher in mice that consumed LDD for 3 weeks prior. This effect was larger in females than in males, as shown by the faster starch oxidation kinetics of LDD vs HDD females during the early postprandial period, not present in males. However, LDD males exhibited a constantly higher RER, both in fasting conditions and during the postprandial period after consumption of the starch bolus. Small intestinal amylase levels did not explain the higher starch oxidation of LDD vs HDD females. From these data we concluded that short-term consumption of lowly digestible starch enhances the oxidation of the starch molecule, especially in females. In Chapter 5, we aimed to retest some of the direct effects of starch digestibility and to test whether LDD vs HDD consumed in early post-weaning can programme metabolism in the long-term in both sexes. Direct exposure to LDD led to smaller adipocyte sizes and lower inflammation in the gonadal adipose compartment of females, and decreased body fat mass in males. In both sexes, digestible energy intake and H2 production were directly increased by LDD. Adult females on a HFD that were exposed to LDD during post-weaning had a better metabolic flexibility and lower macrophage infiltration in the gonadal adipose depot. In males, no programming effects on metabolic flexibility and other metabolic outcomes like body composition and fasting glucose, insulin, and adipokine levels were observed. We concluded that the metabolic programming effects of starches consumed in the post-weaning period are subtle and sexually dimorphic. I discuss the findings of my thesis and provide implications and suggestions for further research in Chapter 6. To conclude, we have shown the added value of integrating new gas sensors into a commercial InCa system (Chapters 2 and 3). This technological extension yields real-time, continuous, and automated data that can inform microbiology and nutritional studies and beyond. By providing additional information with minimal discomfort to the animal, eInCa can contribute to the replacement, reduction, and refinement (3 Rs) of animal experimentation.
Duration of breastfeeding is positively associated with decreased adiposity and increased metabolic health in later life, which might be related to galactose. The aim of this study was to investigate if partial replacement of glucose with galactose in the postweaning diet had a metabolic programming effect. Male and female mice (C57BL/6JRccHsd) received an isocaloric diet (16 energy% fat; 64 energy% carbohydrates; 20 energy% protein) with either glucose (32 energy%) (GLU) or glucose + galactose (GLU + GAL, 16 energy% each) for 3 wk postweaning. Afterwards, all mice were switched to the same 40 energy% high-fat diet (HFD) for 9 wk to evaluate potential programming effects in an obesogenic environment. Data were analyzed within sex. Female body weight (−14%) and fat mass (−47%) were significantly lower at the end of the HFD period (both P < 0.001) among those fed GLU + GAL than among those fed GLU; effects in males were in line with these findings but nonsignificant. Food intake was affected in GLU + GAL–fed females (+8% on postweaning diet, −9% on HFD) compared with GLU-fed females, but not for hypothalamic transcript levels at endpoint. Also, in GLU + GAL–fed females, serum insulin concentrations (−48%, P < 0.05) and the associated homeostasis model assessment of insulin resistance (HOMA-IR) were significantly lower ( P < 0.05) at endpoint, but there were no changes in pancreas morphology. In GLU + GAL–fed females, expression of insulin receptor substrate 2 (Irs2) (−27%, P < 0.01 ; −44%, P < 0.001) and the adipocyte size markers leptin (Lep) (−40%, P < 0.05; −63% , P < 0.05) and mesoderm-specific transcript homolog protein (Mest) (−80%, P < 0.05; −72%, P < 0.05) was lower in gonadal and subcutaneous white adipose tissue (WAT), respectively. Expression of insulin receptor substrate1 (Irs1) (−24%, P < 0.05) was only lower in subcutaneous WAT in GLU + GAL–fed females. Partial replacement of glucose with galactose, resulting in a 1:1 ratio mimicking lactose, in a 3-wk postweaning diet lowered body weight, adiposity, HOMA-IR, and expression of WAT insulin signaling in HFD-challenged female mice in later life. This suggests that prolonged galactose intake may improve metabolic and overall health in later life.
Abstract Indirect calorimetry (InCa) estimates whole-body energy expenditure and total substrate oxidation based on O 2 consumption and CO 2 production, but does not allow for the quantification of oxidation of exogenous substrates with time. To achieve this, we incorporated 13 CO 2 and 12 CO 2 gas sensors into a commercial InCa system and aimed to demonstrate their performance and added value. As a performance indicator, we showed the discriminative oscillations in 13 CO 2 enrichment associated with food intake in mice fed diets containing naturally low (wheat) vs high (maize) 13 C enrichment. To demonstrate the physiological value, we quantified exogenous vs total carbohydrate and fat oxidation continuously, in real time in mice varying in fat mass. Diet-induced obese mice were fed a single liquid mixed meal containing 13 C-isotopic tracers of glucose or palmitate. Over 13 h, ~70% glucose and ~48% palmitate ingested were oxidised. Exogenous palmitate oxidation depended on body fat mass, which was not the case for exogenous glucose oxidation. We conclude that extending an InCa system with 13 CO 2 and 12 CO 2 sensors provides an accessible and powerful technique for real-time continuous quantification of exogenous and whole-body substrate oxidation in mouse models of human metabolic physiology.
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