Inge van der Stelt

Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux

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., Klaus, S. Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux. FASEB J. 29, 1314‐1328 (2015). www.fasebj.org

Quercetin tests negative for genotoxicity in transcriptome analyses of liver and small intestine of mice

Given the positive results of quercetin in in vitro genotoxicity studies, the in vivo genotoxic properties of this important dietary flavonoid warrant testing, especially considering possible high intake via widely available food supplements. Here, this was done by transcriptome analyses of the most relevant tissues, liver and small intestine, of quercetin supplemented mice. Quercetin (0.33%) supplemented to a high-fat diet was administered to mice during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase levels revealed no indications for hepatotoxicity. Microarray pathway analysis of liver and small intestine showed no regulation of genotoxicity related pathways. Analysis of DNA damage related genes also did not point at genotoxicity. Furthermore, a published classifier set of transcripts for identifying genotoxic compounds did not indicate genotoxicity. Only two transcripts of the classifier set were regulated, but in the opposite direction compared with the genotoxic compounds 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1). Based on the weight of evidence of three different types of analysis, we conclude that supplementation with quercetin at ~350 mg/kg bw/day for 12 weeks in mice showed no up-regulation of genotoxicity related pathways in liver and small intestine.

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

ABSTRACT 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 (Prdx3), thioredoxin reductase 1 (Txnrd1), and uncoupling protein 2 (Ucp2) and a downward trend in glutathione peroxidase 3 (Gpx3) and glutathione S-transferase mu 2 (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.

Mitochondrial ATP Depletion Disrupts Caco-2 Monolayer Integrity and Internalizes Claudin 7

Objective: In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Methods: Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed. Results: Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins (TJP) 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Conclusions: Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the distribution of claudin 7.

Metabolic response of visceral white adipose tissue of obese mice exposed for 5 days to human room temperature compared to mouse thermoneutrality

Housing of laboratory mice at room temperature (22°C) might be considered a constant cold stress, which induces a thermogenic program in brown adipose tissue (BAT). However, the early adaptive response of white adipose tissue (WAT), the fat storage organ of the body, to a change from thermoneutrality to room temperature is not known. This was investigated here for various WAT depots, focusing on epididymal WAT (eWAT), widely used as reference depot. Male adult diet-induced obese (DIO) C57BL/6JOlaHsd mice housed at thermoneutrality (29°C), were for 5 days either switched to room temperature (22°C) or remained at thermoneutrality. Energy metabolism was continuously measured using indirect calorimetry. At the end of the study, serum metabolomics and WAT transcriptomics were performed. We confirmed activation of the thermogenic program in 22°C housed mice. Body weight and total fat mass were reduced. Whole body energy expenditure (EE) was increased, with a higher fatty acid to carbohydrate oxidation ratio and increased serum acylcarnitine levels, while energy intake was not significantly different between the two groups. Transcriptome analysis of eWAT identified tissue remodeling and inflammation as the most affected processes. Expression of pro-inflammatory M1 macrophage-related genes, and M1 over M2 macrophage ratio were decreased, which might be linked to an increased insulin sensitivity. Markers of thermogenesis were not altered in eWAT. Decreased expression of tryptophan hydroxylase 2 (Tph2) and cholecystokinin (Cck) might represent altered neuroendocrine signaling. eWAT itself does not show increased fatty acid oxidation. The three measured WATs, epididymal, mesenteric, and retroperitoneal, showed mainly similar responses; reduced inflammation (s100a8), decreased carbohydrate oxidation, and no or small differences in fatty acid oxidation. However, Ucp1 was only expressed and increased in rWAT in 22°C housed mice. Cck expression was decreased in the three WATs, significantly in eWAT and rWAT, in contrast to Tph2, which was decreased in eWAT while not expressed in mWAT and rWAT. Our data show that tissue remodeling, inflammation and neuroendocrine signaling are early responses in WAT to a moderate decrease in environmental temperature.

No Adverse Programming by Post-Weaning Dietary Fructose of Body Weight, Adiposity, Glucose Tolerance or Metabolic Flexibility

Scope 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. Methods and results 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. Conclusions 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.

Preantral follicular atresia occurs mainly through autophagy, while antral follicles degenerate mostly through apoptosis

Abstract There is a general agreement that granulosa cell apoptosis is the cause of antral follicle attrition. Less clear is whether this pathway is also activated in case of preantral follicle degeneration, as several reports mention that the incidence of granulosa cell apoptosis in preantral follicles is negligible. Our objective is therefore to determine which cell-death pathways are involved in preantral and antral follicular degeneration. Atretic preantal and antral follicles were investigated using immunohistochemistry and lasercapture microdissection followed by quantitative real-time reverse transcription polymerase chain reaction. Microtubule-associated light-chain protein 3 (LC3), sequestosome 1 (SQSTM1/P62), Beclin1, autophagy-related protein 7 (ATG7), and cleaved caspase 3 (cCASP3) were used as markers for autophagy and apoptosis, respectively. P62 immunostaining was far less intense in granulosa cells of atretic compared to healthy preantral follicles, while no difference in LC3 and BECLIN1 immunostaining intensity was observed. This difference in P62 immunostaining was not observed in atretic antral follicles. mRNA levels of LC3 and P62 were not different between healthy and atretic (pre)antral follicles. ATG7 immunostaining was observed in granulosa cells of preantral atretic follicles, not in granulosa cells of degenerating antral follicles. The number of cCASP3-positive cells was negligible in preantral atretic follicles, while numerous in atretic antral follicles. Taken together, we conclude that preantral and antral follicular atresia is the result of activation of different cell-death pathways as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy. Summary Sentence Preantral and antral follicles make use of different cell-death pathways, as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia is mainly dependent on enhanced granulosa cell autophagy.

Muscle mitochondrial stress adaptation operates independently of endogenous FGF21 action

Objective: Fibroblast growth factor 21 (FGF21) was recently discovered as stress-induced myokine during mitochondrial disease and proposed as key metabolic mediator of the integrated stress response (ISR) presumably causing systemic metabolic improvements. Curiously, the precise cell-non-autonomous and cell-autonomous relevance of endogenous FGF21 action remained poorly understood. Methods: We made use of the established UCP1 transgenic (TG) mouse, a model of metabolic perturbations made by a specific decrease in muscle mitochondrial efficiency through increased respiratory uncoupling and robust metabolic adaptation and muscle ISR-driven FGF21 induction. In a cross of TG with Fgf21-knockout (FGF21 / ) mice, we determined the functional role of FGF21 as a muscle stress-induced myokine under low and high fat feeding conditions. Results: Here we uncovered that FGF21 signaling is dispensable for metabolic improvements evoked by compromised mitochondrial function in skeletal muscle. Strikingly, genetic ablation of FGF21 fully counteracted the cell-non-autonomous metabolic remodeling and browning of subcutaneous white adipose tissue (WAT), together with the reduction of circulating triglycerides and cholesterol. Brown adipose tissue activity was similar in all groups. Remarkably, we found that FGF21 played a negligible role in muscle mitochondrial stress-related improved obesity resistance, glycemic control and hepatic lipid homeostasis. Furthermore, the protective cell-autonomous muscle mitohormesis and metabolic stress adaptation, including an increased muscle proteostasis via mitochondrial unfolded protein response (UPR) and amino acid biosynthetic pathways did not require the presence of FGF21. Conclusions: Here we demonstrate that although FGF21 drives WAT remodeling, the adaptive pseudo-starvation response under elevated muscle mitochondrial stress conditions operates independently of both WAT browning and FGF21 action. Thus, our findings challenge FGF21 as key metabolic mediator of the mitochondrial stress adaptation and powerful therapeutic target during muscle mitochondrial disease. 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).