RESUMEN
p16 inhibits cyclin-dependent kinases and regulates senescence-mediated arrest as well as p21. Nuclear p16 promotes G1 cell cycle arrest and cellular senescence. In various glomerular diseases, nuclear p16 expression is associated with disease progression. Therefore, the location of p16 is important. However, the mechanism of p16 trafficking between the nucleus and cytoplasm is yet to be fully investigated. TGF-ß1, a major cytokine involved in the development of kidney diseases, can upregulate p21 expression. However, the relationship between TGF-ß1 and p16 is poorly understood. Here, we report the role of podocyte TGF-ß1 in regulating the p16 behavior in glomerular endothelial cells. We analyzed podocyte-specific TGF-ß1 overexpression mice. Although p16 was found in the nuclei of glomerular endothelial cells and led to endothelial cellular senescence, the expression of p16 did not increase in glomeruli. In cultured endothelial cells, TGF-ß1 induced nuclear translocation of p16 without increasing its expression. Among human glomerular diseases, p16 was detected in the nuclei of glomerular endothelial cells. In summary, we demonstrated the novel role of podocyte TGF-ß1 in managing p16 behavior and cellular senescence in glomeruli, which has clinical relevance for the progression of human glomerular diseases.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Línea Celular , Senescencia Celular/fisiología , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Células Endoteliales/metabolismo , Femenino , Genes p16/fisiología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos ICR , Podocitos/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The relationship between cellular senescence and fibrosis in the kidney is being elucidated and we have identified it as therapeutic target in recent studies. Chronic kidney disease has also become a lifestyle disease, often developing on the background of hypertension and dyslipidemia. In this study, we clarify the effect of interaction between these two conditions on kidney fibrosis and senescence. Wild type mice (WT), apolipoprotein E-/- mice (ApoEKO), and endothelial nitric oxide synthase (eNOS)-/- ApoE-/- mice (DKO) were obtained by breeding. Unilateral ureteral obstruction (UUO) was performed on 8-10 week old male mice and the degree of renal tubular injury, fibrosis and kidney senescence were evaluated. DKO manifested elevated blood pressure, higher total cholesterol and lower HDL than WT. DKO showed sustained kidney injury molecule-1 protein expression. Kidney fibrosis was significantly higher in ApoEKO and DKO. mRNA expression of genes related to kidney fibrosis was the highest in DKO. The mRNA expression of Zinc-α2-Glycoprotein and heme oxygenase-1 were significantly decreased in DKO. Furthermore, mRNA expression of p53, p21 and p16 were increased both in ApoEKO and DKO, with DKO being the highest. Senescence associated ß-gal positive tubule area was significantly increased in DKO. Increased DNA damage and target of rapamycin-autophagy spatial coupling compartments (TASCCs) formation was found in DKO. Mice with endothelial dysfunction and dyslipidemia developed kidney fibrosis and accelerated senescence even in young mice after injury. These data highlight the fact managing lifestyle-related diseases from a young age is important for CKD prevention.
Asunto(s)
Apolipoproteínas E/deficiencia , Senescencia Celular/genética , Fibrosis/genética , Eliminación de Gen , Riñón/patología , Óxido Nítrico Sintasa de Tipo III/deficiencia , Insuficiencia Renal Crónica/genética , Animales , Apolipoproteínas E/genética , Autofagia , Presión Sanguínea , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Daño del ADN/genética , Genes p16 , Genes p53 , Humanos , Riñón/lesiones , Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Diabetic nephropathy (DN) is among the most serious complications of diabetes mellitus, and often leads to end-stage renal disease ultimately requiring dialysis or renal transplantation. The loss of podocytes has been reported to have a role in the onset and progression of DN. Here, we addressed the activation mechanism of Smad3 signaling in podocytes. Expression of RII and activation of Smad3 were induced by AGE exposure (P<0.05). Reduction of the activation of RII-Smad3 signaling ameliorated podocyte injuries in Smad3-knockout diabetic mice. The bone morphogenetic protein 4 (BMP4) significantly regulated activation of RII-Smad3 signalings (P<0.05). Moreover, the epithelium-specific transcription factor, Elf3was induced by AGE stimulation and, subsequently, upregulated RII expression in cultured podocytes. Induction of Elf3 and activation of RII-Smad3 signaling, leading to a decrease in WT1 expression, were observed in podocytes in diabetic human kidneys. Moreover, AGE treatment induced the secretion of Elf3-containing exosomes from cultured podocytes, which was dependent on the activation of the TGF-ß-Smad3 signaling pathway. In addition, exosomal Elf3 protein in urine could be measured only in urinary exosomes from patients with DN. The appearance of urinary exosomal Elf3 protein in patients with DN suggested the existence of irreversible injuries in podocytes. The rate of decline in the estimated Glomerular Filtration Rate (eGFR) after measurement of urinary exosomal Elf3 protein levels in patients with DN (R2 = 0.7259) might be useful as an early non-invasive marker for podocyte injuries in DN.
Asunto(s)
Proteínas de Unión al ADN/orina , Nefropatías Diabéticas/orina , Exosomas/metabolismo , Podocitos/metabolismo , Transducción de Señal , Proteína smad3/orina , Factores de Transcripción/orina , Animales , Biomarcadores/orina , Nefropatías Diabéticas/patología , Exosomas/patología , Tasa de Filtración Glomerular , Masculino , Ratones , Podocitos/patologíaRESUMEN
The glomerulus is a network of capillaries known as a tuft, located at the beginning of a nephron in the kidney. Here we describe a novel method for the induction of a macroscopically visible three-dimensional glomerulus-like sphere (GLS). This procedure did not require any additional cytokines and completed the formation of spheres within 24â¯h. After the formation was complete, GLS maintained a steady state for at least five days without proliferation and without a decrease in viability. Therefore, this procedure assists various assays for a prolong period of time. Overall, our protocol allows for a very simple mixing of cells from different sources to obtain fine-grained and highly dispersed GLSs. The kidney filtration barrier is a unique structure characterized by a complex three-dimensional framework of podocytes and endothelial cells. GLS exhibited the induction of many podocyte-specific gene profiles similar to those in adult human kidneys, suggesting that the sphere formation process is important for the maturation of podocytes. Focal segmental glomerulosclerosis (FSGS) is one of the major causes of steroid-resistant nephrotic syndrome, and some circulating permeability factors in the patient's serum FSGS have been implicated in the pathogenesis of the disease. Serum from patients with FSGS induced the collapse of GLS, which imitates the appearance of glomerulosclerosis in patients. In conclusion, the investigation and use of GLS may provide a novel method to elucidate the molecular mechanisms underlying complicated and unexplained events in glomeruli in a similar condition in adult kidneys.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomérulos Renales/metabolismo , Células Cultivadas , Glomeruloesclerosis Focal y Segmentaria/sangre , HumanosRESUMEN
Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with increased morbidity and mortality as compared to other causes of renal disease. Albuminuria is often the first clinical indicator of the presence of DN. However, albuminuria or proteinuria is a common symptom in patients with various renal disorders. Therefore, specific biomarkers for the diagnosis of DN are required. A primary hallmark of DN is the progressive damage and death of glomerular podocytes, resulting in the leaking of proteins into the urine. Urinary exosomes released by podocytes are microvesicles containing information of the originated cells. Podocyte-derived signal transduction factors (PDSTFs) are good candidates to assess podocyte injuries. The profile of PDSTFs in urinary exosomes from patients with DN is different from that from patients with minimal change nehrotic syndrome. In addition, PDSTFs molecules in exosomes were derived from primary murine podocytes under high glucose conditions. Among PDSTFs in urinary exosomes, Wilms tumor 1 (WT1) levels reflected damage of diabetic glomeruli in the patients. Urinary exosomal WT1 can predict the decline in eGFR for the following several years. In conclusion, urinary exosomal WT1 is a useful biomarker to improve risk stratification in patients with DN. J. Med. Invest. 65:208-215, August, 2018.
Asunto(s)
Nefropatías Diabéticas/diagnóstico , Genes del Tumor de Wilms , ARN Mensajero/genética , ARN Mensajero/orina , Adolescente , Adulto , Biomarcadores/orina , Estudios de Casos y Controles , Células Cultivadas , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/orina , Exosomas/genética , Marcadores Genéticos , Humanos , Persona de Mediana Edad , Nefrosis Lipoidea/diagnóstico , Nefrosis Lipoidea/genética , Nefrosis Lipoidea/orina , Podocitos/metabolismo , Pronóstico , Proteínas WT1/genética , Proteínas WT1/metabolismo , Adulto JovenRESUMEN
Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with increased morbidity and mortality compared with other causes of renal diseases. We previously found that Smad1 plays a critical role in the development of DN both in vitro and in vivo. However, functional interaction between Smad1 and Smad3 signaling in DN is unclear. Here, we addressed the molecular interplay between Smad1 and Smad3 signaling under a diabetic condition by using Smad3-knockout diabetic mice. Extracellular matrix (ECM) protein overexpression and Smad1 activation were observed in the glomeruli of db/db mice but were suppressed in the glomeruli of Smad3+/-; db/db mice. Smad3 activation enhanced the phosphorylation of Smad1 C-terminal domain but decreased the phosphorylation of linker domain, thus regulating Smad1 activation in advanced glycation end product-treated mesangial cells (MCs). However, forced phosphorylation of the Smad1 linker domain did not affect Smad3 activation in MCs. Phosphorylation of the Smad1 linker domain increased in Smad3+/-; db/db mice and probucol-treated db/db mice, which was consistent with the attenuation of ECM overproduction. These results indicate that Smad3 expression and activation or probucol treatment alters Smad1 phosphorylation, thus suggesting new molecular mechanisms underlying DN development and progression.
Asunto(s)
Nefropatías Diabéticas/patología , Productos Finales de Glicación Avanzada/metabolismo , Proteína Smad1/metabolismo , Proteína smad3/metabolismo , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Células Cultivadas , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Mesangio Glomerular/citología , Mesangio Glomerular/patología , Productos Finales de Glicación Avanzada/sangre , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Probucol/farmacología , Probucol/uso terapéutico , Dominios Proteicos , Proteína smad3/genéticaRESUMEN
Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Oligopéptidos/metabolismo , Proteínas Proto-Oncogénicas c-cbl/química , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Células HEK293 , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Modelos Moleculares , Oligopéptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Ubiquitinación/efectos de los fármacosRESUMEN
BACKGROUND: In recent years, the number of bedridden people is rapidly increasing due to aging or lack of exercise in Japan. This problem is becoming more serious, since there is no countermeasure against it. In the present study, we designed to investigate whether dietary proteins, especially soy, had beneficial effects on skeletal muscle in 59 volunteers with various physical activities. METHODS: We subjected 59 volunteers with various physical activities to meal intervention examination. Persons with low and high physical activities were divided into two dietary groups, the casein diet group and the soy diet group. They ate daily meals supplemented with 7.8 g of powdered casein or soy protein isolate every day for 30 days. Bedridden patients in hospitals were further divided into three dietary groups: the no supplementation diet group, the casein diet group and the soy diet group. They were also subjected to a blood test, a urinalysis, magnetic resonance imaging analysis and muscle strength test of the knee before and after the meal intervention study. RESULTS: Thirty-day soy protein supplementation significantly increased skeletal muscle volume in participants with low physical activity, compared with 30-day casein protein supplementation. Both casein and soy protein supplementation increased the volume of quadriceps femoris muscle in bedridden patients. Consistently, soy protein significantly increased their extension power of the knee, compared with casein protein. Although casein protein increased skeletal muscle volume more than soy protein in bedridden patients, their muscle strength changes by soy protein supplementation were bigger than those by casein protein supplementation. CONCLUSIONS: The supplementation of soy protein would be one of the effective foods which prevent the skeletal muscle atrophy caused by immobilization or unloading.
Asunto(s)
Proteínas en la Dieta/administración & dosificación , Ejercicio Físico , Fuerza Muscular , Músculo Esquelético/anatomía & histología , Proteínas de Soja/administración & dosificación , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Anciano , Anciano de 80 o más Años , Desoxiguanosina/análogos & derivados , Desoxiguanosina/orina , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana EdadRESUMEN
A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120 µM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glucocorticoides/efectos adversos , Músculo Esquelético/metabolismo , Péptidos/química , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Sistema Libre de Células , Femenino , Células HEK293 , Humanos , Proteínas Sustrato del Receptor de Insulina/química , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efectos de los fármacos , Atrofia Muscular/inducido químicamente , Ácido Mirístico/química , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Ubiquitina/químicaRESUMEN
Skeletal muscle atrophy occurs when the rate of protein degradation exceeds that of protein synthesis in various catabolic conditions, such as fasting, disuse, aging, and chronic diseases. Insulin-like growth factor-1 (IGF-1) signaling stimulates muscle growth and suppresses muscle protein breakdown. In atrophied muscles, ubiquitin ligase, Cbl-b, increases and stimulates the ubiquitination and degradation of IRS-1, an intermediate in IGF-1 signaling pathway, resulting in IGF-1 resistance. In this study, we evaluated the efficacy of atelocollagen (ATCOL)-transported anti-ubiquitination oligopeptide (Cblin: Cbl-b inhibitor) (consisting of tyrosine phosphorylation domain of IRS-1) in starved C2C12 myotubes. The amount of IRS-1 protein was lower in starved versus unstarved myotubes. The Cblin-ATCOL complex inhibited IRS-1 degradation in a concentration-dependent manner. Myotubes incubated with Cblin-ATCOL complex showed significant resistance to starvation-induced atrophy (p<0.01). Furthermore, the Cblin-ATCOL complex significantly inhibited any decrease in Akt phosphorylation (p<0.01) and localization of FOXO3a to the nucleus in starved myotubes. These results suggest that Cblin prevented starvation-induced C2C12 myotube atrophy by maintaining the IGF-1/Akt/FOXO signaling. Therefore, attachment of anti-ubiquitination oligopeptide, Cblin, to ATCOL enhances its delivery to myotubes and could be a potentially effective strategy in the treatment of atrophic myopathies.
Asunto(s)
Colágeno/farmacología , Músculo Esquelético/patología , Atrofia Muscular/prevención & control , Oligopéptidos/farmacología , Ubiquitinación/efectos de los fármacos , Animales , Medio de Cultivo Libre de Suero , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-cbl , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Obesity causes type 2 diabetes, atherosclerosis and cardiovascular diseases by inducing systemic insulin resistance. It is now recognized that obesity is related to chronic low-grade inflammation in adipose tissue. Specifically, activated immune cells infiltrate adipose tissue and cause inflammation. There is increasing evidence that activated macrophages accumulate in the hypertrophied adipose tissue of rodents and humans and induce systemic insulin resistance by secreting inflammatory cytokines. Accordingly, a better understanding of the molecular mechanisms underlying macrophage activation in adipose tissue will facilitate the development of new therapeutic strategies. Currently, little is known about the regulation of macrophage activation, although E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl)-b was identified recently as a novel negative regulator of macrophage activation in adipose tissue. Cbl-b, which is a suppressor of T- and B-cell activation, inhibits intracellular signal transduction by targeting some tyrosine kinases. Notably, preventing Cbl-b-mediated macrophage activation improves obesity-induced insulin resistance in mice. c-Cbl is another member of the Cbl family that is associated with insulin resistance in obesity. These reports suggest that Cbl-b and c-Cbl are potential therapeutic targets for treating obesity-induced insulin resistance. In this review, we focus on the importance of Cbl-b in macrophage activation in aging-induced and high-fat diet-induced obesity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Resistencia a la Insulina/genética , Obesidad/metabolismo , Proteínas Proto-Oncogénicas c-cbl/fisiología , Envejecimiento/fisiología , Animales , Dieta Alta en Grasa , Humanos , Sistema Inmunológico/enzimología , Sistema Inmunológico/metabolismo , Activación de Macrófagos/genética , Ratones , Obesidad/complicaciones , Obesidad/genéticaRESUMEN
Proinflammatory cytokines are factors that induce ubiquitin-proteasome-dependent proteolysis in skeletal muscle, causing muscle atrophy. Although isoflavones, as potent antioxidative nutrients, have been known to reduce muscle damage during the catabolic state, the non-antioxidant effects of isoflavones against muscle atrophy are not well known. Here we report on the inhibitory effects of isoflavones such as genistein and daidzein on muscle atrophy caused by tumor necrosis factor (TNF)-α treatment. In C2C12 myotubes, TNF-α treatment markedly elevated the expression of the muscle-specific ubiquitin ligase MuRF1, but not of atrogin-1, leading to myotube atrophy. We found that MuRF1 promoter activity was mediated by acetylation of p65, a subunit of NFκB, a downstream target of the TNF-α signaling pathway; increased MuRF1 promoter activity was abolished by SIRT1, which is associated with deacetylation of p65. Of interest, isoflavones induced expression of SIRT1 mRNA and phosphorylation of AMP kinase, which is well known to stimulate SIRT1 expression, although there was no direct effect on SIRT1 activation. Moreover, isoflavones significantly suppressed MuRF1 promoter activity and myotube atrophy induced by TNF-α in C2C12 myotubes. These results suggest that isoflavones suppress myotube atrophy in skeletal muscle cells through activation of SIRT1 signaling. Thus, the efficacy of isoflavones could provide a novel therapeutic approach against inflammation-related muscle atrophy.
Asunto(s)
Glycine max/química , Isoflavonas/uso terapéutico , Fibras Musculares Esqueléticas/efectos de los fármacos , Proteínas Musculares/metabolismo , Atrofia Muscular/prevención & control , Fitoterapia , Sirtuina 1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Acetilación , Adenilato Quinasa/metabolismo , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Línea Celular , Inflamación/complicaciones , Inflamación/metabolismo , Inflamación/prevención & control , Isoflavonas/farmacología , Ratones , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Atrofia Muscular/metabolismo , FN-kappa B/metabolismo , Fosforilación , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal , Sirtuina 1/genética , Factor de Transcripción ReIA/metabolismo , Proteínas de Motivos Tripartitos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA), IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.
RESUMEN
We previously reported the potential involvement of casitas B-cell lymphoma-b (Cbl-b) in aging-related murine insulin resistance. Because obesity also induces macrophage recruitment into adipose tissue, we elucidated here the role of Cbl-b in obesity-related insulin resistance. Cbl-b(+/+) and Cbl-b(-/-) mice were fed a high-fat diet (HFD) and then examined for obesity-related changes in insulin signaling. The HFD caused recruitment of macrophages into adipose tissue and increased inflammatory reaction in Cbl-b(-/-) compared with Cbl-b(+/+) mice. Peritoneal macrophages from Cbl-b(-/-) mice and Cbl-b-overexpressing RAW264.7 macrophages were used to examine the direct effect of saturated fatty acids (FAs) on macrophage activation. In macrophages, Cbl-b suppressed saturated FA-induced Toll-like receptor 4 (TLR4) signaling by ubiquitination and degradation of TLR4. The physiological role of Cbl-b in vivo was also examined by bone marrow transplantation and Eritoran, a TLR4 antagonist. Hematopoietic cell-specific depletion of the Cbl-b gene induced disturbed responses on insulin and glucose tolerance tests. Blockade of TLR4 signaling by Eritoran reduced fasting blood glucose and serum interleukin-6 levels in obese Cbl-b(-/-) mice. These results suggest that Cbl-b deficiency could exaggerate HFD-induced insulin resistance through saturated FA-mediated macrophage activation. Therefore, inhibition of TLR4 signaling is an attractive therapeutic strategy for treatment of obesity-related insulin resistance.