Matrisome, innervation and oxidative metabolism affected in older compared with younger males with similar physical activity.

BACKGROUND: Due to the interaction between skeletal muscle ageing and lifestyle factors, it is often challenging to attribute the decline in muscle mass and quality to either changes in lifestyle or to advancing age itself. Because many of the physiological factors affecting muscle mass and quality are modulated by physical activity and physical activity declines with age, the aim of this study is to better understand the effects of early ageing on muscle function by comparing a population of healthy older and young males with similar physical activity patterns. METHODS: Eighteen older (69 ± 2.0 years) and 20 young (22 ± 2.0 years) males were recruited based on similar self-reported physical activity, which was verified using accelerometry measurements. Gene expression profiles of vastus lateralis biopsies obtained by RNA sequencing were compared, and key results were validated using quantitative polymerase chain reaction and western blot. RESULTS: Total physical activity energy expenditure was similar between the young and old group (404 ± 215 vs. 411 ± 189 kcal/day, P = 0.11). Three thousand seven hundred ninety-seven differentially expressed coding genes (DEGs) were identified (adjusted P-value cut-off of <0.05), of which 1891 were higher and 1906 were lower expressed in the older muscle. The matrisome, innervation and inflammation were the main upregulated processes, and oxidative metabolism was the main downregulated process in old compared with young muscle. Lower protein levels of mitochondrial transcription factor A (TFAM, P = 0.030) and mitochondrial respiratory Complexes IV and II (P = 0.011 and P = 0.0009, respectively) were observed, whereas a trend was observed for Complex I (P = 0.062), in older compared with young muscle. Protein expression of Complexes I and IV was significantly correlated to mitochondrial capacity in the vastus lateralis as measured in vivo (P = 0.017, R2  = 0.42 and P = 0.030, R2  = 0.36). A trend for higher muscle-specific receptor kinase (MUSK) protein levels in the older group was observed (P = 0.08). CONCLUSIONS: There are clear differences in the transcriptome signatures of the vastus lateralis muscle of healthy older and young males with similar physical activity levels, including significant differences at the protein level. By disentangling physical activity and ageing, we appoint early skeletal muscle ageing processes that occur despite similar physical activity. Improved understanding of these processes will be key to design targeted anti-ageing therapies.

Pseudo-Starvation Driven Energy Expenditure Negatively Affects Ovarian Follicle Development.

In the present investigation, we examined whether a change in whole body energy fluxes could affect ovarian follicular development, employing mice ectopically expressing uncoupling protein 1 in skeletal muscle (UCP1-TG). Female UCP1-TG and wild-type (WT) mice were dissected at the age of 12 weeks. Energy intake and expenditure, activity, body weight and length, and body composition were measured. Plasma insulin, glucose, leptin, plasma fibroblast growth factor 21 (FGF21) and plasma insulin-like growth factor 1 (IGF1) levels were analyzed and ovarian follicle and corpus luteum numbers were counted. IGF1 signaling was analyzed by immunohistochemical staining for the activation of insulin receptor substrate 1/2 (IRS1/2) and AKT. UCP1-TG female mice had increased energy expenditure, reduced body size, maintained adiposity, and decreased IGF1 concentrations compared to their WT littermates, while preantral and antral follicle numbers were reduced by 40% and 60%, respectively. Corpora lutea were absent in 40% of the ovaries of UCP1-TG mice. Phospho-IRS1, phospho-AKT -Ser473 and -Thr308 immunostaining was present in the granulosa cells of antral follicles in WT ovaries, but faint to absent in the antral follicles of UCP1-TG mice. In conclusion, the reduction in circulating IGF1 levels due to the ectopic expression of UCP1 is associated with reduced immunostaining of the IRS1-PI3/AKT pathway, which may negatively affect ovarian follicle development and ovulation.

Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells.

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.

Omega-3 Phospholipids from Krill Oil Enhance Intestinal Fatty Acid Oxidation More Effectively than Omega-3 Triacylglycerols in High-Fat Diet-Fed Obese Mice.

Antisteatotic effects of omega-3 fatty acids (Omega-3) in obese rodents seem to vary depending on the lipid form of their administration. Whether these effects could reflect changes in intestinal metabolism is unknown. Here, we compare Omega-3-containing phospholipids (krill oil; ω3PL-H) and triacylglycerols (ω3TG) in terms of their effects on morphology, gene expression and fatty acid (FA) oxidation in the small intestine. Male C57BL/6N mice were fed for 8 weeks with a high-fat diet (HFD) alone or supplemented with 30 mg/g diet of ω3TG or ω3PL-H. Omega-3 index, reflecting the bioavailability of Omega-3, reached 12.5% and 7.5% in the ω3PL-H and ω3TG groups, respectively. Compared to HFD mice, ω3PL-H but not ω3TG animals had lower body weight gain (-40%), mesenteric adipose tissue (-43%), and hepatic lipid content (-64%). The highest number and expression level of regulated intestinal genes was observed in ω3PL-H mice. The expression of FA ω-oxidation genes was enhanced in both Omega-3-supplemented groups, but gene expression within the FA ß-oxidation pathway and functional palmitate oxidation in the proximal ileum was significantly increased only in ω3PL-H mice. In conclusion, enhanced intestinal FA oxidation could contribute to the strong antisteatotic effects of Omega-3 when administered as phospholipids to dietary obese mice.

Presence of anti-Müllerian hormone (AMH) during follicular development in the porcine ovary.

BACKGROUND: Anti-Müllerian hormone (AMH) is expressed by granulosa cells of developing follicles and plays an inhibiting role in the cyclic process of follicular recruitment by determining follicle-stimulating hormone threshold levels. Knowledge of AMH expression in the porcine ovary is important to understand the reproductive efficiency in female pigs. RESEARCH AIM: In the present study we investigated the expression of AMH during follicular development in prepubertal and adult female pigs by immunohistochemistry, laser capture micro-dissection and RT-qPCR. RESULTS AND CONCLUSION: Although in many aspects the immunohistochemical localization of AMH in the porcine ovary does not differ from other species, there are also some striking differences. As in most species, AMH appears for the first time during porcine follicular development in the fusiform granulosa cells of recruited primordial follicles and continues to be present in granulosa cells up to the antral stage. By the time follicles reach the pre-ovulatory stage, AMH staining intensity increases significantly, and both protein and gene expression is not restricted to granulosa cells; theca cells now also express AMH. AMH continues to be expressed after ovulation in the luteal cells of the corpus luteum, a phenomenon unique to the porcine ovary. The physiological function of AMH in the corpus luteum is at present not clear. One can speculate that it may contribute to the regulation of the cyclic recruitment of small antral follicles. By avoiding premature exhaustion of the ovarian follicular reserve, AMH may contribute to optimization of reproductive performance in female pigs.

Dietary-Induced Chronic Hypothyroidism Negatively Affects Rat Follicular Development and Ovulation Rate and Is Associated with Oxidative Stress.

The long-term effects of chronic hypothyroidism on ovarian follicular development in adulthood are not well known. Using a rat model of chronic diet-induced hypothyroidism initiated in the fetal period, we investigated the effects of prolonged reduced plasma thyroid hormone concentrations on the ovarian follicular reserve and ovulation rate in prepubertal (12-day-old) and adult (64-day-old and 120-day-old) rats. Besides, antioxidant gene expression, mitochondrial density and the occurrence of oxidative stress were analyzed. Our results show that continuous hypothyroidism results in lower preantral and antral follicle numbers in adulthood, accompanied by a higher percentage of atretic follicles, when compared to euthyroid age-matched controls. Not surprisingly, ovulation rate was lower in the hypothyroid rats. At the age of 120 days, the mRNA and protein content of superoxide dismutase 1 (SOD1) were significantly increased while catalase (CAT) mRNA and protein content was significantly decreased, suggesting a disturbed antioxidant defense capacity of ovarian cells in the hypothyroid animals. This was supported by a significant reduction in the expression of peroxiredoxin 3 ( ITALIC! Prdx3), thioredoxin reductase 1 ( ITALIC! Txnrd1), and uncoupling protein 2 ( ITALIC! Ucp2) and a downward trend in glutathione peroxidase 3 ( ITALIC! Gpx3) and glutathione S-transferase mu 2 ( ITALIC! Gstm2) expression. These changes in gene expression were likely responsible for the increased immunostaining of the oxidative stress marker 4-hydroxynonenal. Together these results suggest that chronic hypothyroidism initiated in the fetal/neonatal period results in a decreased ovulation rate associated with a disturbance of the antioxidant defense system in the ovary.

Improved muscle function and quality after diet intervention with leucine-enriched whey and antioxidants in antioxidant deficient aged mice.

Antioxidant (AOX) deficiencies are commonly observed in older adults and oxidative stress has been suggested to contribute to sarcopenia. Here we investigate if 1) low levels of dietary antioxidants had a negative impact on parameters of muscle mass, function and quality, and 2) to study if nutritional interventions with AOX and/or leucine-enriched whey protein could improve these muscle parameters in aged mice. 18-months-old mice were fed a casein-based antioxidant-deficient (lowox) diet or a casein-based control-diet (CTRL) for 7 months. During the last 3 months, lowox-mice were subjected to either: a) continued lowox, b) supplementation with vitamin A/E, Selenium and Zinc (AOX), c) substitution of casein with leucine-enriched whey protein (PROT) or d) a combination of both AOX and PROT (TOTAL). After 7 months lowox-mice displayed lower muscle strength and more muscle fatigue compared to CTRL. Compared to lowox-mice, PROT-mice showed improved muscle power, grip strength and less muscle fatigue. AOX-mice showed improved oxidative status, less muscle fatigue, improved grip strength and mitochondrial dynamics compared to lowox-mice. The TOTAL-mice showed the combined effects of both interventions compared to lowox-mice. In conclusion, nutritional intervention with AOX and/or leucine-enriched whey protein can play a role in improving muscle health in a AOX-deficient mouse model.

Dynamic changes in energy metabolism upon embryonic stem cell differentiation support developmental toxicant identification.

Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST.

Role of Frizzled6 in the molecular mechanism of beta-carotene action in the lung.

ß-Carotene (BC) is omnipresent in our diet, both as natural food component as well as an additive. BC and its metabolites have important biological functions. For this reason, BC is generally considered to be a health promoting compound. Two human trials, however, have described adverse effects in lung tissue, increasing the risk of lung cancer. We previously applied transcriptomic analyses in a unique animal model, beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1(-/-)) mice that are, like humans, able to accumulate intact BC. In our search to unravel the molecular action of BC in the lung, we previously identified two genes particularly strongly down-regulated by BC in lung tissue of the male Bcmo1(-/-) mice: frizzled homologue 6 (Fzd6) and collagen triple helix repeat containing 1 (Cthrc1). In the present study, our aim was to further elucidate the role of FZD6 in lung epithelial cells and to provide a mechanistic explanation for BC increased lung cancer risk in humans. We performed whole genome microarray analysis on silenced FZD6 in non-tumor human type II bronchial epithelial BEAS-2B cells using RNAi. To directly link FZD6 to BC-effects on the lung, we compared the FZD6-silenced BEAS-2B gene expression profile to the BC-dependent gene expression profile of Bcmo1(-/-) mouse lungs. A number of relevant genes were regulated in the same direction in FZD6(-) BEAS-2B and in BC-exposed lungs of Bcmo1(-/-) mice and revealed enrichment of the Gene Ontology terms "oncogenes", "cell proliferation" and "cell cycle", which suggests a mediating role of FZD6 in BC-induced uncontrolled proliferation of lung cells.

Thermoneutrality results in prominent diet-induced body weight differences in C57BL/6J mice, not paralleled by diet-induced metabolic differences.

SCOPE: Mice are usually housed at 20-24 °C. At thermoneutrality (28 °C) larger diet-induced differences in obesity are seen. We tested whether this leads to large differences in metabolic health parameters. METHODS AND RESULTS: We performed a 14-wk dietary intervention in C57BL/6J mice at 28 °C and assessed adiposity and metabolic health parameters for a semipurified low fat (10 energy%) diet and a moderate high fat (30 energy%) diet. A large and significant diet-induced differential increase in body weight, adipose tissue mass, adipocyte size, serum leptin level, and, to some extent, cholesterol level was observed. No adipose tissue inflammation was seen. No differential effect of the diets on serum glucose, free fatty acids, triacylglycerides, insulin, adiponectin, resistin, PAI-1, MMP-9, sVCAM-1, sICAM-1, sE-selectin, IL-6, ApoE, fibrinogen levels, or HOMA index was observed. Also in muscle no differential effect on mitochondrial density, mitochondrial respiratory control ratio, or mRNA expression of metabolic genes was found. Finally, in liver no differential effect on weight, triacylglycerides level, aconitase/citrate synthase activity ratio was seen. CONCLUSION: Low fat diet and moderate high fat diet induce prominent body weight differences at thermoneutrality, which is not paralleled by metabolic differences. Our data rather suggest that thermoneutrality alters metabolic homeostasis.

Effects of chocolate supplementation on metabolic and cardiovascular parameters in ApoE3L mice fed a high-cholesterol atherogenic diet.

SCOPE: Dietary intake of cocoa and/or chocolate has been suggested to exhibit protective cardiovascular effects although this is still controversial. The aim of this study was to investigate the effects of chocolate supplementation on metabolic and cardiovascular parameters. METHODS AND RESULTS: Four groups of ApoE*3Leiden mice were exposed to the following diet regimens. Group 1: cholesterol-free control diet (CO). Group 2: high-dose (1.0% w/w) control cholesterol (CC). Group 3: CC supplemented chocolate A (CCA) and Group 4: CC supplemented chocolate B (CCB). Both chocolates differed in polyphenol and fiber content, CCA had a relatively high-polyphenol and low-fiber content compared to CCB. Mice fed a high-cholesterol diet showed increased plasma-cholesterol and developed atherosclerosis. Both chocolate treatments, particularly CCA, further increased plasma-cholesterol and increased atherosclerotic plaque formation. Moreover, compared to mice fed a high-cholesterol diet, both chocolate-treated groups displayed increased liver injury. Mice on high-cholesterol diet had elevated plasma levels of sVCAM-1, sE-selectin and SAA, which was further increased in the CCB group. Similar effects were observed for renal inflammation markers. CONCLUSION: The two chocolate preparations showed unfavorable, but different effects on cardiometabolic health in E3L mice, which dissimilarities may be related to differences in chocolate composition. We conclude that discrepancies reported on the effects of chocolate on cardiometabolic health may at least partly be due to differences in chocolate composition.

Quercetin induces hepatic lipid omega-oxidation and lowers serum lipid levels in mice.

Elevated circulating lipid levels are known risk factors for cardiovascular diseases (CVD). In order to examine the effects of quercetin on lipid metabolism, mice received a mild-high-fat diet without (control) or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and (1)H nuclear magnetic resonance were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify possible mechanisms underlying altered circulating lipid levels. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, triglycerides (TG) were decreased with 14% (p<0.001) and total poly unsaturated fatty acids (PUFA) were increased with 13% (p<0.01). Palmitic acid, oleic acid, and linoleic acid were all decreased by 9-15% (p<0.05) in quercetin-fed mice. Both palmitic acid and oleic acid can be oxidized by omega (ω)-oxidation. Gene expression profiling showed that quercetin increased hepatic lipid metabolism, especially ω-oxidation. At the gene level, this was reflected by the up-regulation of cytochrome P450 (Cyp) 4a10, Cyp4a14, Cyp4a31 and Acyl-CoA thioesterase 3 (Acot3). Two relevant regulators, cytochrome P450 oxidoreductase (Por, rate limiting for cytochrome P450s) and the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3) were also up-regulated in the quercetin-fed mice. We conclude that quercetin intake increased hepatic lipid ω-oxidation and lowered corresponding circulating lipid levels, which may contribute to potential beneficial effects on CVD.

Preservation of metabolic flexibility in skeletal muscle by a combined use of n-3 PUFA and rosiglitazone in dietary obese mice.

Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients.

Dietary restriction of mice on a high-fat diet induces substrate efficiency and improves metabolic health.

High energy intake and, specifically, high dietary fat intake challenge the mammalian metabolism and correlate with many metabolic disorders such as obesity and diabetes. However, dietary restriction (DR) is known to prevent the development of metabolic disorders. The current western diets are highly enriched in fat, and it is as yet unclear whether DR on a certain high-fat (HF) diet elicits similar beneficial effects on health. In this research, we report that HF-DR improves metabolic health of mice compared with mice receiving the same diet on an ad libitum basis (HF-AL). Already after five weeks of restriction, the serum levels of cholesterol and leptin were significantly decreased in HF-DR mice, whereas their glucose sensitivity and serum adiponectin levels were increased. The body weight and measured serum parameters remained stable in the following 7 weeks of restriction, implying metabolic adaptation. To understand the molecular events associated with this adaptation, we analyzed gene expression in white adipose tissue (WAT) with whole genome microarrays. HF-DR strongly influenced gene expression in WAT; in total, 8643 genes were differentially expressed between both groups of mice, with a major role for genes involved in lipid metabolism and mitochondrial functioning. This was confirmed by quantitative real-time reverse transcription-PCR and substantiated by increase in mitochondrial density in WAT of HF-DR mice. These results provide new insights in the metabolic flexibility of dietary restricted animals and suggest the development of substrate efficiency.

Glycemic index differences of high-fat diets modulate primarily lipid metabolism in murine adipose tissue.

A low vs. high glycemic index of a high-fat (HF) diet (LGI and HGI, respectively) significantly retarded adverse health effects in adult male C57BL/6J mice, as shown recently (Van Schothorst EM, Bunschoten A, Schrauwen P, Mensink RP, Keijer J. FASEB J 23: 1092-1101, 2009). The LGI diet enhanced whole body insulin sensitivity and repressed HF diet-induced body and white adipose tissue (WAT) weight gain, resulting in significantly reduced serum leptin and resistin levels and increased adiponectin levels. We questioned how WAT is modulated and characterized the molecular mechanisms underlying the glycemic index-mediated effects using whole genome microarrays. This showed that the LGI diet mainly exerts its beneficial effects via substrate metabolism, especially fatty acid metabolism. In addition, cell adhesion and cytoskeleton remodeling showed reduced expression, in line with lower WAT mass. An important transcription factor showing enhanced expression is PPAR-γ. Furthermore, serum levels of triglycerides, total cholesterol, and HDL- and LDL-cholesterol were all significantly reduced by LGI diet, and simultaneously muscle insulin sensitivity was significantly increased as analyzed by protein kinase B/Akt phosphorylation. Cumulatively, even though these mice were fed an HF diet, the LGI diet induced significantly favorable changes in metabolism in WAT. These effects suggest a partial overlap with pharmacological approaches by thiazolidinediones to treat insulin resistance and statins for hypercholesterolemia. It is therefore tempting to speculate that such a dietary approach might beneficially support pharmacological treatment of insulin resistance or hypercholesterolemia in humans.

Dietary restriction of mice on a high-fat diet induces substrate efficiency and improves metabolic health.

High energy intake and, specifically, high dietary fat intake challenge the mammalian metabolism and correlate with many metabolic disorders such as obesity and diabetes. However, dietary restriction (DR) is known to prevent the development of metabolic disorders. The current western diets are highly enriched in fat, and it is as yet unclear whether DR on a certain high-fat (HF) diet elicits similar beneficial effects on health. In this research, we report that HF-DR improves metabolic health of mice compared with mice receiving the same diet on an ad libitum basis (HF-AL). Already after five weeks of restriction, the serum levels of cholesterol and leptin were significantly decreased in HF-DR mice, whereas their glucose sensitivity and serum adiponectin levels were increased. The body weight and measured serum parameters remained stable in the following 7 weeks of restriction, implying metabolic adaptation. To understand the molecular events associated with this adaptation, we analyzed gene expression in white adipose tissue (WAT) with whole genome microarrays. HF-DR strongly influenced gene expression in WAT; in total, 8643 genes were differentially expressed between both groups of mice, with a major role for genes involved in lipid metabolism and mitochondrial functioning. This was confirmed by quantitative real-time reverse transcription-PCR and substantiated by increase in mitochondrial density in WAT of HF-DR mice. These results provide new insights in the metabolic flexibility of dietary restricted animals and suggest the development of substrate efficiency.

Downregulation of Fzd6 and Cthrc1 and upregulation of olfactory receptors and protocadherins by dietary beta-carotene in lungs of Bcmo1-/- mice.

An ongoing controversy exists on beneficial versus harmful effects of high beta-carotene (BC) intake, especially for the lung. To elucidate potential mechanisms, we studied effects of BC on lung gene expression. We used a beta-carotene 15,15'-monooxygenase 1 (Bcmo1) knockout mouse (Bcmo1(-/-)) model, unable to convert BC to retinoids, and wild-type mice (Bcmo1(+/+)) mice to dissect the effects of intact BC from effects of BC metabolites. As expected, BC supplementation resulted in a higher BC accumulation in lungs of Bcmo1(-/-) mice than in lungs of Bcmo1(+/+) mice. Whole mouse genome transcriptome analysis on lung tissue revealed that more genes were regulated in Bcmo1(-/-) mice than Bcmo1(+/+) mice upon BC supplementation. Frizzled homolog 6 (Fzd6) and collagen triple helix repeat containing 1 (Cthrc1) were significantly downregulated (fold changes -2.99 and -2.60, respectively, false discovery rate < 0.05) by BC in Bcmo1(-/-). Moreover, many olfactory receptors and many members of the protocadherin family were upregulated. Since both olfactory receptors and protocadherins have an important function in sensory nerves and Fzd6 and Cthrc1 are important in stem cell development, we hypothesize that BC might have an effect on the highly innervated pulmonary neuroendocrine cell (PNEC) cluster. PNECs are highly associated with sensory nerves and are important cells in the control of stem cells. A role for BC in the innervated PNEC cluster might be of particular importance in smoke-induced carcinogenesis since PNEC-derived lung cancer is highly associated with tobacco smoke.

Transcriptome analysis in benefit-risk assessment of micronutrients and bioactive food components.

The establishment of functional effects due to variation in concentrations of micronutrients in our diet is difficult since they are often not immediately recognized as being healthy or unhealthy. Indeed, effects induced by micronutrients are hard to identify and therefore the establishment of the recommended daily intake, the optimal intake and the upper limit pose a challenge. For bioactive food components this is even more complicated. Whole-genome transcriptome analysis is highly suitable to obtain unbiased information on potential affected biological processes on a whole-genome level. Here, we will describe and discuss several aspects of transcriptome analysis in benefit-risk assessment, including effect size, sensitivity and statistical power, that have to be taken into account to faithfully identify functional effects of micronutrients and bioactive food components.

Induction of lipid oxidation by polyunsaturated fatty acids of marine origin in small intestine of mice fed a high-fat diet.

BACKGROUND: Dietary polyunsaturated fatty acids (PUFA), in particular the long chain marine fatty acids docosahexaenoic (DHA) and eicosapentaenoic (EPA), are linked to many health benefits in humans and in animal models. Little is known of the molecular response to DHA and EPA of the small intestine, and the potential contribution of this organ to the beneficial effects of these fatty acids. Here, we assessed gene expression changes induced by DHA and EPA in the wildtype C57BL/6J murine small intestine using whole genome microarrays and functionally characterized the most prominent biological process. RESULTS: The main biological process affected based on gene expression analysis was lipid metabolism. Fatty acid uptake, peroxisomal and mitochondrial beta-oxidation, and omega-oxidation of fatty acids were all increased. Quantitative real time PCR, and -- in a second animal experiment -- intestinal fatty acid oxidation measurements confirmed significant gene expression differences and showed in a dose-dependent manner significant changes at biological functional level. Furthermore, no major changes in the expression of lipid metabolism genes were observed in the colon. CONCLUSION: We show that marine n-3 fatty acids regulate small intestinal gene expression and increase fatty acid oxidation. Since this organ contributes significantly to whole organism energy use, this effect on the small intestine may well contribute to the beneficial physiological effects of marine PUFAs under conditions that will normally lead to development of obesity, insulin resistance and diabetes.

Effects of a high-fat, low- versus high-glycemic index diet: retardation of insulin resistance involves adipose tissue modulation.

Beneficial effects of low glycemic index (GI) diets in rodents have been studied using healthy low-fat diets, while the effects might be different on high-fat diets inducing progression of insulin resistance. We fed C57BL/6J male mice high-fat low/high-GI (LGI/HGI) diets for 13 wk. Glucose and insulin tolerance and serum substrates, including adipokines, were measured longitudinally. The LGI group showed a significantly higher glucose tolerance from wk 2 onwards, which was supported by lower serum insulin and free fatty acids levels at 8 wk, and a tendency for lower leptin levels, while resistin levels remained similar. At 11 wk, when differences in serum resistin started to increase, differences in serum insulin were diminished. Although food intake was similar throughout the study, body weights and epididymal adipose tissue mass became significantly lower in the LGI group at necropsy. Several serum substrates and adipose tissue leptin mRNA levels, as analyzed by Q-PCR, were, again, significantly lower, whereas adiponectin mRNA levels were higher. Taken together, an LGI high-fat diet maintains higher glucose tolerance and insulin sensitivity via adipose tissue modulation solely because of a difference in the type of carbohydrate, supporting a nutritional approach in the fight against insulin resistance.