UCP1-Tg mice with ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) are a model of improved substrate metabolism and increased longevity. Analysis of myokine expression showed an induction of fibroblast growth factor 21 (FGF21) in SM, resulting in approximately fivefold elevated circulating FGF21 in UCP1-Tg mice. Despite a reduced muscle mass, UCP1-Tg mice showed no evidence for a myopathy or muscle autophagy deficiency but an activation of integrated stress response (ISR; eIF2α/ATF4) in SM. Targeting mitochondrial function in vitro by treating C2C12 myoblasts with the uncoupler FCCP resulted in a dose-dependent activation of ISR, which was associated with increased expression of FGF21, which was also observed by treatment with respiratory chain inhibitors antimycin A and myxothiazol. The cofactor required for FGF21 action, β-klotho, was expressed in white adipose tissue (WAT) of UCP1-Tg mice, which showed an increased browning of WAT similar to what occurred in altered adipocyte morphology, increased brown adipocyte markers (UCP1, CIDEA), lipolysis (HSL phosphorylation), and respiratory capacity. Importantly, treatment of primary white adipocytes with serum of transgenic mice resulted in increased UCP1 expression. Additionally, UCP1-Tg mice showed reduced body length through the suppressed IGF-I-GH axis and decreased bone mass. We conclude that the induction of FGF21 as a myokine is coupled to disturbance of mitochondrial function and ISR activation in SM. FGF21 released from SM has endocrine effects leading to increased browning of WAT and can explain the healthy metabolic phenotype of UCP1-Tg mice. These results confirm muscle as an important endocrine regulator of whole body metabolism.

Brown adipose tissue (BAT)-dependent thermogenesis and its suggested augmenting hormone, FGF21, are potential therapeutic targets in current obesity and diabetes research. Here, we studied the role of UCP1 and FGF21 for metabolic homeostasis in the cold and dissected underlying molecular mechanisms using UCP1-FGF21 double-knockout mice. We report that neither UCP1 nor FGF21, nor even compensatory increases of FGF21 serum levels in UCP1 knockout mice, are required for defense of body temperature or for maintenance of energy metabolism and body weight. Remarkably, cold-induced browning of inguinal white adipose tissue (iWAT) is FGF21 independent. Global RNA sequencing reveals major changes in response to UCP1- but not FGF21-ablation in BAT, iWAT, and muscle. Markers of mitochondrial failure and inflammation are observed in BAT, but in particular the enhanced metabolic reprogramming in iWAT supports the thermogenic role of UCP1 and excludes an important thermogenic role of endogenous FGF21 in normal cold acclimation.

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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 NADPHgenerating 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 stresssignaling 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 Key Words: amino acid metabolism • metabolic reprogramming • mitochondrial myopathy • oxidative stress tolerance • polyamines SKELETAL MUSCLE (SM) WASTING, or atrophy, occurs as a physiologic response to muscle disuse, fasting, starvation, and aging; it also occurs in a wide range of systemic diseases, including cancer, denervation, and diabetes mellitus (1-4). Numerous studies connected alterations in mitochondrial function to muscle atrophy, focusing on mitochondrial respiratory chain dysfunction or formation of reactive oxygen species (ROS) (5), as mitochondria represent an important site of cellular ROS production (6). Mitochondria are key dynamic organelles that not only provide ATP via oxidative phosphorylation but also are involved in the cellular integrated stress response (7) and in mitohormesis, a molecular adaptation and retrograde response resulting in stress resistance (8).We previously demonstrated in a transgenic mouse model with alterations in SM mitochondrial function (uncoupling protein 1 [UCP1] transgenic [TG] mice [UCP1-TG], with ectopic expression of UCP1 in SM) an elevated endogenous antioxidant defense system, induced integrated stress response but also compromised mitochondrial respiratory chain capacity (9) and a strong reduction in SM mass (10). In contrast...

Mitochondrial dysfunction promotes metabolic stress responses in a cell‐autonomous as well as organismal manner. The wasting hormone growth differentiation factor 15 (GDF15) is recognized as a biomarker of mitochondrial disorders, but its pathophysiological function remains elusive. To test the hypothesis that GDF15 is fundamental to the metabolic stress response during mitochondrial dysfunction, we investigated transgenic mice (Ucp1‐TG) with compromised muscle‐specific mitochondrial OXPHOS capacity via respiratory uncoupling. Ucp1‐TG mice show a skeletal muscle‐specific induction and diurnal variation of GDF15 as a myokine. Remarkably, genetic loss of GDF15 in Ucp1‐TG mice does not affect muscle wasting or transcriptional cell‐autonomous stress response but promotes a progressive increase in body fat mass. Furthermore, muscle mitochondrial stress‐induced systemic metabolic flexibility, insulin sensitivity, and white adipose tissue browning are fully abolished in the absence of GDF15. Mechanistically, we uncovered a GDF15‐dependent daytime‐restricted anorexia, whereas GDF15 is unable to suppress food intake at night. Altogether, our evidence suggests a novel diurnal action and key pathophysiological role of mitochondrial stress‐induced GDF15 in the regulation of systemic energy metabolism.

The activity of uncoupling protein-1 (UCP1) is rate-limiting for nonshivering thermogenesis and diet-induced thermogenesis. Characteristically, this activity is inhibited by GDP experimentally and presumably mainly by cytosolic ATP within brown-fat cells. The issue as to whether UCP1 has a residual proton conductance even when fully saturated with GDP/ATP (as has recently been suggested) has not only scientific but also applied interest, since a residual proton conductance would make overexpressed UCP1 weight-reducing even without physiological/pharmacological activation. To examine this question, we have here established optimal conditions for studying the bioenergetics of wild-type and UCP1-/- brown-fat mitochondria, analysing UCP1-mediated differences in parallel preparations of brown-fat mitochondria from both genotypes. Comparing different substrates, we find that pyruvate (or palmitoyl-L-carnitine) shows the largest relative coupling by GDP. Comparing albumin concentrations, we find the range 0.1-0.6% optimal; higher concentrations are inhibitory. Comparing basic medium composition, we find 125 mM sucrose optimal; an ionic medium (50-100 mM KCl) functions for wild-type but is detrimental for UCP1-/- mitochondria. Using optimal conditions, we find no evidence for a residual proton conductance (not a higher post-GDP respiration, a lower membrane potential or an altered proton leak at highest common potential) with either pyruvate or glycerol-3-phosphate as substrates, nor by a 3-4-fold alteration of the amount of UCP1. We could demonstrate that certain experimental conditions, due to respiratoty inhibition, could lead to the suggestion that UCP1 possesses a residual proton conductance but find that under optimal conditions our experiments concur with implications from physiological observations that in the presence of inhibitory nucleotides, UCP1 is not leaky.

ObjectiveFibroblast 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.MethodsWe 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.ResultsHere 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 (UPRmt) and amino acid biosynthetic pathways did not require the presence of FGF21.ConclusionsHere 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.

Keipert S, Ost M, Chadt A, Voigt A, Ayala V, Portero-Otin M, Pamplona R, Al-Hasani H, Klaus S. Skeletal muscle uncouplinginduced longevity in mice is linked to increased substrate metabolism and induction of the endogenous antioxidant defense system. Am J Physiol Endocrinol Metab 304: E495-E506, 2013. First published December 31, 2012 doi:10.1152/ajpendo.00518.2012.-Ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) mitochondria increases lifespan considerably in high-fat diet-fed UCP1 Tg mice compared with wild types (WT). To clarify the underlying mechanisms, we investigated substrate metabolism as well as oxidative stress damage and antioxidant defense in SM of low-fatand high-fat-fed mice. Tg mice showed an increased protein expression of phosphorylated AMP-activated protein kinase, markers of lipid turnover (p-ACC, FAT/CD36), and an increased SM ex vivo fatty acid oxidation. Surprisingly, UCP1 Tg mice showed elevated lipid peroxidative protein modifications with no changes in glycoxidation or direct protein oxidation. This was paralleled by an induction of catalase and superoxide dismutase activity, an increased redox signaling (MAPK signaling pathway), and increased expression of stress-protective heat shock protein 25. We conclude that increased skeletal muscle mitochondrial uncoupling in vivo does not reduce the oxidative stress status in the muscle cell. Moreover, it increases lipid metabolism and reactive lipid-derived carbonyls. This stress induction in turn increases the endogenous antioxidant defense system and redox signaling. Altogether, our data argue for an adaptive role of reactive species as essential signaling molecules for health and longevity.uncoupling protein 1; AMP-activated protein kinase; oxidative stress; redox signaling; lipid metabolism AGING IS A VERY COMPLEX PROCESS driven by numerous molecular pathways and biochemical events. Regarding the relationship between energy metabolism and longevity, there is a hypothesis termed "uncoupling to survive," which suggests that increased mitochondrial uncoupling and thus increased energy expenditure might increase longevity by preventing the formation of reactive oxygen species (ROS) (6). Previously, we showed that transgenic (Tg) mice with an ectopic expression of the uncoupling protein 1 (UCP1) in skeletal muscle (UCP1 Tg mice) showed a delayed development of obesity, improved glucose tolerance, and a 42% increased median lifespan compared with their wild-type (WT) littermates when exposed to a high-fat diet (30). However, the molecular mechanisms for these beneficial health effects of skeletal muscle uncoupling are not yet clarified.One hallmark of UCP1 Tg mice is an increased insulin sensitivity independent of body weight. Insulin resistance plays a crucial part in the pathogenesis of the metabolic syndrome and is characterized by a reduced substrate metabolism and impaired defense against stress in skeletal muscle. Skeletal muscle (SM) uncoupling increases SM glucose uptake and activates the AMPactivated protein k...