Cyclo His-Pro Attenuates Muscle Degeneration in Murine Myopathy Models.

Among the inherited myopathies, a group of muscular disorders characterized by structural and metabolic impairments in skeletal muscle, Duchenne muscular dystrophy (DMD) stands out for its devastating progression. DMD pathogenesis is driven by the progressive degeneration of muscle fibers, resulting in inflammation and fibrosis that ultimately affect the overall muscle biomechanics. At the opposite end of the spectrum of muscle diseases, age-related sarcopenia is a common condition that affects an increasing proportion of the elderly. Although characterized by different pathological mechanisms, DMD and sarcopenia share the development of progressive muscle weakness and tissue inflammation. Here, the therapeutic effects of Cyclo Histidine-Proline (CHP) against DMD and sarcopenia are evaluated. In the mdx mouse model of DMD, it is shown that CHP restored muscle contractility and force production, accompanied by the reduction of fibrosis and inflammation in skeletal muscle. CHP furthermore prevented the development of cardiomyopathy and fibrosis in the diaphragm, the two leading causes of death for DMD patients. CHP also attenuated muscle atrophy and functional deterioration in a mouse model of age-related sarcopenia. These findings from two different models of muscle dysfunction hence warrant further investigation into the effects of CHP on muscle pathologies in animal models and eventually in patients.

Inhibiting de novo ceramide synthesis restores mitochondrial and protein homeostasis in muscle aging.

Disruption of mitochondrial function and protein homeostasis plays a central role in aging. However, how these processes interact and what governs their failure in aging remain poorly understood. Here, we showed that ceramide biosynthesis controls the decline in mitochondrial and protein homeostasis during muscle aging. Analysis of transcriptome datasets derived from muscle biopsies obtained from both aged individuals and patients with a diverse range of muscle disorders revealed that changes in ceramide biosynthesis, as well as disturbances in mitochondrial and protein homeostasis pathways, are prevalent features in these conditions. By performing targeted lipidomics analyses, we found that ceramides accumulated in skeletal muscle with increasing age across Caenorhabditis elegans, mice, and humans. Inhibition of serine palmitoyltransferase (SPT), the rate-limiting enzyme of the ceramide de novo synthesis, by gene silencing or by treatment with myriocin restored proteostasis and mitochondrial function in human myoblasts, in C. elegans, and in the skeletal muscles of mice during aging. Restoration of these age-related processes improved health and life span in the nematode and muscle health and fitness in mice. Collectively, our data implicate pharmacological and genetic suppression of ceramide biosynthesis as potential therapeutic approaches to delay muscle aging and to manage related proteinopathies via mitochondrial and proteostasis remodeling.

Violacein switches off low molecular weight tyrosine phosphatase and rewires mitochondria in colorectal cancer cells.

In the last decade, emerging evidence has shown that low molecular weight protein tyrosine phosphatase (LMWPTP) not only contributes to the progression of cancer but is associated with prostate low survival rate and colorectal cancer metastasis. We report that LMWPTP favors the glycolytic profile in some tumors. Therefore, the focus of the present study was to identify metabolic enzymes that correlate with LMWPTP expression in patient samples. Exploratory data analysis from RNA-seq, proteomics, and histology staining, confirmed the higher expression of LMWPTP in CRC. Our descriptive statistical analyses indicate a positive expression correlation between LMWPTP and energy metabolism enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In addition, we examine the potential of violacein to reprogram energetic metabolism and LMWPTP activity. Violacein treatment induced a shift of glycolytic to oxidative metabolism associated with alteration in mitochondrial efficiency, as indicated by higher oxygen consumption rate. Particularly, violacein treated cells displayed higher proton leak and ATP-linked oxygen consumption rate (OCR) as an indicator of the OXPHOS preference. Notably, violacein is able to bind and inhibit LMWPTP. Since the LMWPTP acts as a hub of signaling pathways that offer tumor cells invasive advantages, such as survival and the ability to migrate, our findings highlight an unexplored potential of violacein in circumventing the metabolic plasticity of tumor cells.

Rosiglitazone protects INS-1E cells from human islet amyloid polypeptide toxicity.

Human islet amyloid polypeptide (hIAPP or amylin) is a hormone co-secreted with insulin by pancreatic ß-cells, and is the main component of islet amyloid. Islet amyloid is found in the pancreas of patients with type 2 diabetes and may be involved in ß-cell dysfunction and death, observed in this disease. Thus, counteracting islet amyloid toxicity represents a therapeutic approach to preserve ß-cell mass and function. In this sense, thiazolidinediones (TZDs), as rosiglitazone, have shown protective effects against other harmful insults to ß-cells. For this reason, we investigated whether rosiglitazone could protect ß-cells from hIAPP-induced cell death and the underlying mechanisms mediating such effect. Here, we show that rosiglitazone improved the viability of hIAPP-exposed INS-1E cells. This benefit is not dependent on the insulin-degrading enzyme (IDE) since rosiglitazone did not modulate IDE protein content and activity. However, rosiglitazone inhibited hIAPP fibrillation and decreased hIAPP-induced expression of C/EBP homologous protein (CHOP) (CTL 100.0 ± 8.4; hIAPP 182.7 ± 19.1; hIAPP + RGZ 102.8 ± 9.5), activating transcription factor-4 (ATF4) (CTL 100.0 ± 3.1; hIAPP 234.9 ± 19.3; hIAPP + RGZ 129.6 ± 3.0) and phospho-eukaryotic initiation factor 2-alpha (p-eIF2α) (CTL 100.0 ± 31.1; hIAPP 234.1 ± 36.2; hIAPP + RGZ 150.4 ± 18.0). These findings suggest that TZDs treatment may be a promising approach to preserve ß-cell mass and function by inhibiting islet amyloid formation and decreasing endoplasmic reticulum stress hIAPP-induced.

Offspring from trained male mice inherit improved muscle mitochondrial function through PPAR co-repressor modulation.

Aim Investigate whether inheritance of improved skeletal muscle mitochondrial function and its association with glycemic control are multigenerational benefits of exercise. MAIN METHODS: Male Swiss mice were subjected to 8 weeks of endurance training and mated with untrained females. KEY FINDINGS: Trained fathers displayed typical endurance training-induced adaptations. Remarkably, offspring from trained fathers also exhibited higher endurance performance, mitochondrial oxygen consumption, glucose tolerance and insulin sensitivity. However, PGC-1α expression was not increased in the offspring. In the offspring, the expression of the co-repressor NCoR1 was reduced, increasing activation of PGC-1α target genes. These effects correlated with higher DNA methylation at the NCoR1 promoter in both, the sperm of trained fathers and in the skeletal muscle of their offspring. SIGNIFICANCE: Higher skeletal muscle mitochondrial function is inherited by epigenetic de-activation of a key PGC-1α co-repressor.

Alternative human eIF5A protein isoform plays a critical role in mitochondria.

The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein containing the amino acid residue hypusine, essential for its activity. Hypusine residue is produced by a posttranslational modification involving deoxyhypusine synthetase and deoxyhypusine hydroxylase. Herein, we aimed to describe the role of the alternative human isoform A on mitochondrial processes. Isoform A depletion modulates oxidative metabolism in association with the downregulation of mitochondrial biogenesis-related genes. Through positive feedback, it increases cell respiration leading to highly reactive oxygen species production, which impacts mitochondrial bioenergetics. These metabolic changes compromise mitochondrial morphology, increasing its electron density and fission, observed by transmission electron microscopy. This set of changes leads the cells to apoptosis, evidenced by increased DNA fragmentation and proapoptotic BAK protein content increase. Thus, we show that the alternative eIF5A isoform A is crucial for energy metabolism controlled by mitochondria and cellular survival.

Regulation of Lin28a-miRNA let-7b-5p pathway in skeletal muscle cells by peroxisome proliferator-activated receptor delta.

Lin28a/miRNA let-7b-5p pathway has emerged as a key regulators of energy homeostasis in the skeletal muscle. However, the mechanism through which this pathway is regulated in the skeletal muscle has remained unclear. We have found that 8 wk of aerobic training (Tr) markedly decreased let-7b-5p expression in murine skeletal muscle, whereas high-fat diet (Hfd) increased its expression. Conversely, Lin28a expression, a well-known inhibitor of let-7b-5p, was induced by Tr and decreased by Hfd. Similarly, in human muscle biopsies, Tr increased LIN28 expression and decreased let-7b-5p expression. Bioinformatics analysis of LIN28a DNA sequence revealed that its enrichment in peroxisome proliferator-activated receptor delta (PPARδ) binding sites, which is a well-known metabolic regulator of exercise. Treatment of primary mouse skeletal muscle cells or C2C12 cells with PPARδ activators GW501516 and AICAR increased Lin28a expression. Lin28a and let-7b-5p expression was also regulated by PPARδ coregulators. While PPARγ coactivator-1α (PGC1α) increased Lin28a expression, corepressor NCoR1 decreased its expression. Furthermore, PGC1α markedly reduced the let-7b-5p expression. PGC1α-mediated induction of Lin28a expression was blocked by the PPARδ inhibitor GSK0660. In agreement, Lin28a expression was downregulated in PPARδ knocked-down cells leading to increased let-7b-5p expression. Finally, we show that modulation of the Lin28a-let-7b-5p pathway in muscle cells leads to changes in mitochondrial metabolism in PGC1α dependent fashion. In summary, we demonstrate that Lin28a-let-7b-5p is a direct target of PPARδ in the skeletal muscle, where it impacts mitochondrial respiration.

NEK10 interactome and depletion reveal new roles in mitochondria.

BACKGROUND: Members of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions. METHODS: HEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established. RESULTS: Here, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content. CONCLUSIONS: Taken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.

Leucine-rich diet induces a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, reducing glucose consumption and metastasis in Walker-256 tumour-bearing rats.

Leucine can stimulate protein synthesis in skeletal muscle, and recent studies have shown an increase in leucine-related mitochondrial biogenesis and oxidative phosphorylation capacity in muscle cells. However, leucine-related effects in tumour tissues are still poorly understood. Thus, we described the effects of leucine in both in vivo and in vitro models of a Walker-256 tumour. Tumour-bearing Wistar rats were randomly distributed into a control group (W; normoprotein diet) and leucine group (LW; leucine-rich diet [normoprotein + 3% leucine]). After 20 days of tumour evolution, the animals underwent 18-fludeoxyglucose positron emission computed tomography (18F-FDG PET-CT) imaging, and after euthanasia, fresh tumour biopsy samples were taken for oxygen consumption rate measurements (Oroboros Oxygraph), electron microscopy analysis and RNA and protein extraction. Our main results from the LW group showed no tumour size change, lower tumour glucose (18F-FDG) uptake, and reduced metastatic sites. Furthermore, leucine stimulated a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, higher mRNA and protein expression of oxidative phosphorylation components, and enhanced mitochondrial density/area even though the leucine-treated tumour had a higher number of apoptotic nuclei with increased oxidative stress. In summary, a leucine-rich diet directed Walker-256 tumour metabolism to a less glycolytic phenotype profile in which these metabolic alterations were associated with a decrease in tumour aggressiveness and reduction in the number of metastatic sites in rats fed a diet supplemented with this branched-chain amino acid.

Opposing action of NCoR1 and PGC-1α in mitochondrial redox homeostasis.

The ability to respond to fluctuations of reactive oxygen species (ROS) within the cell is a central aspect of mammalian physiology. This dynamic process depends on the coordinated action of transcriptional factors to promote the expression of genes encoding for antioxidant enzymes. Here, we demonstrate that the transcriptional coregulators, PGC-1α and NCoR1, are essential mediators of mitochondrial redox homeostasis in skeletal muscle cells. Our findings reveal an antagonistic role of these coregulators in modulating mitochondrial antioxidant induction through Sod2 transcriptional control. Importantly, the activation of this mechanism by either PGC-1α overexpression or NCoR1 knockdown attenuates mitochondrial ROS levels and prevents cell death caused by lipid overload in skeletal muscle cells. The opposing actions of coactivators and corepressors, therefore, exert a commanding role over cellular antioxidant capacity.

Essential role of the PGC-1α/PPARß axis in Ucp3 gene induction.

KEY POINTS: We report that the peroxisome proliferator-activated receptor (PPAR)γ coactivator 1-α (PGC-1α)/PPARß axis is a crucial mediator of uncoupling protein 3 (UCP3) expression in skeletal muscle cells via the transactivativation of a distal PPAR response element at the Ucp3 gene promoter. This mechanism is activated during the myogenic process and by high concentrations of fatty acids independent of PGC-1α protein levels. Ucp3 is essential for PGC-1α-induced oxidative capacity and the adaptive mitochondrial response to fatty acid exposure. These findings provide further evidence for the broad spectrum of the coactivator action in mitochondrial homeostasis, positioning the PGC-1ɑ/PPARß axis as an essential component of the molecular regulation of Ucp3 gene in skeletal muscle cells. ABSTRACT: Uncoupling protein 3 (UCP3) has an essential role in fatty acid metabolism and mitochondrial redox regulation in skeletal muscle. However, the molecular mechanisms involved in the expression of Ucp3 are poorly known. In the present study, we show that the peroxisome proliferator-activated receptor (PPAR)γ coactivator 1-α (PGC-1α)/PPARß axis is a crucial mediator of Ucp3 expression in skeletal muscle cells. In silico analysis of the UCP3 promoter and quantitative chromatin immunoprecipitation experiments revealed that the induction of the UCP3 transcript is mediated by the transactivation of a distal PPAR response element at the Ucp3 gene promoter by the coactivator PGC-1α. This mechanism is activated during myogenesis and during metabolic stress induced by fatty acids independent of PGC-1α protein levels. We also provide evidence that Ucp3 is essential for PGC-1α-induced oxidative capacity. Taken together, our results highlight PGC-1ɑ/PPARß as an essential component of the molecular regulation of Ucp3 gene in skeletal muscle cells.

Abnormal brown adipose tissue mitochondrial structure and function in IL10 deficiency.

BACKGROUND: Inflammation is the most relevant mechanism linking obesity with insulin-resistance and metabolic disease. It impacts the structure and function of tissues and organs involved in metabolism, such as the liver, pancreatic islets and the hypothalamus. Brown adipose tissue has emerged as an important component of whole body energy homeostasis, controlling caloric expenditure through the regulation of non-shivering thermogenesis. However, little is known about the impact of systemic inflammation on the structure and function of brown adipose tissue. METHODS: The relations between IL10 and mitochondria structure/function and also with thermogenesis were evaluated by bioinformatics using human and rodent data. Real-time PCR, immunoblot, fluorescence and transmission electron microscopy were employed to determine the effect of IL10 in the brown adipose tissue of wild type and IL10 knockout mice. FINDINGS: IL10 knockout mice, a model of systemic inflammation, present severe structural abnormalities of brown adipose tissue mitochondria, which are round-shaped with loss of cristae structure and increased fragmentation. IL10 deficiency leads to newborn cold intolerance and impaired UCP1-dependent brown adipose tissue mitochondrial respiration. The reduction of systemic inflammation with an anti-TNFα monoclonal antibody partially rescued the structural but not the functional abnormalities of brown adipose tissue mitochondria. Using bioinformatics analyses we show that in both humans and mice, IL10 transcripts correlate with mitochondrial lipid metabolism and caspase gene expression. INTERPRETATION: IL10 and systemic inflammation play a central role in the regulation of brown adipose tissue by controlling mitochondrial structure and function. FUND: Sao Paulo Research Foundation grant 2013/07607-8.

Role of NCoR1 in mitochondrial function and energy metabolism.

Mitochondrial number and shape are constantly changing in response to increased energy demands. The ability to synchronize mitochondrial pathways to respond to energy fluctuations within the cell is a central aspect of mammalian homeostasis. This dynamic process depends on the coordinated activation of transcriptional complexes to promote the expression of genes encoding for mitochondrial proteins. Recent evidence has shown that the nuclear corepressor NCoR1 is an essential metabolic switch which acts on oxidative metabolism signaling. Here, we provide an overview of the emerging role of NCoR1 in the transcriptional control of energy metabolism. The identification and characterization of NCoR1 as a central, evolutionary conserved player in mitochondrial function have revealed a novel layer of metabolic control. Defining the precise mechanisms by which NCoR1 acts on energy homeostasis will ultimately contribute towards the development of novel therapies for the treatment of metabolic diseases such as obesity and type 2 diabetes.

A microplate assay for measuring cell death in C2C12 cells.

The main goal of this study was to develop a straightforward and rapid microplate assay for measuring propidium iodide (PI) in C2C12 cells. The PI method has proven to be an efficient quantitative assay for analyzing cell viability through PI fluorescence analysis. Importantly, the protocol takes less than 30 min and the results are reproducible. C2C12 cells were exposed to an increasing concentration of palmitate for a period of 24 h to induce cell death, and the PI fluorescence increased in a concentration-dependent manner. Evaluation of mitochondrial function and the production of reactive oxygen species confirmed the deleterious effects of palmitate. Also, the microplate PI assay demonstrated high sensitivity, as indicated by the detection of modest fluctuations in cell viability in response to catalase overexpression in palmitate-treated cells. The microplate PI assay, therefore, offers an accurate method for use in in-vitro studies.

Role of microRNAs on the Regulation of Mitochondrial Biogenesis and Insulin Signaling in Skeletal Muscle.

Mitochondria play a critical role in several cellular processes and cellular homeostasis. Mitochondrion dysfunction has been correlated with numerous metabolic diseases such as obesity and type 2 diabetes. MicroRNAs are non-coding RNAs that have emerged as key regulators of cell metabolism. The microRNAs act as central regulators of metabolic gene networks by leading to the degradation of their target messenger RNA or repression of protein translation. In addition, vesicular and non-vesicular circulating miRNAs exhibit a potential role as mediators of the cross-talk between the skeletal muscle and other tissues/organs. In this review, we will focus on the emerging knowledge of miRNAs controlling mitochondrial function and insulin signaling in skeletal muscle cells. J. Cell. Physiol. 232: 958-966, 2017. © 2016 Wiley Periodicals, Inc.