RESUMO
In diabetes, macrophages and inflammation are increased in the islets, along with ß-cell dysfunction. Here, we demonstrate that galectin-3 (Gal3), mainly produced and secreted by macrophages, is elevated in islets from both high-fat diet (HFD)-fed and diabetic db/db mice. Gal3 acutely reduces glucose-stimulated insulin secretion (GSIS) in ß-cell lines and primary islets in mice and humans. Importantly, Gal3 binds to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) and inhibits calcium influx via the cytomembrane and subsequent GSIS. ß-Cell CACNG1 deficiency phenocopies Gal3 treatment. Inhibition of Gal3 through either genetic or pharmacologic loss of function improves GSIS and glucose homeostasis in both HFD-fed and db/db mice. All animal findings are applicable to male mice. Here we show a role of Gal3 in pancreatic ß-cell dysfunction, and Gal3 could be a therapeutic target for the treatment of type 2 diabetes.
Assuntos
Dieta Hiperlipídica , Galectina 3 , Secreção de Insulina , Células Secretoras de Insulina , Animais , Humanos , Masculino , Camundongos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica/efeitos adversos , Galectina 3/metabolismo , Galectina 3/genética , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Follicle-stimulating hormone (FSH) is involved in mammalian reproduction via binding to FSH receptor (FSHR). However, several studies have found that FSH and FSHR play important roles in extragonadal tissue. Here, we identified the expression of FSHR in human and mouse pancreatic islet ß-cells. Blocking FSH signaling by Fshr knock-out led to impaired glucose tolerance owing to decreased insulin secretion, while high FSH levels caused insufficient insulin secretion as well. In vitro, we found that FSH orchestrated glucose-stimulated insulin secretion (GSIS) in a bell curve manner. Mechanistically, FSH primarily activates Gαs via FSHR, promoting the cAMP/protein kinase A (PKA) and calcium pathways to stimulate GSIS, whereas high FSH levels could activate Gαi to inhibit the cAMP/PKA pathway and the amplified effect on GSIS. Our results reveal the role of FSH in regulating pancreatic islet insulin secretion and provide avenues for future clinical investigation and therapeutic strategies for postmenopausal diabetes.
Assuntos
Hormônio Foliculoestimulante , Ilhotas Pancreáticas , Camundongos , Animais , Humanos , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/metabolismo , Secreção de Insulina , Glucose/farmacologia , Glucose/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Ilhotas Pancreáticas/metabolismo , Transdução de Sinais , Insulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Mamíferos/metabolismoRESUMO
Cleavage under target and tagment (CUT&Tag) is a technology that utilizes the fusion protein of Tn5 transposase and protein A/G which can guide Tn5 enzyme to the antibody bound to target protein and cleave the chromatin regions adjacent to target protein. Chromatin libraries are then tagged and sequenced by the high-throughput sequencing to obtain chromatin information at specific sites or protein binding locations. CUT&Tag technology plays an important role in the research of DNA and protein interactions. It can be used to understand the modifications of histone and the bindings of transcription factors. Compared with the traditional chromatin immunoprecipitation-sequencing (ChIP-seq) technology, the CUT&Tag has the strengths of high signal-to-noise ratio, good repeatability, short experimental period, and low cell input. It shows great advantages in early embryonic development, stem cells, cancer, epigenetics and other research fields. In this article, we described the protocol of CUT&Tag for metabolic tissue cells (mouse primary islet cells), to provide an epigenetic method for studying metabolic cells.
Assuntos
Cromatina , Histonas , Camundongos , Animais , Imunoprecipitação da Cromatina/métodos , Análise de Sequência de DNA/métodos , Histonas/metabolismo , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Adipose tissue macrophage (ATM) has been shown to play a key role in the pathogenesis of obesity-associated adipose tissue inflammation and metabolic diseases. However, the upstream factors that integrate the environmental signals to control ATM activation and adipose inflammation in obesity remain elusive. Here, we identify BAF60a, a subunit of the switch/sucrose-nonfermentable (SWI/SNF) chromatin remodeling complexes, as the central checkpoint regulator of obesity-induced ATM activation, adipose tissue inflammation, and systemic metabolic impairment. BAF60a expression was robustly downregulated in the adipose tissue stromal vascular fractions in type 2 diabetic mice. Myeloid-specific BAF60a knockout (BaMKO) promotes ATM proinflammatory activation, exacerbating diet-induced obesity, insulin resistance, and metabolic dysfunction. Conversely, myeloid-specific overexpression of BAF60a in mice attenuates macrophage proinflammatory activation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that BAF60a inactivation triggers the expression of proinflammatory gene program through chromatin remodeling. Moreover, motif analysis of ATAC-Seq and CUT&Tag-Seq data identifies the transcription factor Atf3 that physically interacts with BAF60a to suppress the proinflammatory gene expression, thereby controlling ATM activation and metabolic inflammation in obesity. Consistently, myeloid-specific Atf3 deficiency also promotes the proinflammatory activation of macrophage. This work uncovers BAF60a/Atf3 axis as the key regulator in obesity-associated ATM activation, adipose tissue inflammation, and metabolic diseases.
Assuntos
Diabetes Mellitus Experimental , Resistência à Insulina , Tecido Adiposo/metabolismo , Animais , Cromatina/metabolismo , Proteínas Cromossômicas não Histona , Diabetes Mellitus Experimental/metabolismo , Dieta , Inflamação/genética , Inflamação/metabolismo , Resistência à Insulina/genética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/metabolismo , Sacarose/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD), whose pathogenesis remains unelucidated, has become an increasingly prevalent disease globally requiring novel treatment strategies. This study aims to explore the role of leukocyte cell-derived chemotaxin 2 (LECT2), one of the known hepatokines, in the development of NAFLD. METHODS: The serum LECT2 level was evaluated in patients with NAFLD and male C57BL/6 mice fed a high-fat diet (HFD) for 8 weeks. Tail intravenous injection of adeno-associated virus that contained Lect2 short hairpin RNA or Lect2 overexpression plasmid was administered to mice to inhibit or increase hepatic Lect2 expression. Hepatic steatosis was evaluated by histological staining with haematoxylin and eosin and Oil Red O, and also by quantitative hepatic triglyceride measurements. RNA-seq was performed to discover the specific targets of LECT2 on NAFLD. RESULTS: Serum and hepatic LECT2 levels were elevated in NAFLD patients and HFD-fed mice. Inhibition of hepatic Lect2 expression alleviated HFD-induced hepatic steatosis and inflammation, whereas hepatic overexpression of Lect2 aggravated HFD-induced hepatic steatosis and inflammation. RNA-seq and bioinformatical analysis suggested that the signal transducers and activators of transcription-1 (STAT-1) pathway might play an indispensable role in the interaction between LECT2 and NAFLD. A STAT-1 inhibitor could reverse the accumulation of hepatic lipids caused by Lect2 overexpression. CONCLUSION: LECT2 expression is significantly elevated in NAFLD. LECT2 induces the occurrence and development of NAFLD through the STAT-1 pathway. LECT2 may be a potential therapeutic target for NAFLD.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Hepatopatia Gordurosa não Alcoólica , Animais , Fatores Quimiotáticos , Dieta Hiperlipídica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Leucócitos , Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , TransdutoresRESUMO
BACKGROUND & AIMS: Our previous study found that platelet counts were positively associated with body fat percentage in human. In the present study, we conducted a reverse translational study to explore the role of platelets in modulating pre-adipocyte proliferation in mice. METHODS: Mouse pre-adipocyte cell line (3T3-L1) and human pre-adipocytes harvested from female subcutaneous fat were used. Pre-adipocytes were co-cultured with platelets or platelet releasate, which were isolated from mice or humans. The cell viability and proliferative ability of the pre-adipocytes were examined by MTT and flow cytometry assays. Western blotting analysis was used to determine the phosphorylation levels of proteins in the mTOR pathway. RESULTS: The number of platelets in the adipose tissues from obese mice was significantly higher than that from lean mice. Platelets and collagen-activated platelet releasate stimulated the proliferation of human pre-adipocytes and 3T3-L1 cells in vitro. Besides, platelets from obese mice were more potent in stimulating pre-adipocyte proliferation than those from lean control mice. Mechanistically, platelets enhanced pre-adipocyte proliferation through the acceleration of cell cycle progression from G0/G1 to S phase cell cycle progression. At the molecular level, platelets promoted pre-adipocyte proliferation through mTOR pathway-mediated upregulation of cyclin D1 expression. CONCLUSION: In conclusion, platelets and platelet releasate play an important role in the proliferation of pre-adipocytes. Our study may provide new clues and the molecular mechanism of the causal pathways between platelets and body fat to explain the finding we observed in population study.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/fisiopatologia , Plaquetas/fisiologia , Células 3T3-L1 , Tecido Adiposo/patologia , Animais , Apoptose , Plaquetas/patologia , Comunicação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Ciclina D1/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/patologia , Obesidade/fisiopatologia , Contagem de Plaquetas , Organismos Livres de Patógenos Específicos , Gordura Subcutânea , Serina-Treonina Quinases TOR/fisiologia , Pesquisa Translacional BiomédicaRESUMO
Diabetes mellitus is a serious metabolic condition associated with a multitude of cardiovascular complications. Moreover, the prevalence of diabetes in heart failure populations is higher than that in control populations. However, the role of cardiomyocyte alterations in type 2 diabetes mellitus (T2DM) has not been well characterized and the underlying mechanisms remain elusive. In this study, two patients who were diagnosed as T2DM were recruited and patient-specific induced pluripotent stem cells (iPSCs) were generated from urine epithelial cells using nonintegrated Sendai virus. The iPSC lines derived from five healthy subjects were used as controls. All iPSCs were differentiated into cardiomyocytes (iPSC-CMs) using the monolayer-based differentiation protocol. T2DM iPSC-CMs exhibited various disease phenotypes, including cellular hypertrophy and lipid accumulation. Moreover, T2DM iPSC-CMs exhibited higher susceptibility to high-glucose/high-lipid challenge than control iPSC-CMs, manifesting an increase in apoptosis. RNA-Sequencing analysis revealed a differential transcriptome profile and abnormal activation of TGFß signaling pathway in T2DM iPSC-CMs. We went on to show that inhibition of TGFß significantly rescued the hypertrophic phenotype in T2DM iPSC-CMs. In conclusion, we demonstrate that the iPSC-CM model is able to recapitulate cellular phenotype of T2DM. Our results indicate that iPSC-CMs can therefore serve as a suitable model for investigating molecular mechanisms underlying diabetic cardiomyopathies and for screening therapeutic drugs.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Apoptose/genética , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Biomarcadores , Estudos de Casos e Controles , Diferenciação Celular/genética , Células Cultivadas , Diabetes Mellitus Tipo 2/etiologia , Células Epiteliais/metabolismo , Glucose/metabolismo , Humanos , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/citologia , Metabolismo dos Lipídeos , Miócitos Cardíacos/citologia , TranscriptomaRESUMO
Rab5 is a master regulator for endosome biogenesis and transport while its in vivo physiological function remains elusive. Here, we find that Rab5a is upregulated in several in vivo and in vitro myogenesis models. By generating myogenic Rab5a-deficient mice, we uncover the essential roles of Rab5a in regulating skeletal muscle regeneration. We further reveal that Rab5a promotes myoblast differentiation and directly interacts with insulin receptor substrate 1 (IRS1), an essential scaffold protein for propagating IGF signaling. Rab5a interacts with IRS1 in a GTP-dependent manner and this interaction is enhanced upon IGF-1 activation and myogenic differentiation. We subsequently identify that the arginine 207 and 222 of IRS1 and tyrosine 82, 89, and 90 of Rab5a are the critical amino acid residues for mediating the association. Mechanistically, Rab5a modulates IRS1 activation by coordinating the association between IRS1 and the IGF receptor (IGFR) and regulating the intracellular membrane targeting of IRS1. Both myogenesis-induced and IGF-evoked AKT-mTOR signaling are dependent on Rab5a. Myogenic deletion of Rab5a also reduces the activation of AKT-mTOR signaling during skeletal muscle regeneration. Taken together, our study uncovers the physiological function of Rab5a in regulating muscle regeneration and delineates the novel role of Rab5a as a critical switch controlling AKT-mTOR signaling by activating IRS1.
Assuntos
Diferenciação Celular , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Esquelético/fisiologia , Mioblastos/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração/fisiologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Células HEK293 , Membro Posterior/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/genética , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
The initiation and development of diabetes are mainly ascribed to the loss of functional ß-cells. Therapies designed to regenerate ß-cells provide great potential for controlling glucose levels and thereby preventing the devastating complications associated with diabetes. This requires detailed knowledge of the molecular events and underlying mechanisms in this disorder. Here, we report that expression of microRNA-223 (miR-223) is up-regulated in islets from diabetic mice and humans, as well as in murine Min6 ß-cells exposed to tumor necrosis factor α (TNFα) or high glucose. Interestingly, miR-223 knockout (KO) mice exhibit impaired glucose tolerance and insulin resistance. Further analysis reveals that miR-223 deficiency dramatically suppresses ß-cell proliferation and insulin secretion. Mechanistically, using luciferase reporter gene assays, histological analysis, and immunoblotting, we demonstrate that miR-223 inhibits both forkhead box O1 (FOXO1) and SRY-box 6 (SOX6) signaling, a unique bipartite mechanism that modulates expression of several ß-cell markers (pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), and urocortin 3 (UCN3)) and cell cycle-related genes (cyclin D1, cyclin E1, and cyclin-dependent kinase inhibitor P27 (P27)). Importantly, miR-223 overexpression in ß-cells could promote ß-cell proliferation and improve ß-cell function. Taken together, our results suggest that miR-223 is a critical factor for maintaining functional ß-cell mass and adaptation during metabolic stress.
Assuntos
Proteína Forkhead Box O1/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição SOXD/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Proliferação de Células , Ciclina D1/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Proteína Forkhead Box O1/química , Proteína Forkhead Box O1/genética , Teste de Tolerância a Glucose , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Ratos , Fatores de Transcrição SOXD/química , Fatores de Transcrição SOXD/genética , Transdução de Sinais , Transativadores/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Skeletal muscle is a major site of postprandial glucose disposal. Inadequate insulin action in skeletal myocytes contributes to hyperglycemia in diabetes. Although glucose is known to stimulate insulin secretion by ß cells, whether it directly engages nutrient signaling pathways in skeletal muscle to maintain systemic glucose homeostasis remains largely unexplored. Here we identified the Baf60c-Deptor-AKT pathway as a target of muscle glucose sensing that augments insulin action in skeletal myocytes. Genetic activation of this pathway improved postprandial glucose disposal in mice, whereas its muscle-specific ablation impaired insulin action and led to postprandial glucose intolerance. Mechanistically, glucose triggers KATP channel-dependent calcium signaling, which promotes HDAC5 phosphorylation and nuclear exclusion, leading to Baf60c induction and insulin-independent AKT activation. This pathway is engaged by the anti-diabetic sulfonylurea drugs to exert their full glucose-lowering effects. These findings uncover an unexpected mechanism of glucose sensing in skeletal myocytes that contributes to homeostasis and therapeutic action.
Assuntos
Glicemia/metabolismo , Metabolismo Energético , Fibras Musculares Esqueléticas/metabolismo , Transdução de Sinais , Animais , Glicemia/efeitos dos fármacos , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Homeostase , Humanos , Hipoglicemiantes/farmacologia , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Canais KATP/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Período Pós-Prandial , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Compostos de Sulfonilureia/farmacologia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Conventional gold standard histopathologic diagnosis requires information of both high resolution structural and chemical changes in tissue. Providing optical information at ultrasonic resolution, photoacoustic (PA) technique could provide highly sensitive and highly accurate tissue characterization noninvasively in the authentic in vivo environment, offering a replacement for histopathology. A two-dimensional (2D) physio-chemical spectrogram (PCS) combining micrometer to centimeter morphology and chemical composition simultaneously can be generated for each biological sample with PA measurements at multiple optical wavelengths. This spectrogram presents a unique 2D "physio-chemical signature" for any specific type of tissue. Comprehensive analysis of PCS, termed PA physio-chemical analysis (PAPCA), can lead to very rich diagnostic information, including the contents of all relevant molecular and chemical components along with their corresponding histological microfeatures, comparable to those accessible by conventional histology. PAPCA could contribute to the diagnosis of many diseases involving diffusive patterns such as fatty liver.
Assuntos
Fígado Gorduroso/patologia , Animais , Biópsia , Dieta Hiperlipídica , Modelos Animais de Doenças , Fígado Gorduroso/diagnóstico por imagem , Fígado Gorduroso/metabolismo , Fibrose , Camundongos , Camundongos Endogâmicos C57BL , Técnicas Fotoacústicas , Máquina de Vetores de SuporteRESUMO
Brown fat activates uncoupled respiration in response to cold temperature and contributes to systemic metabolic homeostasis. To date, the metabolic action of brown fat has been primarily attributed to its role in fuel oxidation and uncoupling protein 1 (UCP1)-mediated thermogenesis. Whether brown fat engages other tissues through secreted factors remains largely unexplored. Here we show that neuregulin 4 (Nrg4), a member of the epidermal growth factor (EGF) family of extracellular ligands, is highly expressed in adipose tissues, enriched in brown fat and markedly increased during brown adipocyte differentiation. Adipose tissue Nrg4 expression was reduced in rodent and human obesity. Gain- and loss-of-function studies in mice demonstrated that Nrg4 protects against diet-induced insulin resistance and hepatic steatosis through attenuating hepatic lipogenic signaling. Mechanistically, Nrg4 activates ErbB3 and ErbB4 signaling in hepatocytes and negatively regulates de novo lipogenesis mediated by LXR and SREBP1c in a cell-autonomous manner. These results establish Nrg4 as a brown fat-enriched endocrine factor with therapeutic potential for the treatment of obesity-associated disorders, including type 2 diabetes and nonalcoholic fatty liver disease (NAFLD).
Assuntos
Tecido Adiposo Marrom/metabolismo , Lipogênese , Fígado/metabolismo , Neurregulinas/genética , Obesidade/genética , RNA Mensageiro/genética , Receptor ErbB-3/metabolismo , Receptor ErbB-4/metabolismo , Células 3T3-L1 , Adipócitos Marrons/metabolismo , Adipogenia , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Células HEK293 , Humanos , Resistência à Insulina , Receptores X do Fígado , Camundongos , Neurregulinas/metabolismo , Obesidade/metabolismo , Receptores Nucleares Órfãos/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Skeletal muscle insulin resistance in type 2 diabetes is associated with a shift from oxidative to glycolytic metabolism in myofibers. However, whether this metabolic switch is detrimental or adaptive for metabolic homeostasis has not been resolved. We recently demonstrated that the Baf60c/Deptor pathway promotes glycolytic metabolism in the muscle and protects mice from diet-induced insulin resistance. However, the nature of the signals that impinge on this pathway and the role of Baf60c in glucose homeostasis in the severe insulin-resistant state remain unknown. Here we show that expression of Baf60c and Deptor was downregulated in skeletal muscle in obesity, accompanied by extracellular signal-related kinase (ERK) activation. In cultured myotubes, inhibition of ERK, but not Jun NH2-terminal kinase and IκB kinase, blocked the downregulation of Baf60c and Deptor by the proinflammatory cytokine tumor necrosis factor-α. Treatment of obese mice with the ERK inhibitor U0126 rescued Baf60c and Deptor expression in skeletal muscle and lowered blood glucose. Transgenic rescue of Baf60c in skeletal muscle restored Deptor expression and Akt phosphorylation and ameliorated insulin resistance in ob/ob mice. This study identifies the Baf60c/Deptor pathway as a target of proinflammatory signaling in skeletal muscle that may link meta-inflammation to skeletal myofiber metabolism and insulin resistance.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Glucose/metabolismo , Homeostase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Animais , Butadienos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Inibidores Enzimáticos/farmacologia , Teste de Tolerância a Glucose , Homeostase/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Insulina/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Nitrilas/farmacologia , Obesidade/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A shift from oxidative to glycolytic metabolism has been associated with skeletal muscle insulin resistance in type 2 diabetes. However, whether this metabolic switch is deleterious or adaptive remains under debate, in part because of a limited understanding of the regulatory network that directs the metabolic and contractile specification of fast-twitch glycolytic muscle. Here we show that Baf60c (also called Smarcd3), a transcriptional cofactor enriched in fast-twitch muscle, promotes a switch from oxidative to glycolytic myofiber type through DEP domain-containing mTOR-interacting protein (Deptor)-mediated Akt activation. Muscle-specific transgenic expression of Baf60c activates a program of molecular, metabolic and contractile changes characteristic of glycolytic muscle. In addition, Baf60c is required for maintaining glycolytic capacity in adult skeletal muscle in vivo. Baf60c expression is significantly lower in skeletal muscle from obese mice compared to that from lean mice. Activation of the glycolytic muscle program by transgenic expression of Baf60c protects mice from diet-induced insulin resistance and glucose intolerance. Further mechanistic studies revealed that Deptor is induced by the Baf60c-Six4 transcriptional complex and mediates activation of Akt and glycolytic metabolism by Baf60c in a cell-autonomous manner. This work defines a fundamental mechanism underlying the specification of fast-twitch glycolytic muscle and illustrates that the oxidative-to-glycolytic metabolic shift in skeletal muscle is potentially adaptive and beneficial in the diabetic state.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Glucose/metabolismo , Glicólise , Proteínas Musculares/fisiologia , Músculos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/fisiologia , Animais , Proteínas Cromossômicas não Histona/metabolismo , Análise por Conglomerados , Ativação Enzimática , Homeostase , Humanos , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , TransgenesRESUMO
The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes. DOI:http://dx.doi.org/10.7554/eLife.00444.001.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Colesterol/sangue , Dislipidemias/enzimologia , Retículo Endoplasmático/enzimologia , Fígado/enzimologia , Pró-Proteína Convertases/sangue , Serina Endopeptidases/sangue , Proteínas de Transporte Vesicular/deficiência , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Dislipidemias/sangue , Dislipidemias/genética , Retículo Endoplasmático/metabolismo , Epistasia Genética , Genótipo , Células HEK293 , Humanos , Fígado/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/metabolismo , Transporte Proteico , Receptores de LDL/genética , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Transfecção , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
BACKGROUND & OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPARgamma) is known to be highly expressed in breast cancer tissues. Thiazolidinediones (TZD), the specific ligands for PPARgamma, can inhibit cell proliferation and induce apoptosis. This study was to investigate the possibility of using troglitazone, one of TZD, as the sensitizer of epirubicin in the treatment of estrogen receptor (ER) negative breast cancer. METHODS: MTT assay and flow cytometry were used to examine the cell proliferation and apoptosis in two ER negative breast cancer cell line MDA-MB-435S and MDA-MB-231. Cells were treated with either troglitazone or epirubicin alone or co-treated with troglitazone and epirubicin. Western blot analysis was used to assess the expression level of Bcl-2. The migration potential of cells with different treatments was analyzed by the wound healing assay. RESULTS: The effect of epirubicin on inhibiting cell proliferation of breast cancer cells was enhanced by co-treatment with troglitazone in the range of 4 micromol/L to 24 micromol/L. The 50% inhibitory concentration (IC(50)) of epirubicin was reduced to 60% when combined with troglitazone compared to the treatment with epirubicin only. Treatment of cells with troglitazone or epirubicin alone could not induce significant apoptosis. However, the apoptotic indexes of MDA-MB-435S and MDA-MB-231 cells co-treated with troglitazone and epirubicin were (5.48+/-0.45)% and (10.08+/-1.89)%, respectively. Co-treatment with troglitazone and epirubicin further downregulated the expression level of Bcl-2 and inhibited cell migration simultaneously. CONCLUSIONS: Troglitazone could not only augment the effect of epirubicin on inhibiting cell proliferation and inducing apoptosis, but also suppress the migration of breast cancer cells. Troglitazone may sensitize the effect of epirubicin on breast cancer cells.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Cromanos/farmacologia , Epirubicina/farmacologia , Tiazolidinedionas/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Sinergismo Farmacológico , Feminino , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Estrogênio/análise , TroglitazonaRESUMO
Peroxisome proliferator-activated receptor (PPAR) isoforms (alpha and gamma) are known to be expressed in pancreatic islets as well as in insulin-producing cell lines. Ligands of PPAR have been shown to enhance glucose-induced insulin secretion in rat pancreatic islets. However, their effect on insulin secretion is still unclear. To understand the molecular mechanism by which PPARgamma exerts its effect on glucose-induced insulin secretion, we examined the endogenous activity of PPAR isoforms, and studied the PPARgamma function and its target gene expression in INS-1 cells. We found that: (1) endogenous PPARg was activated in a ligand-dependent manner in INS-1 cells; (2) overexpression of PPARgamma in the absence of PPARgamma ligands enhanced glucose-induced insulin secretion, which indicates that the increased glucose-induced insulin secretion is a PPARgamma-mediated event; (3) the addition of both PPARgamma and retinoid X receptor (RXR) ligands showed a synergistic effect on the augmentation of reporter activity, suggesting that the hetero-dimerization of PPARgamma and RXR is required for the regulation of the target genes; (4) PPARs upregulated both the glucose transporter 2 (GLUT2) and Cb1-associated protein (CAP) genes in INS-1 cells. Our findings suggest an important mechanistic pathway in which PPARgamma enhances glucose-induced insulin secretion by activating the expression of GLUT2 and CAP genes in a ligand-dependent manner.