AMPK-dependent and -independent coordination of mitochondrial function and muscle fiber type by FNIP1.

Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1Tg) mice. Fnip1Tg mice were crossed with Fnip1-knockout (Fnip1KO) mice to generate Fnip1TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness.

Annonaceous Acetogenin Mimic AA005 Inhibits the Growth of TNBC MDA-MB-468 Cells by Altering Cell Energy Metabolism.

Triple-negative breast cancer (TNBC) is a serious health issue for women worldwide and there is still no suitable treatment option. AA005, a structurally simplified mimic of natural Annonaceous acetogenins, presents outstanding properties with impressive cytotoxicity and cell-type selective actions. The present study was aimed at evaluating the potential of AA005 as a therapeutic agent for TNBC. AA005 potently inhibited the growth of TNBC cells at 50 nM level. Inspired by the finding of the phosphatase and tensin homologue (PTEN) tumor suppressor, the effect of AA005 on aerobic glycolysis was investigated in TNBC MDA-MB-468 cells. A short-term AA005 exposure markedly suppressed mitochondrial function in MDA-MB-468 cells, thus activating the aerobic glycolysis to lessen the risk of decreased ATP generation in mitochondria. Prolonging the incubation time of AA005 clearly weakened the aerobic glycolysis in the cells. This was in part attributed to the PI3K-AKT pathway inactivation and subsequent declined glucose uptake. As a consequence, the energy supply was completely cut from the two major energy-producing pathways. Further experiments showed that AA005 resulted in irreversible damage on cell activity including cell cycle and growth, inducing mitochondrial oxidative stress and ultimately leading to cell death. In addition, the in vivo therapeutic efficacy of AA005 was proved on 4T1 xenograft tumor mice model. Our data demonstrate that AA005 exhibited a great potential for future clinical applications in TNBC therapy.

Increased glycolysis in skeletal muscle coordinates with adipose tissue in systemic metabolic homeostasis.

Insulin-independent glucose metabolism, including anaerobic glycolysis that is promoted in resistance training, plays critical roles in glucose disposal and systemic metabolic regulation. However, the underlying mechanisms are not completely understood. In this study, through genetically manipulating the glycolytic process by overexpressing human glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and 6-phosphofructo-2-kinase-fructose-2,6-biphosphatase 3 (PFKFB3) in mouse skeletal muscle, we examined the impact of enhanced glycolysis in metabolic homeostasis. Enhanced glycolysis in skeletal muscle promoted accelerated glucose disposal, a lean phenotype and a high metabolic rate in mice despite attenuated lipid metabolism in muscle, even under High-Fat diet (HFD). Further study revealed that the glucose metabolite sensor carbohydrate-response element-binding protein (ChREBP) was activated in the highly glycolytic muscle and stimulated the elevation of plasma fibroblast growth factor 21 (FGF21), possibly mediating enhanced lipid oxidation in adipose tissue and contributing to a systemic effect. PFKFB3 was critically involved in promoting the glucose-sensing mechanism in myocytes. Thus, a high level of glycolysis in skeletal muscle may be intrinsically coupled to distal lipid metabolism through intracellular glucose sensing. This study provides novel insights for the benefit of resistance training and for manipulating insulin-independent glucose metabolism.

Exercise Inducible Lactate Dehydrogenase B Regulates Mitochondrial Function in Skeletal Muscle.

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate, which are critical fuel metabolites of skeletal muscle particularly during exercise. However, the physiological relevance of LDH remains poorly understood. Here we show that Ldhb expression is induced by exercise in human muscle and negatively correlated with changes in intramuscular pH levels, a marker of lactate production, during isometric exercise. We found that the expression of Ldhb is regulated by exercise-induced peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Ldhb gene promoter reporter studies demonstrated that PGC-1α activates Ldhb gene expression through multiple conserved estrogen-related receptor (ERR) and myocyte enhancer factor 2 (MEF2) binding sites. Transgenic mice overexpressing Ldhb in muscle (muscle creatine kinase (MCK)-Ldhb) exhibited increased exercise performance and enhanced oxygen consumption during exercise. MCK-Ldhb muscle was shown to have enhanced mitochondrial enzyme activity and increased mitochondrial gene expression, suggesting an adaptive oxidative muscle transformation. In addition, mitochondrial respiration capacity was increased and lactate production decreased in MCK-Ldhb skeletal myotubes in culture. Together, these results identified a previously unrecognized Ldhb-driven alteration in muscle mitochondrial function and suggested a mechanism for the adaptive metabolic response induced by exercise training.

Muscle-bone cross-talk through the FNIP1-TFEB-IGF2 axis is associated with bone metabolism in human and mouse.

Clinical evidence indicates a close association between muscle dysfunction and bone loss; however, the underlying mechanisms remain unclear. Here, we report that muscle dysfunction-related bone loss in humans with limb-girdle muscular dystrophy is associated with decreased expression of folliculin-interacting protein 1 (FNIP1) in muscle tissue. Supporting this finding, murine gain- and loss-of-function genetic models demonstrated that muscle-specific ablation of FNIP1 caused decreased bone mass, increased osteoclastic activity, and mechanical impairment that could be rescued by myofiber-specific expression of FNIP1. Myofiber-specific FNIP1 deficiency stimulated expression of nuclear translocation of transcription factor EB, thereby activating transcription of insulin-like growth factor 2 (Igf2) at a conserved promoter-binding site and subsequent IGF2 secretion. Muscle-derived IGF2 stimulated osteoclastogenesis through IGF2 receptor signaling. AAV9-mediated overexpression of IGF2 was sufficient to decrease bone volume and impair bone mechanical properties in mice. Further, we found that serum IGF2 concentration was negatively correlated with bone health in humans in the context of osteoporosis. Our findings elucidate a muscle-bone cross-talk mechanism bridging the gap between muscle dysfunction and bone loss. This cross-talk represents a potential target to treat musculoskeletal diseases and osteoporosis.

FNIP1 abrogation promotes functional revascularization of ischemic skeletal muscle by driving macrophage recruitment.

Ischaemia of the heart and limbs attributable to compromised blood supply is a major cause of mortality and morbidity. The mechanisms of functional angiogenesis remain poorly understood, however. Here we show that FNIP1 plays a critical role in controlling skeletal muscle functional angiogenesis, a process pivotal for muscle revascularization during ischemia. Muscle FNIP1 expression is down-regulated by exercise. Genetic overexpression of FNIP1 in myofiber causes limited angiogenesis in mice, whereas its myofiber-specific ablation markedly promotes the formation of functional blood vessels. Interestingly, the increased muscle angiogenesis is independent of AMPK but due to enhanced macrophage recruitment in FNIP1-depleted muscles. Mechanistically, myofiber FNIP1 deficiency induces PGC-1α to activate chemokine gene transcription, thereby driving macrophage recruitment and muscle angiogenesis program. Furthermore, in a mouse hindlimb ischemia model of peripheral artery disease, the loss of myofiber FNIP1 significantly improved the recovery of blood flow. Thus, these results reveal a pivotal role of FNIP1 as a negative regulator of functional angiogenesis in muscle, offering insight into potential therapeutic strategies for ischemic diseases.

Proteolytic rewiring of mitochondria by LONP1 directs cell identity switching of adipocytes.

Mitochondrial proteases are emerging as key regulators of mitochondrial plasticity and acting as both protein quality surveillance and regulatory enzymes by performing highly regulated proteolytic reactions. However, it remains unclear whether the regulated mitochondrial proteolysis is mechanistically linked to cell identity switching. Here we report that cold-responsive mitochondrial proteolysis is a prerequisite for white-to-beige adipocyte cell fate programming during adipocyte thermogenic remodelling. Thermogenic stimulation selectively promotes mitochondrial proteostasis in mature white adipocytes via the mitochondrial protease LONP1. Disruption of LONP1-dependent proteolysis substantially impairs cold- or ß3 adrenergic agonist-induced white-to-beige identity switching of mature adipocytes. Mechanistically, LONP1 selectively degrades succinate dehydrogenase complex iron sulfur subunit B and ensures adequate intracellular succinate levels. This alters the histone methylation status on thermogenic genes and thereby enables adipocyte cell fate programming. Finally, augmented LONP1 expression raises succinate levels and corrects ageing-related impairments in white-to-beige adipocyte conversion and adipocyte thermogenic capacity. Together, these findings reveal that LONP1 links proteolytic surveillance to mitochondrial metabolic rewiring and directs cell identity conversion during adipocyte thermogenic remodelling.

FNIP1 regulates adipocyte browning and systemic glucose homeostasis in mice by shaping intracellular calcium dynamics.

Metabolically beneficial beige adipocytes offer tremendous potential to combat metabolic diseases. The folliculin interacting protein 1 (FNIP1) is implicated in controlling cellular metabolism via AMPK and mTORC1. However, whether and how FNIP1 regulates adipocyte browning is unclear. Here, we demonstrate that FNIP1 plays a critical role in controlling adipocyte browning and systemic glucose homeostasis. Adipocyte-specific ablation of FNIP1 promotes a broad thermogenic remodeling of adipocytes, including increased UCP1 levels, high mitochondrial content, and augmented capacity for mitochondrial respiration. Mechanistically, FNIP1 binds to and promotes the activity of SERCA, a main Ca2+ pump responsible for cytosolic Ca2+ removal. Loss of FNIP1 resulted in enhanced intracellular Ca2+ signals and consequential activation of Ca2+-dependent thermogenic program in adipocytes. Furthermore, mice lacking adipocyte FNIP1 were protected against high-fat diet-induced insulin resistance and liver steatosis. Thus, these findings reveal a pivotal role of FNIP1 as a negative regulator of beige adipocyte thermogenesis and unravel an intriguing functional link between intracellular Ca2+ dynamics and adipocyte browning.

Mitochondrial proteostasis stress in muscle drives a long-range protective response to alleviate dietary obesity independently of ATF4.

Mitochondrial quality in skeletal muscle is crucial for maintaining energy homeostasis during metabolic stresses. However, how muscle mitochondrial quality is controlled and its physiological impacts remain unclear. Here, we demonstrate that mitoprotease LONP1 is essential for preserving muscle mitochondrial proteostasis and systemic metabolic homeostasis. Skeletal muscle-specific deletion of Lon protease homolog, mitochondrial (LONP1) impaired mitochondrial protein turnover, leading to muscle mitochondrial proteostasis stress. A benefit of this adaptive response was the complete resistance to diet-induced obesity. These favorable metabolic phenotypes were recapitulated in mice overexpressing LONP1 substrate ΔOTC in muscle mitochondria. Mechanistically, mitochondrial proteostasis imbalance elicits an unfolded protein response (UPRmt) in muscle that acts distally to modulate adipose tissue and liver metabolism. Unexpectedly, contrary to its previously proposed role, ATF4 is dispensable for the long-range protective response of skeletal muscle. Thus, these findings reveal a pivotal role of LONP1-dependent mitochondrial proteostasis in directing muscle UPRmt to regulate systemic metabolism.

Disuse-associated loss of the protease LONP1 in muscle impairs mitochondrial function and causes reduced skeletal muscle mass and strength.

Mitochondrial proteolysis is an evolutionarily conserved quality-control mechanism to maintain proper mitochondrial integrity and function. However, the physiological relevance of stress-induced impaired mitochondrial protein quality remains unclear. Here, we demonstrate that LONP1, a major mitochondrial protease resides in the matrix, plays a role in controlling mitochondrial function as well as skeletal muscle mass and strength in response to muscle disuse. In humans and mice, disuse-related muscle loss is associated with decreased mitochondrial LONP1 protein. Skeletal muscle-specific ablation of LONP1 in mice resulted in impaired mitochondrial protein turnover, leading to mitochondrial dysfunction. This caused reduced muscle fiber size and strength. Mechanistically, aberrant accumulation of mitochondrial-retained protein in muscle upon loss of LONP1 induces the activation of autophagy-lysosome degradation program of muscle loss. Overexpressing a mitochondrial-retained mutant ornithine transcarbamylase (ΔOTC), a known protein degraded by LONP1, in skeletal muscle induces mitochondrial dysfunction, autophagy activation, and cause muscle loss and weakness. Thus, these findings reveal a role of LONP1-dependent mitochondrial protein quality-control in safeguarding mitochondrial function and preserving skeletal muscle mass and strength, and unravel a link between mitochondrial protein quality and muscle mass maintenance during muscle disuse.

Histone methyltransferase MLL4 controls myofiber identity and muscle performance through MEF2 interaction.

Skeletal muscle depends on the precise orchestration of contractile and metabolic gene expression programs to direct fiber-type specification and to ensure muscle performance. Exactly how such fiber type-specific patterns of gene expression are established and maintained remains unclear, however. Here, we demonstrate that histone monomethyl transferase MLL4 (KMT2D), an enhancer regulator enriched in slow myofibers, plays a critical role in controlling muscle fiber identity as well as muscle performance. Skeletal muscle-specific ablation of MLL4 in mice resulted in downregulation of the slow oxidative myofiber gene program, decreased numbers of type I myofibers, and diminished mitochondrial respiration, which caused reductions in muscle fatty acid utilization and endurance capacity during exercise. Genome-wide ChIP-Seq and mRNA-Seq analyses revealed that MLL4 directly binds to enhancers and functions as a coactivator of the myocyte enhancer factor 2 (MEF2) to activate transcription of slow oxidative myofiber genes. Importantly, we also found that the MLL4 regulatory circuit is associated with muscle fiber-type remodeling in humans. Thus, our results uncover a pivotal role for MLL4 in specifying structural and metabolic identities of myofibers that govern muscle performance. These findings provide therapeutic opportunities for enhancing muscle fitness to combat a variety of metabolic and muscular diseases.

Targeted reversal and phosphorescence lifetime imaging of cancer cell metabolism via a theranostic rhenium(I)-DCA conjugate.

Cancer cell metabolism is quite different from normal cells. Targeting cancer metabolism and untuning the tumor metabolic machine has emerged as a promising strategy for cancer therapy. We have developed a multi-functional Re-dca conjugate (Re-dca 2) by conjugating the metabolic modulator dichloroacetate (DCA) to mitochondria-targeted rhenium(I) complex, allowing its efficient penetration into cancer cells and selective accumulation in mitochondria, thus achieving the cancer cell metabolism reversal from glycolysis to glucose oxidation at pharmacologically relevant DCA doses. Mechanism studies confirm the inhibition effect of Re-dca 2 on the activity of pyruvate dehydrogenase kinase (PDK) and capture the metabolic reversal window in Re-dca 2 treated NCI-1229 cells at the early stage of drug treatment, resulting in selective killing of malignant cells cocultured with normal cells, significant inhibition of cancer cell metastasis and invasion, as well as excellent anti-angiogenesis activities in zebrafish embryos. By comparison, DCA-free Re(I) analogue is also investigated under the same conditions. Although this analogue also exhibits cytotoxicity due to the Re(I) core, metabolic reversal is not induced by this analogue and its anti-metastasis activity is much lower than Re-dca 2, indicating the synergistic effect of Re(I) core and DCA moiety on cancer therapy. In vivo anti-cancer investigations also indicate that the mitochondria-targeted Re-dca 2 can effectively inhibit the tumor growth without affecting the body weight of nude mice, and the therapeutic effect is much better than the DCA-free Re(I) analogue 2a. Simultaneously, the O2-sensitive phosphorescent lifetimes of Re-dca 2 can be utilized for PLIM imaging of intracellular oxygen consumption, thus reflecting the Re-dca 2 induced glycolysis-to-glucose oxidation reversal at the early drug treatment stage. The excellent phosphorescence of Re-dca 2 can also be utilized for real-time tracking of mitochondrial morphological changes during treatment. In a word, rational design of phosphorescent metallodrug and metabolic modulator conjugates for synergistic treatment is a promising strategy for simultaneous untuning and tracking tumor metabolic machine, thus providing new clues for cancer therapy and mechanisms.

Mitophagy Directs Muscle-Adipose Crosstalk to Alleviate Dietary Obesity.

The quality of mitochondria in skeletal muscle is essential for maintaining metabolic homeostasis during adaptive stress responses. However, the precise control mechanism of muscle mitochondrial quality and its physiological impacts remain unclear. Here, we demonstrate that FUNDC1, a mediator of mitophagy, plays a critical role in controlling muscle mitochondrial quality as well as metabolic homeostasis. Skeletal-muscle-specific ablation of FUNDC1 in mice resulted in LC3-mediated mitophagy defect, leading to impaired mitochondrial energetics. This caused decreased muscle fat utilization and endurance capacity during exercise. Interestingly, mice lacking muscle FUNDC1 were protected against high-fat-diet-induced obesity with improved systemic insulin sensitivity and glucose tolerance despite reduced muscle mitochondrial energetics. Mechanistically, FUNDC1 deficiency elicited a retrograde response in muscle that upregulated FGF21 expression, thereby promoting the thermogenic remodeling of adipose tissue. Thus, these findings reveal a pivotal role of FUNDC1-dependent mitochondrial quality control in mediating the muscle-adipose dialog to regulate systemic metabolism.

Simultaneously Inducing and Tracking Cancer Cell Metabolism Repression by Mitochondria-Immobilized Rhenium(I) Complex.

Mitochondrial metabolism is essential for tumorigenesis, and the development of cancer is usually accompanied by alternations of mitochondrial function. Emerging studies have demonstrated that targeting mitochondria and mitochondrial metabolism is an effective strategy for cancer therapy. In this work, eight phosphorescent organometallic rhenium(I) complexes have been synthesized and explored as mitochondria-targeted theranostic agents, capable of inducing and tracking the therapeutic effect simultaneously. Complexes 1b-4b can quickly and efficiently penetrate into A549 cells, specifically localizing within mitochondria, and their cytotoxicity is superior to cisplatin against the cancer cells screened. Notably, complex 3b [Re(CO)3(DIP) (py-3-CH2Cl)]+ containing thiol-reactive chloromethylpyridyl moiety for mitochondria immobilization shows higher cytotoxicity and selectivity against cancer cells than other Re(I) complexes without mitochondria-immobilization properties. Mechanistic studies show that complexes 1b-4b induce a cascade of mitochondria-dependent events including mitochondrial damage, mitochondrial respiration inhibition, cellular ATP depletion, reactive oxygen species (ROS) elevation, and caspase-dependent apoptosis. By comparison, mitochondria-immobilized 3b causes more effective repression of mitochondrial metabolism than mitochondrial-nonimmobilized complexes. The excellent phosphorescence and O2-sensitive lifetimes of mitochondria-immobilized 3b can be utilized for real-time tracking of the morphological changes of mitochondria and mitochondrial respiration repression during therapy process, accordingly providing reliable information for understanding anticancer mechanisms.

Coupling of mitochondrial function and skeletal muscle fiber type by a miR-499/Fnip1/AMPK circuit.

Upon adaption of skeletal muscle to physiological and pathophysiological stimuli, muscle fiber type and mitochondrial function are coordinately regulated. Recent studies have identified pathways involved in control of contractile proteins of oxidative-type fibers. However, the mechanism for coupling of mitochondrial function to the muscle contractile machinery during fiber type transition remains unknown. Here, we show that the expression of the genes encoding type I myosins, Myh7/Myh7b and their intronic miR-208b/miR-499, parallels mitochondrial function during fiber type transitions. Using in vivo approaches in mice, we found that miR-499 drives a PGC-1α-dependent mitochondrial oxidative metabolism program to match shifts in slow-twitch muscle fiber composition. Mechanistically, miR-499 directly targets Fnip1, an AMP-activated protein kinase (AMPK)-interacting protein that negatively regulates AMPK, a known activator of PGC-1α. Inhibition of Fnip1 reactivated AMPK/PGC-1α signaling and mitochondrial function in myocytes. Restoration of the expression of miR-499 in the mdx mouse model of Duchenne muscular dystrophy (DMD) reduced the severity of DMD Thus, we have identified a miR-499/Fnip1/AMPK circuit that can serve as a mechanism to couple muscle fiber type and mitochondrial function.