Temperature-flux induced metabolic adjustments in WAT

Mice have their lowest (basal) metabolic rate when housed at thermoneutrality, which starts above 29 degrees C. Although they eat less, and thus reduce their energy intake, their energy balance remains positive leading to an increased adiposity, especially when fed a high fat diet. However, almost all metabolic mouse studies are performed at standard room temperatures ranging between 20 and 22 degrees C. Previously, we showed that housing mice at thermoneutrality lead to massive increased adiposity, while metabolic dysfunction of white adipose tissue (WAT) was absent. Here, we studied whether an increased metabolic flux through WAT induces cellular stress and underlies tissue dysfunction leading to tissue inflammation. C57BL/6JOlaHsd wildtype male mice, aged 9 weeks, were fed purified low fat diet (BIOCLAIMS, Hoevenaars et al., Genes and Nutrition 2012) for 3 weeks to acclimatize, followed by 12 weeks a BIOCLAIMS high-fat diet (HFD), all at thermoneutrality (29 degrees C). Subsequently, mice were divided into different treatment groups: i) control group, which remained at thermoneutrality for 5 days, ii) 5 days normal housing temperature (22 C degrees) while housed continously in an indirect calorimetry system. At the end of the study, after 2 hours food removal at the start of the light phase, mice were killed immediately by decapitation after taking them out of the indirect calorimetry system. After sacrification, epididymal WAT was immediately dissected and snap frozen in liquid nitrogen. Total RNA was isolated, quantified and qualified, and subsequently used for global gene expression profiling using Agilent 8x60K microarrays

Partial replacement of glucose with galactose in the post-weaning diet positively affects markers of liver health

Previous research showed a beneficial programming effect of replacting glucose in the post-weaning diet with galactose on later life adiposity. Here, we studied the direct effect of the diets in the postweaning phase in female mice. In this study, female mice were fed a glucose diet (32 en% glucose; GLU) or a glucose+galactose diet (16 en% glucose and 16 en% galactose; GLU+GAL) postweaning for three weeks, from postnatal day (PN) 21 till PN42. We observed lower circulating insulin levels and lower hepatic triglyceride levels in the females on the GLU+GAL diet. The body weight, fat mass, liver weight and liver glycogen content did not differ between the groups. We next studied hepatic gene expression profiles, because of the altered hepatic triglyceride levels and since the liver is considered the primary site of galactose metabolism. However, detailed analyses including pathway analysis, showed mainly inflammation being reduced by the GLU+GAL treatment. This was confirmed by qPCR of liver tissues and focussed serum protein analysis

DFO-induced hypoxia leading to metabolic dysfunction in WAT

Metabolic dysfunction of white adipose tissue (WAT) is considered to be underlying the comorbidities in obesity, including insulin resistance and tissue inflammation. Moreover, due to the expansion of WAT, local tissue hypoxia has been reported. Whether local tissue hypoxia underlies the metabolic de-arrangements and is the first step in initiation inflammation is questioned here. Desferrioxamine (DFO) is a chemical compound trapping free iron, thus leading to a reduction in oxygen availability within the body, which mimics hypoxia. Therefore, C57BL/6JOlaHsd wildtype male mice, aged 9 weeks, were fed purified low fat diet (BIOCLAIMS, Hoevenaars et al., Genes and Nutrition 2012) for 3 weeks to acclimatize, followed by 12 weeks a BIOCLAIMS high-fat diet (HFD), all at thermoneutrality (29 degrees C) to induce massive expansion of WAT without metabolic dysregulation (Hoevenaars et al., Mol Nutr Food Res 2014). Subsequently, mice were divided into different treatment groups: i) control group, ii) 5 days Desferrioxamine (DFO) daily injections (100 mg/kg body weight). Mice were killed by decapitation at the end of the experiment after 2 hour food removal at the start of the light phase. After sacrification, epididymal WAT was immediately dissected and snap frozen in liquid nitrogen. Total RNA was isolated, quantified and qualified, and subsequently used for global gene expression profiling using Agilent 8x60K microarrays

Partial replacement of glucose with galactose in the post-weaning diet positively affects markers of liver health

Previous research showed a beneficial programming effect of replacting glucose in the post-weaning diet with galactose on later life adiposity. Here, we studied the direct effect of the diets in the postweaning phase in female mice. In this study, female mice were fed a glucose diet (32 en% glucose; GLU) or a glucose+galactose diet (16 en% glucose and 16 en% galactose; GLU+GAL) postweaning for three weeks, from postnatal day (PN) 21 till PN42. We observed lower circulating insulin levels and lower hepatic triglyceride levels in the females on the GLU+GAL diet. The body weight, fat mass, liver weight and liver glycogen content did not differ between the groups. We next studied hepatic gene expression profiles, because of the altered hepatic triglyceride levels and since the liver is considered the primary site of galactose metabolism. However, detailed analyses including pathway analysis, showed mainly inflammation being reduced by the GLU+GAL treatment. This was confirmed by qPCR of liver tissues and focussed serum protein analysis

Temperature-flux induced metabolic adjustments in WAT

Mice have their lowest (basal) metabolic rate when housed at thermoneutrality, which starts above 29 degrees C. Although they eat less, and thus reduce their energy intake, their energy balance remains positive leading to an increased adiposity, especially when fed a high fat diet. However, almost all metabolic mouse studies are performed at standard room temperatures ranging between 20 and 22 degrees C. Previously, we showed that housing mice at thermoneutrality lead to massive increased adiposity, while metabolic dysfunction of white adipose tissue (WAT) was absent. Here, we studied whether an increased metabolic flux through WAT induces cellular stress and underlies tissue dysfunction leading to tissue inflammation. C57BL/6JOlaHsd wildtype male mice, aged 9 weeks, were fed purified low fat diet (BIOCLAIMS, Hoevenaars et al., Genes and Nutrition 2012) for 3 weeks to acclimatize, followed by 12 weeks a BIOCLAIMS high-fat diet (HFD), all at thermoneutrality (29 degrees C). Subsequently, mice were divided into different treatment groups: i) control group, which remained at thermoneutrality for 5 days, ii) 5 days normal housing temperature (22 C degrees) while housed continously in an indirect calorimetry system. At the end of the study, after 2 hours food removal at the start of the light phase, mice were killed immediately by decapitation after taking them out of the indirect calorimetry system. After sacrification, epididymal WAT was immediately dissected and snap frozen in liquid nitrogen. Total RNA was isolated, quantified and qualified, and subsequently used for global gene expression profiling using Agilent 8x60K microarrays

DFO-induced hypoxia leading to metabolic dysfunction in WAT

Metabolic dysfunction of white adipose tissue (WAT) is considered to be underlying the comorbidities in obesity, including insulin resistance and tissue inflammation. Moreover, due to the expansion of WAT, local tissue hypoxia has been reported. Whether local tissue hypoxia underlies the metabolic de-arrangements and is the first step in initiation inflammation is questioned here. Desferrioxamine (DFO) is a chemical compound trapping free iron, thus leading to a reduction in oxygen availability within the body, which mimics hypoxia. Therefore, C57BL/6JOlaHsd wildtype male mice, aged 9 weeks, were fed purified low fat diet (BIOCLAIMS, Hoevenaars et al., Genes and Nutrition 2012) for 3 weeks to acclimatize, followed by 12 weeks a BIOCLAIMS high-fat diet (HFD), all at thermoneutrality (29 degrees C) to induce massive expansion of WAT without metabolic dysregulation (Hoevenaars et al., Mol Nutr Food Res 2014). Subsequently, mice were divided into different treatment groups: i) control group, ii) 5 days Desferrioxamine (DFO) daily injections (100 mg/kg body weight). Mice were killed by decapitation at the end of the experiment after 2 hour food removal at the start of the light phase. After sacrification, epididymal WAT was immediately dissected and snap frozen in liquid nitrogen. Total RNA was isolated, quantified and qualified, and subsequently used for global gene expression profiling using Agilent 8x60K microarrays

Metabolic adaptation of white adipose tissue to acute, short-term environmental oxygen restriction in mice [Mus musculus]

White adipose tissue (WAT) expansion during e.g. obesity reduces oxygen availability in WAT in mice. Little is known on the adaptation of WAT to mild environmental oxygen restriction (OxR). Therefore, we studied metabolic adaptation to acute OxR in fasted, diet-induced moderately obese mice that were exposed to mild hypoxic (12% O2) or normoxic (20.9% O2) conditions for only 6 hours. Adaptation was assessed by determination of amino acids and (acyl)carnitines levels in serum and WAT, and by whole genome expression analysis in WAT. Adaptation was also assessed during the exposure using indirect calorimetry. We found that OxR reduced mitochondrial oxidation at whole-body level, as shown by a reduction in whole-body oxygen consumption and an increase in serum long-chain acylcarnitine levels. WAT did not seem to contribute to this serum profile since only short-chain acylcarnitines were increased in WAT and gene expression analysis indicated an increase in mitochondrial oxidation, based on coordinate down-regulation of Sirt4, Gpam and Chchd3/Minos3. In addition, OxR did not induce oxidative stress in obese WAT, but increased molecular pathways involved in cell growth and proliferation. OxR increased levels of tyrosine, lysine and ornithine in serum and of leucine/isoleucine in WAT. This study shows that OxR limits oxidative phosphorylation at whole-body level, but in WAT compensatory mechanisms seem to operate. The down-regulation of the mitochondria-related genes Sirt4, Gpam, and Chchd3 may be considered as a biomarker profile for WAT mitochondrial reprogramming in response to acute exposure to limited oxygen availabilit

Metabolic adaptation of white adipose tissue to acute, short-term environmental oxygen restriction in mice [Mus musculus]

White adipose tissue (WAT) expansion during e.g. obesity reduces oxygen availability in WAT in mice. Little is known on the adaptation of WAT to mild environmental oxygen restriction (OxR). Therefore, we studied metabolic adaptation to acute OxR in fasted, diet-induced moderately obese mice that were exposed to mild hypoxic (12% O2) or normoxic (20.9% O2) conditions for only 6 hours. Adaptation was assessed by determination of amino acids and (acyl)carnitines levels in serum and WAT, and by whole genome expression analysis in WAT. Adaptation was also assessed during the exposure using indirect calorimetry. We found that OxR reduced mitochondrial oxidation at whole-body level, as shown by a reduction in whole-body oxygen consumption and an increase in serum long-chain acylcarnitine levels. WAT did not seem to contribute to this serum profile since only short-chain acylcarnitines were increased in WAT and gene expression analysis indicated an increase in mitochondrial oxidation, based on coordinate down-regulation of Sirt4, Gpam and Chchd3/Minos3. In addition, OxR did not induce oxidative stress in obese WAT, but increased molecular pathways involved in cell growth and proliferation. OxR increased levels of tyrosine, lysine and ornithine in serum and of leucine/isoleucine in WAT. This study shows that OxR limits oxidative phosphorylation at whole-body level, but in WAT compensatory mechanisms seem to operate. The down-regulation of the mitochondria-related genes Sirt4, Gpam, and Chchd3 may be considered as a biomarker profile for WAT mitochondrial reprogramming in response to acute exposure to limited oxygen availabilit

Amino acid deprivation due to overexpression of UCP1 in skeletal muscle: signalling via FGF-21

Recent studies on mouse and human skeletal muscle (SM) demonstrated the important link between mitochondrial function and the cellular metabolic adaptation. To identify key compensatory molecular mechanisms in response to chronic mitochondrial distress, we analyzed mice with ectopic SM respiratory uncoupling in uncoupling protein 1 transgenic (UCP1-TG) mice as model of muscle-specific compromised mitochondrial function. Here we describe a detailed metabolic reprogramming profile associated with mitochondrial perturbations in SM, triggering an increased protein turnover and amino acid metabolism with induced biosynthetic serine/1-carbon/glycine pathway and the longevity-promoting polyamine spermidine as well as the trans-sulfuration pathway. This is related to an induction of NADPH-generating pathways and glutathione metabolism as an adaptive mitohormetic response and defense against increased oxidative stress. Strikingly, consistent muscle retrograde signaling profiles were observed in acute stress states such as muscle cell starvation and lipid overload, muscle regeneration, and heart muscle inflammation, but not in response to exercise. We provide conclusive evidence for a key compensatory stress-signaling network that preserves cellular function, oxidative stress tolerance, and survival during conditions of increased SM mitochondrial distress, a metabolic reprogramming profile so far only demonstrated for cancer cells and heart muscle.-Ost, M., Keipert, S., van Schothorst, E. M., Donner, V., van der Stelt, I., Kipp, A. P., Petzke, K.-J., Jove, M., Pamplona, R., Portero-Otin, M., Keijer, J., and Klaus, S. Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux

Postnatal induction of muscle fatty acid oxidation in mice differing in propensity to obesity: a role of pyruvate dehydrogenase

Background/objective: Adaptation to the extrauterine environment depends on a switch from glycolysis to catabolism of fatty acids (FA) provided as milk lipids. We sought to learn whether the postnatal induction of muscle FA oxidation in mice could reflect propensity to obesity and to characterize the mechanisms controlling this induction. Methods: Experiments were conducted using obesity-resistant A/J and obesity-prone C57BL/6J (B6) mice maintained at 30 °C, from 5 to 28 days after birth. At day 10, both A/J and B6 mice with genetic ablation (KO) of α2 subunit of AMP-activated protein kinase (AMPK) were also used. In skeletal muscle, expression of selected genes was determined using quantitative real-time PCR, and AMPK subunits content was evaluated using Western blotting. Activities of both AMPK and pyruvate dehydrogenase (PDH), as well as acylcarnitine levels in the muscle were measured. Results: Acylcarnitine levels and gene expression indicated transient increase in FA oxidation during the first 2 weeks after birth, with a stronger increase in A/J mice. These data correlated with (i) the surge in plasma leptin levels, which peaked at day 10 and was higher in A/J mice, and (ii) relatively low activity of PDH linked with up-regulation of PDH kinase 4 gene (Pdk4) expression in the 10-day-old A/J mice. In contrast with the Pdk4 expression, transient up-regulation of uncoupling protein 3 gene was observed in B6 but not A/J mice. AMPK activity changed during the development, without major differences between A/J and B6 mice. Expression of neither Pdk4 nor other muscle genes was affected by AMPK-KO. Conclusions: Our results indicate a relatively strong postnatal induction of FA oxidation in skeletal muscle of the obesity-resistant A/J mice. This induction is transient and probably results from suppression of PDH activity, linked with a postnatal surge in plasma leptin levels, independent of AMPK.</p