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1.
Sci Rep ; 13(1): 18943, 2023 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-37919323

RESUMEN

Skeletal muscle fibers regulate surrounding endothelial cells (EC) via secretion of numerous angiogenic factors, including extracellular vesicles (SkM-EV). Muscle fibers are broadly classified as oxidative (OXI) or glycolytic (GLY) depending on their metabolic characteristics. OXI fibers secrete more pro-angiogenic factors and have greater capillary densities than GLY fibers. OXI muscle secretes more EV than GLY, however it is unknown whether muscle metabolic characteristics regulate EV contents and signaling potential. EVs were isolated from primarily oxidative or glycolytic muscle tissue from mice. MicroRNA (miR) contents were determined and endothelial cells were treated with OXI- and GLY-EV to investigate angiogenic signaling potential. There were considerable differences in miR contents between OXI- and GLY-EV and pathway analysis identified that OXI-EV miR were predicted to positively regulate multiple endothelial-specific pathways, compared to GLY-EV. OXI-EV improved in vitro angiogenesis, which may have been mediated through nitric oxide synthase (NOS) related pathways, as treatment of endothelial cells with a non-selective NOS inhibitor abolished the angiogenic benefits of OXI-EV. This is the first report to show widespread differences in miR contents between SkM-EV isolated from metabolically different muscle tissue and the first to demonstrate that oxidative muscle tissue secretes EV with greater angiogenic signaling potential than glycolytic muscle tissue.


Asunto(s)
Vesículas Extracelulares , MicroARNs , Animales , Ratones , Células Endoteliales/metabolismo , Músculo Esquelético/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Vesículas Extracelulares/metabolismo , Estrés Oxidativo
2.
Mol Metab ; 66: 101618, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36283677

RESUMEN

OBJECTIVES: Insulin treatment remains the sole effective intervention for Type 1 Diabetes. Here, we investigated the therapeutic potential of converting intestinal epithelial cells to insulin-producing, glucose-responsive ß-like cells by targeted inhibition of FOXO1. We have previously shown that this can be achieved by genetic ablation in gut Neurogenin3 progenitors, adenoviral or shRNA-mediated inhibition in human gut organoids, and chemical inhibition in Akita mice, a model of insulin-deficient diabetes. METHODS: We profiled two novel FOXO1 inhibitors in reporter gene assays, and hepatocyte gene expression studies, and in vivo pyruvate tolerance test (PTT) for their activity and specificity. We evaluated their glucose-lowering effect in mice rendered insulin-deficient by administration of streptozotocin. RESULTS: We provide evidence that two novel FOXO1 inhibitors, FBT432 and FBT374 have glucose-lowering and gut ß-like cell-inducing properties in mice. FBT432 is also highly effective in combination with a Notch inhibitor in this model. CONCLUSION: The data add to a growing body of evidence suggesting that FOXO1 inhibition be pursued as an alternative treatment to insulin administration in diabetes.


Asunto(s)
Diabetes Mellitus Experimental , Proteína Forkhead Box O1 , Animales , Ratones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Proteína Forkhead Box O1/antagonistas & inhibidores , Glucosa/metabolismo , Insulina/metabolismo , Estreptozocina
3.
Exp Physiol ; 107(8): 906-918, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35561231

RESUMEN

NEW FINDINGS: What is the central question of this study? Do obesity and acute resistance exercise alter the regulation of muscle intercellular communication pathways consistent with inadequate compensatory angiogenesis in response to muscle loading present in individuals with obesity? What is the main finding and its importance? Obesity is associated with differences in both pro- and anti-angiogenic signalling consistent with lower muscle capillarization. Acute resistance exercise increases the release of skeletal muscle small extracellular vesicles independent of body mass. These results identify new cellular factors associated with impaired angiogenesis in obesity and the positive effects of acute resistance exercise in lean and obese skeletal muscle. ABSTRACT: Obesity (OB) impairs cell-to-cell communication signalling. Small extracellular vesicles (EVs), which include exosomes, are released by skeletal muscle and participate in cell-to-cell communication, including the regulation of angiogenesis. Resistance exercise (REx) increases muscle fibre size and capillarization. Although obesity increases muscle fibre size, there is an inadequate increase in capillarization such that capillary density is reduced. It was hypothesized that REx-induced angiogenic signalling and EV biogenesis would be lower with obesity. Sedentary lean (LN) and OB subjects (n = 8 per group) performed three sets of single-leg knee-extension REx at 80% of maximum. Muscle biopsies were obtained at rest, 15 min and 3 h postexercise and analysed for angiogenic and EV biogenesis mRNA and protein. In OB subjects, muscle fibre size was ∼20% greater and capillary density with type II fibres ∼25% lower compared with LN subjects (P < 0.001). In response to REx, the increase in VEGF mRNA (pro-angiogenic) was similar (3-fold) between groups, while thrombospondin-1 (TSP-1) mRNA (anti-angiogenic) increased ∼2.5-fold in OB subjects only (P = 0.010). miR-130a (pro-angiogenic) was ∼1.4-fold (P = 0.011) and miR-503 (anti-angiogenic) ∼1.8-fold (P = 0.017) greater in OB compared with LN subjects at all time points. In both groups, acute REx decreased the EV surface protein Alix by ∼50%, consistent with the release of exosomes (P = 0.016). Acute REx appears to induce the release of skeletal muscle small EVs independent of body mass. However, with obesity there is predominantly impaired angiogenic signalling, consistent with inadequate angiogenesis in response to basal muscle hypertrophy.


Asunto(s)
Músculo Esquelético , Neovascularización Fisiológica , Obesidad , Entrenamiento de Fuerza , Humanos , MicroARNs/metabolismo , Músculo Esquelético/fisiología , Obesidad/metabolismo , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
4.
Exp Physiol ; 107(5): 462-475, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35293040

RESUMEN

NEW FINDINGS: What is the central question of this study? Is 1 week of exercise training sufficient to reduce local and systemic inflammation? Do obesity and short-term concurrent aerobic and resistance exercise training alter skeletal muscle extracellular vesicle (EV) contents? What is the main finding and its importance? Obesity alters skeletal muscle small EV microRNAs targeting inflammatory and growth pathways. Exercise training alters skeletal muscle small EV microRNAs targeting inflammatory pathways, indicative of reduced inflammation. Our findings provide support for the hypotheses that EVs play a vital role in intercellular communication during health and disease and that EVs mediate many of the beneficial effects of exercise. ABSTRACT: Obesity is associated with chronic inflammation characterized by increased levels of inflammatory cytokines, whereas exercise training reduces inflammation. Small extracellular vesicles (EVs; 30-150 nm) participate in cell-to-cell communication in part through microRNA (miRNA) post-transcriptional regulation of mRNA. We examined whether obesity and concurrent aerobic and resistance exercise training alter skeletal muscle EV miRNA content and inflammatory signalling. Vastus lateralis biopsies were obtained from sedentary individuals with (OB) and without obesity (LN). Before and after 7 days of concurrent aerobic and resistance training, muscle-derived small EV miRNAs and whole-muscle mRNAs were measured. Pathway analysis revealed that obesity alters small EV miRNAs that target inflammatory (SERPINF1, death receptor and Gαi ) and growth pathways (Wnt/ß-catenin, PTEN, PI3K/AKT and IGF-1). In addition, exercise training alters small EV miRNAs in an anti-inflammatory manner, targeting the IL-10, IL-8, Toll-like receptor and nuclear factor-κB signalling pathways. In whole muscle, IL-8 mRNA was reduced by 50% and Jun mRNA by 25% after exercise training, consistent with the anti-inflammatory effects of exercise on skeletal muscle. Obesity and 7 days of concurrent exercise training differentially alter skeletal muscle-derived small EV miRNA contents targeting inflammatory and anabolic pathways.


Asunto(s)
Vesículas Extracelulares , MicroARNs , Ejercicio Físico/fisiología , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Humanos , Inflamación/metabolismo , Interleucina-8/metabolismo , MicroARNs/genética , Músculo Esquelético/fisiología , Obesidad/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Mensajero/metabolismo
5.
Cell Stem Cell ; 29(1): 101-115.e10, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34582804

RESUMEN

Adult stem cells maintain regenerative tissue structure and function by producing tissue-specific progeny, but the factors that preserve their tissue identities are not well understood. The small and large intestines differ markedly in cell composition and function, reflecting their distinct stem cell populations. Here we show that SATB2, a colon-restricted chromatin factor, singularly preserves LGR5+ adult colonic stem cell and epithelial identity in mice and humans. Satb2 loss in adult mice leads to stable conversion of colonic stem cells into small intestine ileal-like stem cells and replacement of the colonic mucosa with one that resembles the ileum. Conversely, SATB2 confers colonic properties on the mouse ileum. Human colonic organoids also adopt ileal characteristics upon SATB2 loss. SATB2 regulates colonic identity in part by modulating enhancer binding of the intestinal transcription factors CDX2 and HNF4A. Our study uncovers a conserved core regulator of colonic stem cells able to mediate cross-tissue plasticity in mature intestines.


Asunto(s)
Colon , Íleon , Animales , Mucosa Intestinal , Ratones , Organoides , Células Madre
6.
Eur J Appl Physiol ; 120(12): 2835-2846, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32989478

RESUMEN

PURPOSE: Obesity is associated with numerous changes in skeletal muscle including greater muscle mass and muscle fiber cross sectional area (FCSA), yet fasted muscle protein synthesis is lower. Activation of the IGF-1/Akt/mTOR pathway is critical for muscle mass maintenance, muscle hypertrophy, and muscle protein regulation. Resistance exercise (RE) increases muscle mass, FCSA, and IGF-1. Persons with obesity have greater skeletal muscle mass and larger skeletal muscle fiber cross sectional area. The IGF-1/Akt/mTOR pathway is critical for the regulation of skeletal muscle mass. Our study found men and women with obesity have lower skeletal muscle IGF-1 mRNA and protein and higher expression of miR-206 an epigenetic regulator of IGF-1, at rest and following an acute bout of resistance exercise. Despite this, Akt mediated signaling was maintained and maintenance of phosphorylation does not appear to be accounted for by compensatory pathways. Our findings suggest a possible negative feedback mechanism via increased miR-206 and in turn decreased IGF-1 to limit further skeletal muscle hypertrophy in persons with obesity. The current work investigated if: (1) obesity dysregulates basal skeletal muscle IGF-1 pathways; and (2) obesity augments the muscle IGF-1 pathway responses to acute RE. METHODS: Eight sedentary (no self-reported physical activity), lean (LN) and eight sedentary subjects with obesity (OB) had vastus lateralis biopsies taken at rest, and 15 min and 3 h post-acute RE for the measurement of the IGF-1 pathway and muscle FCSA. RESULTS: Type II FCSA was larger in OB vs. LN. Skeletal muscle IGF-1 mRNA and IGF-1 protein were lower in OB vs. LN at rest and post-exercise. Acute RE increased IGF-1 protein similarly in both groups. The expression of miR-206, a post-transcriptional inhibitor of IGF-1 expression, was higher in OB vs. LN and linked with lower IGF-1 mRNA (r = - 0.54). CONCLUSION: In spite of greater muscle FCSA, muscle IGF-1 expression was lower in obesity suggesting negative feedback may be limiting muscle mass expansion in obesity.


Asunto(s)
Ejercicio Físico/fisiología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Femenino , Humanos , Masculino , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Fosforilación/fisiología , ARN Mensajero/metabolismo , Entrenamiento de Fuerza/métodos , Transducción de Señal/fisiología
7.
Exp Physiol ; 105(3): 511-521, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31917487

RESUMEN

NEW FINDINGS: What is the central question of this study? What is the impact of acute aerobic and aerobic + resistance (concurrent) exercise on the regulation of multivesicular body formation in human skeletal muscle? What is the main finding and its importance? Gene expression for proteins associated with multivesicular body biogenesis was increased in response to concurrent exercise, and gene expression of microRNA processing (genetic information) was increased in response to aerobic and concurrent exercise. A greater understanding of the processing of multivesicular bodies in response to acute exercise may lead to novel treatments focused on intercellular communication pathways. ABSTRACT: Regular aerobic exercise (AEx) and resistance exercise (REx) promote many beneficial adaptations. Skeletal muscle participates in intercellular communication in part through the release of myokines and extracellular vesicles including exosomes (EXOs), the latter containing mRNA, microRNA (miRNA), lipids and proteins. Exercise-induced regulation of skeletal muscle multivesicular body (MVB) biogenesis leading to EXO formation and release is poorly understood. We hypothesized that acute exercise would increase skeletal muscle MVB biogenesis and EXO release pathways with a greater response to aerobic + resistance exercise (A+REx) than to AEx alone. Twelve sedentary, healthy male subjects exercised on a cycle ergometer for 45 min (AEx) followed by single leg, knee extensor, resistance exercise (A+REx). Vastus lateralis biopsies were obtained at rest and 1 h post-exercise. Key components of the MVB biogenesis, EXO biogenesis and release, and miRNA processing pathways were analysed. Clathrin and Alix mRNA (MVB biogenesis) were increased by A+REx, while DICER and exportin mRNA (miRNA processing) were increased by AEx and A+REx. There were positive relationships between MVBs and miRNA processing genes following both AEx and A+REx consistent with coordinated regulation of these interrelated processes (Alix mRNA increased with Drosha, exportin and Dicer mRNA). Acute exercise increases the regulation of components of MVB and EXO pathways as well as miRNA processing components. A greater understanding of the production and packaging of skeletal muscle MVBs, EXOs and mature miRNA could lead to novel treatments focused on intercellular communication.


Asunto(s)
Ejercicio Físico/fisiología , Exosomas/metabolismo , Exosomas/fisiología , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/fisiología , Transducción de Señal/fisiología , Adolescente , Adulto , Humanos , Masculino , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Músculo Cuádriceps/fisiología , ARN Mensajero/metabolismo , Adulto Joven
8.
J Physiol ; 597(20): 5109-5124, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31471968

RESUMEN

KEY POINTS: Cellular communication occurs between endothelial cells and skeletal muscle satellite cells and is mitogenic for both cell types under normal conditions. Skeletal muscle atrophy and endothelial cell dysfunction occur in tandem in cardiovascular disease, type II diabetes and ageing. The present study investigated how induction of endothelial cell dysfunction via high glucose treatment impacts growth and differentiation of human skeletal muscle satellite cells in vitro. Secreted factors from high glucose treated endothelial cells impaired satellite cell expansion and differentiation via decreased proliferation and dysregulation of p38 mitogen-activated protein kinase in satellite cells committed to myogenesis. These findings highlight a novel potential role for endothelial cells in the development and pathology of skeletal muscle atrophy, which is common in patients with endothelial dysfunction related pathologies. ABSTRACT: Cross-talk between endothelial cells (ECs) and skeletal muscle satellite cells (MuSC) has been identified as an important regulator of cellular functions in both cell types. In healthy conditions, EC secreted factors promote MuSC growth and differentiation. Endothelial and satellite cell dysfunction occur in tandem in many disease states; however, no data exist examining the impact of dysfunctional EC signalling on satellite cells. Therefore, the present study aimed to evaluate the effect that factors secreted from high glucose (HG) treated ECs have on the growth and differentiation of human satellite cells (HMuSC) using a conditioned medium (CM) cell culture model. Satellite cells were isolated from human skeletal muscle and grown in CM from normal or HG treated human umbilical vein ECs (HUVECs). Satellite cells grown in CM from HG treated HUVECs reduced growth (25%), differentiation (25%) and myonuclear fusion (35%). These responses were associated with increased superoxide (50%) and inflammatory cytokines (25-50%) in HG treated HUVECs and HG-CM. Decreased expansion of HG-CM treated HMuSCs was driven by a decrease in proliferation. Impaired gene expression and protein content of myogenic differentiation factors were preceded by decreased phosphorylation of p38 mitogen-activated protein kinase in HMuSC treated with CM from HG treated HUVECs. The results obtained in the present study are the first to show that factors secreted from HG treated ECs cause impairments in human muscle satellite cell growth and differentiation in vitro, highlighting endothelial cell health and secretion as a potential target for treating vascular disease-associated skeletal muscle dysfunction.


Asunto(s)
Glucosa/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Células Satélite del Músculo Esquelético/efectos de los fármacos , Adulto , Diferenciación Celular , Células Cultivadas , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Fibras Musculares Esqueléticas/fisiología , Células Satélite del Músculo Esquelético/fisiología , Adulto Joven
9.
Elife ; 82019 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-31393265

RESUMEN

Muscle development and regeneration require delicate cell cycle regulation of embryonic myoblasts and adult muscle satellite cells (MuSCs). Through analysis of the Polo-like kinase (Plk) family cell-cycle regulators in mice, we show that Plk1's expression closely mirrors myoblast dynamics during embryonic and postnatal myogenesis. Cell-specific deletion of Plk1 in embryonic myoblasts leads to depletion of myoblasts, developmental failure and prenatal lethality. Postnatal deletion of Plk1 in MuSCs does not perturb their quiescence but depletes activated MuSCs as they enter the cell cycle, leading to regenerative failure. The Plk1-null MuSCs are arrested at the M-phase, accumulate DNA damage, and apoptose. Mechanistically, Plk1 deletion upregulates p53, and inhibition of p53 promotes survival of the Plk1-null myoblasts. Pharmacological inhibition of Plk1 similarly inhibits proliferation but promotes differentiation of myoblasts in vitro, and blocks muscle regeneration in vivo. These results reveal for the first time an indispensable role of Plk1 in developmental and regenerative myogenesis.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Desarrollo de Músculos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Regeneración , Animales , Células Cultivadas , Ratones , Mioblastos/fisiología , Células Satélite del Músculo Esquelético/fisiología , Quinasa Tipo Polo 1
10.
J Biol Chem ; 294(37): 13718-13728, 2019 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-31346037

RESUMEN

Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21cLacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. We further show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization.


Asunto(s)
Proteínas del Choque Térmico HSC70/metabolismo , Metiltransferasas/metabolismo , Miofibrillas/metabolismo , Animales , Autofagia , Femenino , Técnicas de Sustitución del Gen/métodos , Células HEK293 , Proteínas del Choque Térmico HSC70/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Factores de Transcripción MEF2/genética , Masculino , Metilación , Metiltransferasas/genética , Ratones , Desarrollo de Músculos/genética , Músculos/metabolismo , Mioblastos/metabolismo , Miofibrillas/genética , Procesamiento Proteico-Postraduccional
11.
FASEB J ; 33(8): 9672-9684, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31162944

RESUMEN

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation.-Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Metiltransferasas/metabolismo , Atrofia Muscular/metabolismo , Miofibrillas/metabolismo , Animales , Western Blotting , Bortezomib/uso terapéutico , Diferenciación Celular/genética , Células Cultivadas , Femenino , Inmunoprecipitación , Metiltransferasas/genética , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Atrofia Muscular/patología , Mutación/genética , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Miofibrillas/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN
12.
J R Soc Interface ; 16(155): 20190079, 2019 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-31213169

RESUMEN

Low-intensity electric fields can induce changes in cell differentiation and cytoskeletal stresses that facilitate manipulation of osteoblasts and mesenchymal stem cells; however, the application times (tens of minutes) are of the order of physiological mechanisms, which can complicate treatment consistency. Intense nanosecond pulsed electric fields (nsPEFs) can overcome these challenges by inducing similar stresses on shorter timescales while additionally inducing plasma membrane nanoporation, ion transport and intracellular structure manipulation. This paper shows that treating myoblasts and osteoblasts with five 300 ns PEFs with intensities from 1.5 to 25 kV cm-1 increased proliferation and differentiation. While nsPEFs above 5 kV cm-1 decreased myoblast population growth, 10 and 20 kV cm-1 trains increased myoblast population by approximately fivefold 48 h after exposure when all cell densities were set to the same level after exposure. Three trials of the PEF-treated osteoblasts showed that PEF trains between 2.5 and 10 kV cm-1 induced the greatest population growth compared to the control 48 h after treatment. Trains of nsPEFs between 1.5 and 5 kV cm-1 induced the most nodule formation in osteoblasts, indicating bone formation. These results demonstrate the potential utility for nsPEFs to rapidly modulate stem cells for proliferation and differentiation and motivate future experiments to optimize PEF parameters for in vivo applications.


Asunto(s)
Diferenciación Celular , Proliferación Celular , Electricidad , Mioblastos/metabolismo , Osteoblastos/metabolismo , Animales , Humanos , Ratones , Mioblastos/citología , Osteoblastos/citología
13.
Exp Physiol ; 104(8): 1262-1273, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31115069

RESUMEN

NEW FINDINGS: What is the central question of this study? Capillary rarefaction is found in diabetic and aged muscle, whereas exercise increases skeletal muscle angiogenesis. The association implies a crosstalk between muscle cells and endothelial cells. The underlying mechanisms mediating the crosstalk between these cells remains to be elucidated fully. What is the main finding and its importance? Endothelial cell functions are regulated by skeletal muscle cell-derived exosomes via a vascular endothelial growth factor-independent pathway. This study reveals a new mechanism mediating the crosstalk between skeletal muscle cells and endothelial cells. ABSTRACT: Loss of skeletal muscle capillarization, known as capillary rarefaction, is found in type 2 diabetes, chronic heart failure and healthy ageing and is associated with impaired delivery of substrates to the muscle. However, the interaction and communication of skeletal muscle with endothelial cells in the regulation of capillaries surrounding the muscle remains elusive. Exosomes are a type of secreted extracellular vesicle containing mRNAs, proteins and, especially, microRNAs that exert paracrine and endocrine effects. In this study, we investigated whether skeletal muscle-derived exosomes (SkM-Exo) regulate the endothelial cell functions of angiogenesis. We demonstrated that C2C12 myotube-derived exosomes improved endothelial cell functions, assessed by the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs), which were increased by 20, 23 and 40%, respectively, after SkM-Exo exposure. The SkM-Exo failed to activate HUVEC vascular endothelial growth factor (VEGF) signalling. The SkM-Exo increased HUVEC reactive oxygen species and activated the nuclear factor-κB pathway, suggesting that SkM-Exo-induced angiogenesis was mediated by a VEGF-independent pathway. In addition, several angiogenic microRNAs were packaged in SkM-Exo, with miR-130a being particularly enriched and successfully transferred from SkM-Exo to HUVECs. Delivery of miRNAs into endothelial cells might explain the enhancement of reactive oxygen species production and angiogenesis by SkM-Exo. The potential angiogenic effect of SkM-Exo could provide an effective therapy for promoting skeletal muscle angiogenesis in diseases characterized by capillary rarefaction or inadequate angiogenesis.


Asunto(s)
Exosomas/metabolismo , Exosomas/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Diabetes Mellitus Tipo 2 , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo
14.
Diabetes ; 67(4): 624-635, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29317435

RESUMEN

Stress responses promote obesity and insulin resistance, in part, by activating the stress-responsive mitogen-activated protein kinases (MAPKs), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Stress also induces expression of MAPK phosphatase-1 (MKP-1), which inactivates both JNK and p38 MAPK. However, the equilibrium between JNK/p38 MAPK and MKP-1 signaling in the development of obesity and insulin resistance is unclear. Skeletal muscle is a major tissue involved in energy expenditure and glucose metabolism. In skeletal muscle, MKP-1 is upregulated in high-fat diet-fed mice and in skeletal muscle of obese humans. Mice lacking skeletal muscle expression of MKP-1 (MKP1-MKO) showed increased skeletal muscle p38 MAPK and JNK activities and were resistant to the development of diet-induced obesity. MKP1-MKO mice exhibited increased whole-body energy expenditure that was associated with elevated levels of myofiber-associated mitochondrial oxygen consumption. miR-21, a negative regulator of PTEN expression, was upregulated in skeletal muscle of MKP1-MKO mice, resulting in increased Akt activity consistent with enhanced insulin sensitivity. Our results demonstrate that skeletal muscle MKP-1 represents a critical signaling node through which inactivation of the p38 MAPK/JNK module promotes obesity and insulin resistance.


Asunto(s)
Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Resistencia a la Insulina , MAP Quinasa Quinasa 4/metabolismo , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Dieta Alta en Grasa , Metabolismo Energético , Humanos , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Mitocondrias Musculares/metabolismo , Consumo de Oxígeno , Transducción de Señal
15.
Diabetes ; 67(3): 400-411, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29298809

RESUMEN

Sucrose nonfermenting-related kinase (SNRK) is a member of the AMPK-related kinase family, and its physiological role in adipose energy homeostasis and inflammation remains unknown. We previously reported that SNRK is ubiquitously and abundantly expressed in both white adipose tissue (WAT) and brown adipose tissue (BAT), but SNRK expression diminishes in adipose tissue in obesity. In this study we report novel experimental findings from both animal models and human genetics. SNRK is essential for survival; SNRK globally deficient pups die within 24 h after birth. Heterozygous mice are characterized by inflamed WAT and less BAT. Adipocyte-specific ablation of SNRK causes inflammation in WAT, ectopic lipid deposition in liver and muscle, and impaired adaptive thermogenesis in BAT. These metabolic disorders subsequently lead to decreased energy expenditure, higher body weight, and insulin resistance. We further confirm the significant association of common variants of the SNRK gene with obesity risk in humans. Through applying a phosphoproteomic approach, we identified eukaryotic elongation factor 1δ and histone deacetylase 1/2 as potential SNRK substrates. Taking these data together, we conclude that SNRK represses WAT inflammation and is essential to maintain BAT thermogenesis, making it a novel therapeutic target for treating obesity and associated metabolic disorders.


Asunto(s)
Adipocitos Marrones/metabolismo , Adipocitos Blancos/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Paniculitis/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adipocitos Marrones/inmunología , Adipocitos Marrones/patología , Adipocitos Marrones/ultraestructura , Adipocitos Blancos/inmunología , Adipocitos Blancos/patología , Adipocitos Blancos/ultraestructura , Animales , Índice de Masa Corporal , Células Cultivadas , Cruzamientos Genéticos , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias/inmunología , Mitocondrias/patología , Mitocondrias/ultraestructura , Obesidad/genética , Obesidad/fisiopatología , Paniculitis/etiología , Paniculitis/inmunología , Paniculitis/patología , Polimorfismo de Nucleótido Simple , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Termogénesis
16.
J Clin Endocrinol Metab ; 103(3): 882-889, 2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29155999

RESUMEN

Context: Almost 50% of type 2 diabetic (T2D) patients are poorly controlled [glycated hemoglobin (HbA1c) ≥ 7%]; however, the mechanisms responsible for progressively worsening glycemic control are poorly understood. Lower skeletal muscle mitochondrial respiratory capacity is associated with low insulin sensitivity and the development of T2D. Objective: We investigated if skeletal muscle insulin sensitivity (SI) was different between well-controlled T2D (WCD) and poorly controlled T2D (PCD) and if the difference was associated with differences resulting from mitochondrial respiratory function. Design: Vastus lateralis muscle mitochondrial respiration, mitochondrial content, mitochondrial enzyme activity, and fatty acid oxidation (FAO) were measured. SI and the acute response to glucose (AIRg) were calculated by MINMOD analysis from glucose and insulin obtained during a modified, frequently sampled, intravenous glucose tolerance test. Results: SI and AIRg were lower in PCD than WCD. Muscle incomplete FAO was greater in PCD than WCD and greater incomplete FAO was associated with lower SI and higher HbA1c. Hydroxyacyl-coenzyme A dehydrogenase expression and activity were greater in PCD than WCD. There was no difference in maximal mitochondrial respiration or content between WCD and PCD. Conclusion: The current results suggest that greater skeletal muscle incomplete FAO in poorly controlled T2D is due to elevated ß oxidation and is associated with worsening muscle SI.


Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Ácidos Grasos/metabolismo , Resistencia a la Insulina/fisiología , Músculo Esquelético/metabolismo , Respiración de la Célula/fisiología , Diabetes Mellitus Tipo 2/sangre , Femenino , Prueba de Tolerancia a la Glucosa , Hemoglobina Glucada/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias Musculares/fisiología , Oxidación-Reducción
17.
EBioMedicine ; 16: 212-223, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-28117277

RESUMEN

Brown adipose tissue (BAT) represents a promising agent to ameliorate obesity and other metabolic disorders. However, the abundance of BAT decreases with age and BAT paucity is a common feature of obese subjects. As brown adipocytes and myoblasts share a common Myf5 lineage origin, elucidating the molecular mechanisms underlying the fate choices of brown adipocytes versus myoblasts may lead to novel approaches to expand BAT mass. Here we identify MyoD as a key negative regulator of brown adipocyte development. CRISPR/CAS9-mediated deletion of MyoD in C2C12 myoblasts facilitates their adipogenic transdifferentiation. MyoD knockout downregulates miR-133 and upregulates the miR-133 target Igf1r, leading to amplification of PI3K-Akt signaling. Accordingly, inhibition of PI3K or Akt abolishes the adipogenic gene expression of MyoD null myoblasts. Strikingly, loss of MyoD converts satellite cell-derived primary myoblasts to brown adipocytes through upregulation of Prdm16, a target of miR-133 and key determinant of brown adipocyte fate. Conversely, forced expression of MyoD in brown preadipocytes blocks brown adipogenesis and upregulates the expression of myogenic genes. Importantly, miR-133a knockout significantly blunts the inhibitory effect of MyoD on brown adipogenesis. Our results establish MyoD as a negative regulator of brown adipocyte development by upregulating miR-133 to suppress Akt signaling and Prdm16.


Asunto(s)
Adipocitos Marrones/metabolismo , Transdiferenciación Celular/genética , Proteína MioD/genética , Mioblastos/metabolismo , Adipocitos Marrones/citología , Adipogénesis/genética , Animales , Secuencia de Bases , Western Blotting , Sistemas CRISPR-Cas , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones Noqueados , MicroARNs/genética , Microscopía Fluorescente , Proteína MioD/metabolismo , Mioblastos/citología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
18.
Nat Commun ; 8: 14328, 2017 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-28094257

RESUMEN

Satellite cells (SCs) are myogenic stem cells required for regeneration of adult skeletal muscles. A proper balance among quiescence, activation and differentiation is essential for long-term maintenance of SCs and their regenerative function. Here we show a function of Pten (phosphatase and tensin homologue) in quiescent SCs. Deletion of Pten in quiescent SCs leads to their spontaneous activation and premature differentiation without proliferation, resulting in depletion of SC pool and regenerative failure. However, prior to depletion, Pten-null activated SCs can transiently proliferate upon injury and regenerate injured muscles, but continually decline during regeneration, suggesting an inability to return to quiescence. Mechanistically, Pten deletion increases Akt phosphorylation, which induces cytoplasmic translocation of FoxO1 and suppression of Notch signalling. Accordingly, constitutive activation of Notch1 prevents SC depletion despite Pten deletion. Our findings delineate a critical function of Pten in maintaining SC quiescence and reveal an interaction between Pten and Notch signalling.


Asunto(s)
Células Madre Adultas/enzimología , Senescencia Celular , Fosfohidrolasa PTEN/metabolismo , Células Satélite del Músculo Esquelético/enzimología , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Femenino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Masculino , Ratones , Ratones Noqueados , Desarrollo de Músculos , Fosfohidrolasa PTEN/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo
19.
Development ; 144(2): 235-247, 2017 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-27993983

RESUMEN

Myogenic regulatory factors (MRFs), including Myf5, MyoD (Myod1) and Myog, are muscle-specific transcription factors that orchestrate myogenesis. Although MRFs are essential for myogenic commitment and differentiation, timely repression of their activity is necessary for the self-renewal and maintenance of muscle stem cells (satellite cells). Here, we define Ascl2 as a novel inhibitor of MRFs. During mouse development, Ascl2 is transiently detected in a subpopulation of Pax7+ MyoD+ progenitors (myoblasts) that become Pax7+ MyoD- satellite cells prior to birth, but is not detectable in postnatal satellite cells. Ascl2 knockout in embryonic myoblasts decreases both the number of Pax7+ cells and the proportion of Pax7+ MyoD- cells. Conversely, overexpression of Ascl2 inhibits the proliferation and differentiation of cultured myoblasts and impairs the regeneration of injured muscles. Ascl2 competes with MRFs for binding to E-boxes in the promoters of muscle genes, without activating gene transcription. Ascl2 also forms heterodimers with classical E-proteins to sequester their transcriptional activity on MRF genes. Accordingly, MyoD or Myog expression rescues myogenic differentiation despite Ascl2 overexpression. Ascl2 expression is regulated by Notch signaling, a key governor of satellite cell self-renewal. These data demonstrate that Ascl2 inhibits myogenic differentiation by targeting MRFs and facilitates the generation of postnatal satellite cells.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Desarrollo de Músculos/genética , Factores Reguladores Miogénicos/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Células Cultivadas , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Factores Reguladores Miogénicos/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/fisiología , Transducción de Señal/genética , Activación Transcripcional/genética
20.
FASEB J ; 30(11): 3745-3758, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27458245

RESUMEN

Exercise promotes multiple beneficial effects on muscle function, including induction of mitochondrial biogenesis. miR-133a is a muscle-enriched microRNA that regulates muscle development and function. The role of miR-133a in exercise tolerance has not been fully elucidated. In the current study, mice that were deficient in miR-133a demonstrated low maximal exercise capacity and low resting metabolic rate. Transcription of the mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-γ coactivator 1-α, peroxisome proliferator-activated receptor-γ coactivator 1-ß, nuclear respiratory factor-1, and transcription factor A, mitochondrial were lower in miR-133a-deficient muscle, which was consistent with lower mitochondrial mass and impaired exercise capacity. Six weeks of endurance exercise training increased the transcriptional level of miR-133a and stimulated mitochondrial biogenesis in wild-type mice, but failed to improve mitochondrial function in miR-133a-deficient mice. Further mechanistic analysis showed an increase in the miR-133a potential target, IGF-1 receptor, along with hyperactivation of Akt signaling, in miR-133a-deficient mice, which was consistent with lower transcription of the mitochondrial biogenesis regulators. These findings indicate an essential role of miR-133a in skeletal muscle mitochondrial biogenesis, exercise tolerance, and response to exercise training.-Nie, Y., Sato, Y., Wang, C., Yue, F., Kuang, S., Gavin, T. P. Impaired exercise tolerance, mitochondrial biogenesis, and muscle fiber maintenance in miR-133a-deficient mice.


Asunto(s)
Tolerancia al Ejercicio/fisiología , MicroARNs/genética , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Biogénesis de Organelos , Animales , Ratones Noqueados , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Condicionamiento Físico Animal/métodos , Resistencia Física/fisiología , Factores de Transcripción/metabolismo
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