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The gut microbiome interacts continuously with the host and its diet. Studying these interactions and their evolution in vivo as soon as they happen have been impossible. Here we develop a method to study microbiota-host-diet interactions continuously, non-invasively, and in real time, by measuring hydrogen (H2) and methane (CH4) production in mice housed in indirect calorimetry chambers., The gut microbiome interacts continuously with the host and its diet. Studying these interactions and their evolution in vivo as soon as they happen have been impossible. Here we develop a method to study microbiota-host-diet interactions continuously, non-invasively, and in real time, by measuring hydrogen (H2) and methane (CH4) production in mice housed in indirect calorimetry chambers.

Background: Body weight (BW) cycling, the yo-yo effect, is generally thought to have adverse effects on human metabolic health. However, human and animal experiments are limited in number and do not provide clear answers, partly due to large variations in experimental design, parameters measured, and definitions of BW cycling. Here, we examined the effect of repetitive BW cycling versus single- and non-cycling control groups, without alterations in diet composition, on steady state BW and metabolic parameters. Methods: We induced well-defined BW cycles on a semi-purified high fat diet in C57BL/6J mice, a well-described animal model for diet-induced obesity, and measured energy expenditure and relevant metabolic parameters. Results: Our setup indeed resulted in the intended BW changes and always reached a stage of energy balance. A history of weight cycling did not result in increased BW or fat mass compared with the control group, nor in deteriorated serum concentrations of glucose, adipokines and serum triglyceride and free fatty acid (FFA) concentrations. If anything, BW tended to be reduced, presumably because of a reduced overall energy intake in BW cycling animals. Conclusion: Repeated cycling in BW without changes in diet composition does not lead to impaired metabolic health nor increased BW (gain).

Indirect calorimetry (InCa) can potentially be used to noninvasively assess metabolic and age-related flexibility. To assess the use of InCa for this purpose, we tested the sensitivity and response stability over time of three InCa-based treatments in old versus adult mice. Diurnal patterns of respiratory exchange ratio were followed for 24 hours under standard conditions (Treatment 1), but the results were not stable between test periods. As a challenge, fasted mice received glucose to test switch-effectiveness from fat to glucose oxidation (Treatment 2). No differences between groups were observed, although old mice showed higher adiposity and lower white adipose tissue (WAT) mitochondrial density, indicative of age-impaired metabolic health. Lastly, adaptation to a challenge of oxygen restriction (OxR, 14.5% O2) was assessed as a novel approach (Treatment 3). This treatment stably detected significant differences: old mice did not maintain reduced oxygen consumption under OxR during both test periods, whereas adult mice did. Further biochemical and gene expression analyses showed that OxR affected glucose and lactate homeostasis in liver and WAT of adult mice, supporting the observed differences in oxygen consumption. In conclusion, InCa analysis of the response to OxR in mice is a sensitive and reproducible treatment to noninvasively measure age-impaired metabolic health.

Poly-unsaturated fatty acids (PUFAs) are considered to be healthier than saturated fatty acids (SFAs), but others postulate that especially the ratio of omega-6 to omega-3 PUFAs (n6/n3 ratio) determines health. Health can be determined with biomarkers, but functional health status is likely better reflected by challenge tests that assess metabolic flexibility. The aim of this study was to determine the effect of high-fat diets with different fatty acid compositions, but similar n6/n3 ratio, on metabolic flexibility. Therefore, adult male mice received isocaloric high-fat diets with either predominantly PUFAs (HFpu diet) or predominantly SFAs (HFs diet) but similar n6/n3 ratio for six months, during and after which several biomarkers for health were measured. Metabolic flexibility was assessed by the response to an oral glucose tolerance test, a fasting and re-feeding test and an oxygen restriction test (OxR; normobaric hypoxia). The latter two are non-invasive, indirect calorimetry-based tests that measure the adaptive capacity of the body as a whole. We found that the HFs diet, compared to the HFpu diet, increased mean adipocyte size, liver damage, and ectopic lipid storage in liver and muscle; although, we did not find differences in body weight, total adiposity, adipose tissue health, serum adipokines, whole body energy balance, or circadian rhythm between HFs and HFpu mice. HFs mice were, furthermore, less flexible in their response to both fasting- re-feeding and OxR, while glucose tolerance was indistinguishable. To conclude, the HFs versus the HFpu diet increased ectopic fat storage, liver damage, and mean adipocyte size and reduced metabolic flexibility in male mice. This study underscores the physiological relevance of indirect calorimetry-based challenge tests.

Dietary flavonoid intake is associated with reduced risk of cardiovascular diseases, possibly by affecting metabolic health. The relative potency of different flavonoids in causing beneficial effects on energy and lipid metabolism has not been investigated. Effects of quercetin, hesperetin, epicatechin, apigenin and anthocyanins in mice fed a high-fat diet (HF) for 12 weeks were compared, relative to normal-fat diet. HF-induced body weight gain was significantly lowered by all flavonoids (17–29 %), but most by quercetin. Quercetin significantly lowered HF-induced hepatic lipid accumulation (71 %). Mesenteric adipose tissue weight and serum leptin levels were significantly lowered by quercetin, hesperetin and anthocyanins. Adipocyte cell size and adipose tissue inflammation were not affected. The effect on body weight and composition could not be explained by individual significant effects on energy intake, energy expenditure or activity. Lipid metabolism was not changed as measured by indirect calorimetry or expression of known lipid metabolic genes in liver and white adipose tissue. Hepatic expression of Cyp2b9 was strongly downregulated by all flavonoids. In conclusion, all flavonoids lowered parameters of HF-induced adiposity, with quercetin being most effective.

Dietary triglycerides are hydrolyzed in the small intestine principally by pancreatic lipase. Following uptake by enterocytes and secretion as chylomicrons, dietary lipids are cleared from the bloodstream via lipoprotein lipase. Whereas lipoprotein lipase is inhibited by several proteins including Angiopoietin-like 4 (Angptl4), no endogenous regulator of pancreatic lipase has yet been identified. Here we present evidence that Angptl4 is an endogenous inhibitor of dietary lipid digestion. Angptl4-/- mice were heavier compared to their wild-type counterparts without any difference in food intake, energy expenditure or locomotor activity. However, Angptl4-/- mice showed decreased lipid content in the stools and increased accumulation of dietary triglycerides in the small intestine, which coincided with elevated luminal lipase activity in Angptl4-/- mice. Furthermore, recombinant Angptl4 reduced the activity of pancreatic lipase as well as the lipase activity in human ileostomy output. In conclusion, our data suggest that Angptl4 is an endogenous inhibitor of intestinal lipase activity.

Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,150-monooxygenase 1 knockout (Bcmo1-/-) mice, which are—like humans—able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1-/- mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1-/- mice. Testosterone levels were higher after BC supplementation only in Bcmo1-/- mice, which had, unlike wild-type (Bcmo1?/?) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice.