RESUMO
BACKGROUND: Biliary atresia (BA) is an intractable disease of unknown cause that develops in the neonatal period. It causes jaundice and liver damage due to the destruction of extrahepatic biliary tracts,. We have found that heterozygous knockout mice of the SRY related HMG-box 17 (Sox17) gene, a master regulator of stem/progenitor cells in the gallbladder wall, exhibit a condition like BA. However, the precise contribution of hypoplastic gallbladder wall to the pathogenesis of hepatobiliary disease in Sox17 heterozygous embryos and human BA remains unclear. METHODS: We employed cholangiography and histological analyses in the mouse BA model. Furthermore, we conducted a retrospective analysis of human BA. RESULTS: We show that gallbladder wall hypoplasia causes abnormal multiple connections between the hilar hepatic bile ducts and the gallbladder-cystic duct in Sox17 heterozygous embryos. These multiple hilar extrahepatic ducts fuse with the developing intrahepatic duct walls and pull them out of the liver parenchyma, resulting in abnormal intrahepatic duct network and severe cholestasis. In human BA with gallbladder wall hypoplasia (i.e., abnormally reduced expression of SOX17), we also identify a strong association between reduced gallbladder width (a morphometric parameter indicating gallbladder wall hypoplasia) and severe liver injury at the time of the Kasai surgery, like the Sox17-mutant mouse model. CONCLUSIONS: Together with the close correlation between gallbladder wall hypoplasia and liver damage in both mouse and human cases, these findings provide an insight into the critical role of SOX17-positive gallbladder walls in establishing functional bile duct networks in the hepatic hilus of neonates.
Biliary atresia (BA) is a disease in newborns that causes a serious liver condition due to damage to the bile ducts (the pathways that carry bile juice). Although reduced function of a key gene called Sox17, which is essential for forming the gallbladder wall, has been observed in some BA cases, the link between gallbladder issues and liver damage is unknown. This study has shown how damage spreads through the bile ducts in the liver around the time of birth when there are problems in the gallbladder wall due to reduced SOX17 function. The findings indicate that proper growth of the gallbladder wall during this critical period is essential for forming a normal network of bile ducts in the developing liver. This discovery is promising for early diagnosis and better treatment of BA in newborns.
RESUMO
In mammalian testes, undifferentiated spermatogonia (Aundiff) undergo differentiation in response to retinoic acid (RA), while their progenitor states are partially maintained by fibroblast growth factors (FGFs). Sertoli valve (SV) is a region located at the terminal end of seminiferous tubule (ST) adjacent to the rete testis (RT), where the high density of Aundiff is constitutively maintained with the absence of active spermatogenesis. However, the molecular and cellular characteristics of SV epithelia still remain unclear. In this study, we first identified the region-specific AKT phosphorylation in the SV Sertoli cells and demonstrated non-cell autonomous specialization of Sertoli cells in the SV region by performing a Sertoli cell ablation/replacement experiment. The expression of Fgf9 was detected in the RT epithelia, while the exogenous administration of FGF9 caused ectopic AKT phosphorylation in the Sertoli cells of convoluted ST. Furthermore, we revealed the SV region-specific expression of Cyp26a1, which encodes an RA-degrading enzyme, and demonstrated that the increased RA levels in the SV region disrupt its pool of Aundiff by inducing their differentiation. Taken together, RT-derived FGFs and low levels of RA signaling contribute to the non-cell-autonomous regionalization of the SV epithelia and its local maintenance of Aundiff in the SV region.
Assuntos
Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Tretinoína/metabolismo , Animais , Diferenciação Celular , Epitélio/fisiologia , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/análise , Regeneração , Ácido Retinoico 4 Hidroxilase/genética , Ácido Retinoico 4 Hidroxilase/metabolismo , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/crescimento & desenvolvimento , Células de Sertoli/fisiologia , Células de Sertoli/transplante , Transdução de Sinais , Espermatogênese , Tretinoína/farmacologia , Regulação para CimaRESUMO
In mouse testes, Sertoli cells support the continuous process of spermatogenesis, which is dependent on seminiferous epithelial cycles along the longitudinal axis of the seminiferous tubule. Sertoli cell function is modulated partly by local cytokines and/or growth factors derived from adjacent tissues such as blood vessels, macrophages, rete testis, etc. However, the spatial activation patterns by local signals in vivo remain unclear. In this study, we focused on Signal Transducers and Activators of Transcription (STAT) signaling in Sertoli cells, because STAT is a major crucial cytokine transducer for somatic cyst cell regulation in Drosophila testis niches. In mouse testes, STAT3 was ubiquitously expressed in Sertoli cells throughout the seminiferous tubules. Phosphorylated STAT3 (p-STAT3) was predominantly observed in the Sertoli cells within the valve-like structure adjacent to the rete testis (i.e., the Sertoli valve [SV]) in the terminal segment of the proximal seminiferous tubules. In the distal seminiferous tubules with active spermatogenesis, most Sertoli cells were negative for anti-p-STAT3 staining. Albeit rarely, a small patch of several p-STAT3-positive Sertoli cells was detected frequently in seminiferous epithelial cycle stages I-VI. Such p-STAT3-positive ratios in the convoluted seminiferous epithelia were significantly increased in germ cell-less testes than in the wild-type testes, but with considerably lower ratios than in the SV region. These findings imply that regionally distinct patterns of STAT3 phosphorylation in the Sertoli cells depend on either location or spermatogenic activity in normal healthy testes in vivo, highlighting a novel entry point to understanding STAT signaling in mammalian spermatogenesis.
Assuntos
Fator de Transcrição STAT3/metabolismo , Epitélio Seminífero/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Masculino , Camundongos , Especificidade de Órgãos , FosforilaçãoRESUMO
RATIONALE: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. OBJECTIVE: The role of vaspin in the diabetic vascular complications remains elusive, and we investigated the effects of vaspin on the vascular function under the diabetic milieu. METHODS AND RESULTS: Adenovirus carrying the full length of the vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon-injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb, and Pdgfrb genes was significantly reduced by the treatment of Vaspin-Ad. In cuff-injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells. Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to human aortic endothelial cells, subjected to tandem tag purification and liquid chromatography-tandem mass spectrometry, and we identified GRP78 (78-kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78, and voltage-dependent anion channel on the plasma membrane was confirmed by the immunoprecipitation studies using aortas of Vaspin Tg mice. The binding assay using (125)I-vaspin in human aortic endothelial cells revealed high-affinity binding (dissociation constant = 0.565×10(-9) m) by the treatment of 5 µM thapsigargin, which recruited GRP78 from the endoplasmic reticulum to plasma membrane by inducing endoplasmic reticulum stress. In human aortic endothelial cells, vaspin induced phosphorylation of Akt and inhibited the kringle 5-induced Ca(2+) influx and subsequent apoptosis. CONCLUSIONS: Vaspin is a novel ligand for the cell-surface GRP78/voltage-dependent anion channel complex in endothelial cells and promotes proliferation, inhibits apoptosis, and protects vascular injuries in diabetes mellitus.
Assuntos
Adipocinas/metabolismo , Apoptose/fisiologia , Endotélio Vascular/patologia , Proteínas de Choque Térmico/metabolismo , Serpinas/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Adenoviridae/genética , Adipocinas/genética , Animais , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Wistar , Serpinas/genética , Estreptozocina/efeitos adversosRESUMO
To understand genetic programs controlling mammalian central nervous system (CNS) development, we have identified one transgene-inserted mutation, which showed embryonic lethality during neurulation. Determination of the transgene integration site and rescue experiments revealed that the Brd2 gene, whose products specifically bind acetylated histone H4 and can mediate transcription, was the cause of this mutation. Expression studies with specific markers demonstrated that cell cycle progression was accelerated and neuronal differentiation as well as cell cycle exit were impaired in Brd2-deficient neruoepithelial cells. To investigate whether Brd2 regulates neuronal differentiation through a E2F1 transcriptional factor, which directly binds Brd2 and controls genes expression for cell cycle progression and exit, we analyzed Brd2;E2F1 double mutant phenotypes and, consequently found that abnormalities in neuronal differentiation and cell cycle progression due to Brd2-deficiency were restored by removing the E2F1 gene. These findings suggest that Brd2 is required for cell cycle exit and neuronal differentiation of neuroepithelial cells through the E2F1 pathway during mouse CNS development.
Assuntos
Ciclo Celular/fisiologia , Sistema Nervoso Central/crescimento & desenvolvimento , Fator de Transcrição E2F1/metabolismo , Células Neuroepiteliais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Proteínas Serina-Treonina Quinases/fisiologia , Alelos , Animais , Ciclo Celular/genética , Diferenciação Celular , Sistema Nervoso Central/metabolismo , Proteínas Cromossômicas não Histona , Fator de Transcrição E2F1/genética , Camundongos , Camundongos Transgênicos , Neurogênese/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de TranscriçãoRESUMO
During mammalian sex determination of XY fetuses, SRY induces SOX9 in Sertoli cells, resulting in formation of testes with seminiferous tubules, interstitial Leydig cells and peritubular myoid cells. Meanwhile XX fetuses without SRY develop ovaries. In cattle, most XX heifers born with a male twin, so-called freemartins, develop nonfunctioning ovaries and genitalia with an intersex phenotype. Interestingly, freemartins sometimes develop highly masculinized gonads with seminiferous tubule-like structures despite the absence of SRY. However, in these cases, the degree of masculinization in each gonadal somatic cell type is unclear. Here, we report a rare case of a freemartin Japanese black calf with almost complete XX sexreversal. Gross anatomical analysis of this calf revealed the presence of a pair of small testis-like gonads with rudimentary epididymides, in addition to highly masculinized genitalia including a pampiniform plexus, scrotum and vesicular gland. Histological and immunohistochemical analyses of these masculinized gonads revealed well-defined seminiferous tubule-like structures throughout the whole gonadal parenchyma. In epithelia of these tubules, SOX9-positive supporting cells (i.e., Sertoli cells) were found to be arranged regularly along the bases of tubules, and they were also positive for GDNF, one of the major factors for spermatogenesis. 3ß-HSD-positive cells (i.e., Leydig cells) and SMA-positive peritubular myoid cells were also identified around tubules. Therefore, for the first time, we found the transdifferentiation of ovarian somatic cells into all testicular somatic cell types in the XX freemartin gonads. These data strongly support the idea of a high sexual plasticity in the ovarian somatic cells of mammalian gonads.
Assuntos
Transdiferenciação Celular , Freemartinismo/patologia , Gônadas/patologia , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Bovinos , Células Epiteliais/metabolismo , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Gônadas/metabolismo , Masculino , Fatores de Transcrição SOX9/metabolismo , Fator Esteroidogênico 1/metabolismoRESUMO
It is unknown whether adipokines derived from adipose tissues modulate endoplasmic reticulum (ER) stress induced in obesity. Here, we show that visceral adipose tissue-derived serine protease inhibitor (vaspin) binds to cell-surface 78-kDa glucose-regulated protein (GRP78), which is recruited from ER to plasma membrane under ER stress. Vaspin transgenic mice were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis, while vaspin-deficient mice developed glucose intolerance associated with upregulation of ER stress markers. With tandem affinity tag purification using HepG2 cells, we identified GRP78 as an interacting molecule. The complex formation of vaspin, GRP78, and murine tumor cell DnaJ-like protein 1 (MTJ-1) (DnaJ homolog, subfamily C, member 1) on plasma membrane was confirmed by cell-surface labeling with biotin and immunoprecipitation in liver tissues and H-4-II-E-C3 cells. The addition of recombinant human vaspin in the cultured H-4-II-E-C3 cells also increased the phosphorylation of Akt and AMP-activated protein kinase (AMPK) in a dose-dependent manner, and anti-GRP78 antibodies completely abrogated the vaspin-induced upregulation of pAkt and pAMPK. Vaspin is a novel ligand for cell-surface GRP78/MTJ-1 complex, and its subsequent signals exert beneficial effects on ER stress-induced metabolic dysfunctions.
Assuntos
Adipocinas/metabolismo , Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Gordura Intra-Abdominal/metabolismo , Proteínas de Neoplasias/metabolismo , Obesidade/metabolismo , Serpinas/metabolismo , Adipocinas/química , Adipocinas/genética , Adipocinas/isolamento & purificação , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP40/antagonistas & inibidores , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Ligantes , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Obesidade/patologia , Transporte Proteico , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serpinas/química , Serpinas/genética , Serpinas/isolamento & purificação , Transdução de Sinais , Regulação para CimaRESUMO
During fasting, induction of hepatic gluconeogenesis is crucial to ensure proper energy homeostasis. Such induction is dysregulated in type 2 diabetes, resulting in the development of fasting hyperglycemia. Hormonal and nutrient regulation of metabolic adaptation during fasting is mediated predominantly by the transcriptional coactivator peroxisome proliferative activated receptor γ coactivator 1α (PGC-1α) in concert with various other transcriptional regulators. Although CITED2 (CBP- and p300-interacting transactivator with glutamic acid- and aspartic acid-rich COOH-terminal domain 2) interacts with many of these molecules, the role of this protein in the regulation of hepatic gluconeogenesis was previously unknown. Here we show that CITED2 is required for the regulation of hepatic gluconeogenesis through PGC-1α. The abundance of CITED2 was increased in the livers of mice by fasting and in cultured hepatocytes by glucagon-cAMP-protein kinase A (PKA) signaling, and the amount of CITED2 in liver was higher in mice with type 2 diabetes than in non-diabetic mice. CITED2 inhibited the acetylation of PGC-1α by blocking its interaction with the acetyltransferase general control of amino acid synthesis 5-like 2 (GCN5). The consequent downregulation of PGC-1α acetylation resulted in an increase in its transcriptional coactivation activity and an increased expression of gluconeogenic genes. The interaction of CITED2 with GCN5 was disrupted by insulin in a manner that was dependent on phosphoinositide 3-kinase (PI3K)-thymoma viral proto-oncogene (Akt) signaling. Our results show that CITED2 functions as a transducer of glucagon and insulin signaling in the regulation of PGC-1α activity that is associated with the transcriptional control of gluconeogenesis and that this function is mediated through the modulation of GCN5-dependent PGC-1α acetylation. We also found that loss of hepatic CITED2 function suppresses gluconeogenesis in diabetic mice, suggesting it as a therapeutic target for hyperglycemia.
Assuntos
Gluconeogênese/fisiologia , Glucose/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Acetilação/efeitos dos fármacos , Acetiltransferases/metabolismo , Adenoviridae/genética , Animais , Células Cultivadas , Cromonas/farmacologia , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Gluconeogênese/genética , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores para Leptina/deficiência , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirtuínas/genética , Sirtuínas/metabolismo , Transativadores/genética , Fatores de Transcrição , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Acyl-CoA:monoacylglycerol acyltransferase (MGAT) plays a predominant role in the resynthesis of triacylglycerol in the small intestine, but its contribution to triacylglycerol synthesis in other tissues, such as the liver, is not clear. In this study, we identified a novel MGAT gene, which is identical with lysophosphatidylglycerol acyltransferase1 (LPGAT1). Mouse LPGAT1 is expressed in a number of tissues and most highly expressed in the liver. Hepatic LPGAT1 expression in diabetic db/db mice is higher than that in the control db/m mouse, which is consistent with increased hepatic MGAT activity in db/db mouse. To elucidate the role of LPGAT1 gene in lipid metabolism in db/db mice, we constructed an adenovirus of short hairpin RNA (shRNA) targeting LPGAT1 to selectively knockdown LPGAT1 gene expression in the liver. Hepatic MGAT activity and LPGAT1 expression in db/db mice infected with LPGAT1 shRNA adenovirus were significantly lower than those in mice infected with the control virus. Notably, treatment with LPGAT1 shRNA adenovirus caused a marked reduction in serum triacylglycerol and cholesterol levels and a significant increase in hepatic cholesterol level. These findings indicate that LPGAT1, a newly identified MGAT enzyme, plays a significant role in hepatic triacylglycerol synthesis and secretion in db/db mice.
Assuntos
Aciltransferases/metabolismo , Fígado/metabolismo , Triglicerídeos/metabolismo , Aciltransferases/deficiência , Aciltransferases/genética , Adenoviridae/genética , Animais , Células CHO , Cricetinae , Cricetulus , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Sequências Repetidas Invertidas , Metabolismo dos Lipídeos/genética , Fígado/enzimologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triglicerídeos/sangueRESUMO
Physical exercise ameliorates metabolic disorders such as type 2 diabetes mellitus and obesity, but the molecular basis of these effects remains elusive. In the present study, we found that exercise up-regulates heparin-binding epidermal growth factor-like growth factor (HB-EGF) in skeletal muscle. To address the metabolic consequences of such gain of HB-EGF function, we generated mice that overexpress this protein specifically in muscle. The transgenic animals exhibited a higher respiratory quotient than did wild-type mice during indirect calorimetry, indicative of their selective use of carbohydrate rather than fat as an energy substrate. They also showed substantial increases in glucose tolerance, insulin sensitivity, and glucose uptake by skeletal muscle. These changes were accompanied by increased kinase activity of Akt in skeletal muscle and consequent inhibition of Forkhead box O1-dependent expression of the pyruvate dehydrogenase kinase 4 gene. Furthermore, mice with a high level of transgene expression were largely protected from obesity, hepatic steatosis, and insulin resistance, even when maintained on a high-fat diet. Our results suggest that HB-EGF produced by contracting muscle acts as an insulin sensitizer that facilitates peripheral glucose disposal.
Assuntos
Glucose/metabolismo , Resistência à Insulina/fisiologia , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Fígado Gorduroso/prevenção & controle , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Homeostase/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/prevenção & controle , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The transcriptional regulator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1alpha expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1alpha transcript (designated PGC-1alpha-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1alpha-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca(2+)- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1alpha-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1alpha expression in contracting muscle.
Assuntos
Proteínas de Choque Térmico/genética , Contração Muscular/genética , Músculo Esquelético/fisiologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Transcrição Gênica , Ativação Transcricional , Sequência de Bases , Cálcio/farmacologia , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
In patients with various catabolic conditions, glucocorticoid excess induces skeletal muscle wasting by accelerating protein degradation via the ubiquitin-proteasome pathway. Although the transcriptional coactivator p300 has been implicated in this pathological process, regulatory mechanisms and molecular targets of its action remain unclear. Here we show that CREB-binding protein (CBP)/p300-interacting transactivator with ED-rich tail 2 (Cited2), which binds to the cysteine-histidine-rich region 1 of p300 and CBP, regulates muscle mass in vitro. Adenovirus-mediated overexpression of wild-type Cited2 significantly blocked morphological alterations of C2C12 myotubes with a concomitant decrease in myosin heavy chain protein in response to synthetic glucocorticoid dexamethasone, which were attributable to the reduced induction of atrophy-related ubiquitin ligases MuRF1 and MAFbx. These myotube-sparing effects were less pronounced, however, with a carboxyl-terminally truncated mutant of Cited2 that lacked the ability to bind p300. These results suggest that the gain of Cited2 function counteracts glucocorticoid-induced muscle atrophy through inhibition of proteolysis mediated by p300-dependent gene transcription.
Assuntos
Dexametasona/toxicidade , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Proteínas Repressoras/fisiologia , Transativadores/fisiologia , Animais , Glucocorticoides/toxicidade , Humanos , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/metabolismo , Atrofia Muscular/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Proteínas Ligases SKP Culina F-Box/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Gene related to anergy in lymphocytes (GRAIL) is an E3 ubiquitin ligase that regulates energy in T-lymphocytes. Whereas, the relevance of GRAIL to T lymphocyte function is well established, the role of this protein in other cell types remains unknown. Given that GRAIL is abundant in the liver, we investigated the potential function of GRAIL in nutrient metabolism by generating mice in which the expression of GRAIL is reduced specifically in the liver. Adenovirus-mediated transfer of a short hairpin RNA specific for GRAIL mRNA markedly reduced the amounts of GRAIL mRNA and protein in the liver. Blood glucose levels of the mice with hepatic GRAIL deficiency did not differ from those of control animals in the fasted or fed states. However, these mice manifested glucose intolerance in association with a normal increase in plasma insulin levels during glucose challenge. The mice also manifested an increase in the serum concentration of free fatty acids, whereas the serum levels of cholesterol and triglyceride were unchanged. The hepatic abundance of mRNAs for glucose-6-phosphatase, catalytic (a key enzyme in hepatic glucose production) and for sterol regulatory element-binding transcription factor 1 (an important transcriptional regulator of lipogenesis) was increased in the mice with hepatic GRAIL deficiency, possibly contributing to the metabolic abnormalities of these animals. Our results thus demonstrate that GRAIL in the liver is essential for maintenance of normal glucose and lipid metabolism in living animals.
Assuntos
Anergia Clonal/imunologia , Glucose/metabolismo , Metabolismo dos Lipídeos , Fígado/enzimologia , Linfócitos/enzimologia , Linfócitos/imunologia , Ubiquitina-Proteína Ligases/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) complex. Genetic ablation of p27(Kip1) in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27(Kip1) by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2(-/-) MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), largely restored lipid accumulation and PPARgamma gene expression in Skp2(-/-) MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27(Kip1) expression.
Assuntos
Adipócitos/fisiologia , Adipogenia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fibroblastos/citologia , Proteínas Quinases Associadas a Fase S/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Cromanos/farmacologia , Embrião de Mamíferos/citologia , Camundongos , Camundongos Knockout , Proteínas Quinases Associadas a Fase S/genética , Tiazolidinedionas/farmacologia , TroglitazonaRESUMO
Regulation of hepatic gene expression is largely responsible for the control of nutrient metabolism. We previously showed that the transcription factor STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. However, the role of STAT3 in the control of lipid metabolism has remained unknown. We have now investigated the effects of hepatic overexpression of STAT3, achieved by adenovirus-mediated gene transfer, on glucose and lipid metabolism in insulin-resistant diabetic mice. Forced expression of STAT3 reduced blood glucose and plasma insulin concentrations as well as the hepatic abundance of mRNA for phosphoenolpyruvate carboxykinase. However, it also increased the plasma levels of triglyceride and total cholesterol without affecting those of low density lipoprotein- or high density lipoprotein-cholesterol. The hepatic abundance of mRNAs for fatty acid synthase and acetyl-CoA carboxylase, both of which catalyze the synthesis of fatty acids, was increased by overexpression of STAT3, whereas that of mRNAs for sterol regulatory element-binding proteins 1a, 1c, or 2 was unaffected. Moreover, the amount of mRNA for acyl-CoA oxidase, which contributes to beta-oxidation, was decreased by forced expression of STAT3. These results indicate that forced activation of STAT3 signaling in the liver of insulin-resistant diabetic mice increased the circulating levels of atherogenic lipids through changes in the hepatic expression of genes involved in lipid metabolism. Furthermore, these alterations in hepatic gene expression likely occurred through a mechanism independent of sterol regulatory element-binding proteins.
Assuntos
Metabolismo dos Lipídeos , Fígado/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Glicemia/metabolismo , Regulação da Expressão Gênica , Gluconeogênese/genética , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
Phosphoinositide-dependent kinase-1 (PDK1) is implicated in the metabolic effects of insulin as a key mediator of phosphoinositide 3-kinase-dependent signaling. Here we show that mice with liver-specific PDK1 deficiency manifest various defects in the metabolic actions of insulin in the liver as well as a type 2 diabetes-like phenotype characterized by marked hyperinsulinemia and postprandial hyperglycemia. The hepatic abundance of glucokinase, an important determinant of glucose flux and glucose-evoked signaling in hepatocytes, was substantially reduced in these mice. Restoration of hepatic glucokinase expression, with the use of an adenoviral vector, induced insulin-like effects in the liver and almost completely normalized the fasting hyperinsulinemia and postprandial hyperglycemia in these animals. These results indicate that, if the hepatic abundance of glucokinase is maintained, ingested glucose is normally disposed of even in the absence of acute activation of proximal insulin signaling, such as the activation of Akt, in the liver.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucoquinase/genética , Fígado/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Primers do DNA , Glucose/metabolismo , Teste de Tolerância a Glucose , Insulina/farmacologia , Camundongos , Camundongos Transgênicos , Período Pós-Prandial , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Sox7, Sox17 and Sox18 constitute group F of the Sox family of HMG box transcription factor genes. Dominant-negative mutations in Sox18 underlie the cardiovascular defects observed in ragged mutant mice. By contrast, Sox18(-/-) mice are viable and fertile, and display no appreciable anomaly in their vasculature, suggesting functional compensation by the two other SoxF genes. Here, we provide direct evidence for redundant function of Sox17 and Sox18 in postnatal neovascularization by generating Sox17(+/-) -Sox18(-/-) double mutant mice. Whereas Sox18(-/-) and Sox17(+/-) -Sox18(+/-) mice showed no vascular defects, approximately half of the Sox17(+/-) -Sox18(-/-) pups died before postnatal day 21 (P21). They showed reduced neovascularization in the liver sinusoids and kidney outer medulla vasa recta at P7, which most likely caused the ischemic necrosis observed by P14 in hepatocytes and renal tubular epithelia. Those that survived to adulthood showed similar, but milder, vascular anomalies in both liver and kidney, and females were infertile with varying degrees of vascular abnormalities in the reproductive organs. These anomalies corresponded with sites of expression of Sox7 and Sox17 in the developing postnatal vasculature. In vitro angiogenesis assays, using primary endothelial cells isolated from the P7 livers, showed that the Sox17(+/-) -Sox18(-/-) endothelial cells were defective in endothelial sprouting and remodeling of the vasculature in a phenotype-dependent manner. Therefore, our findings indicate that Sox17 and Sox18, and possibly all three SoxF genes, are cooperatively involved in mammalian vascular development.
Assuntos
Proteínas HMGB/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Neovascularização Fisiológica , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Vasos Sanguíneos/anormalidades , Vasos Sanguíneos/citologia , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Anormalidades Congênitas/genética , Anormalidades Congênitas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Feminino , Proteínas HMGB/genética , Proteínas de Grupo de Alta Mobilidade/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hibridização In Situ , Rim/anormalidades , Rim/patologia , Fígado/anormalidades , Fígado/patologia , Camundongos , Camundongos Knockout , Morfogênese , Fatores de Transcrição SOXF , Distribuição Tecidual , Fatores de Transcrição/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. The mechanism by which hepatic STAT3 is regulated by nutritional or hormonal status has remained unknown, however. Here, we show that an increase in the plasma insulin concentration, achieved either by glucose administration or by intravenous insulin infusion, stimulates tyrosine phosphorylation of STAT3 in the liver. This effect of insulin was mediated by the hormone's effects in the brain, and the increase in hepatic IL-6 induced by the brain-insulin action is essential for the activation of STAT3. The inhibition of hepatic glucose production and of expression of gluconeogenic genes induced by intracerebral ventricular insulin infusion was impaired in mice with liver-specific STAT3 deficiency or in mice with IL-6 deficiency. These results thus indicate that IL-6-STAT3 signaling in the liver contributes to insulin action in the brain, leading to the suppression of hepatic glucose production.
Assuntos
Encéfalo/metabolismo , Glucose/metabolismo , Insulina/fisiologia , Fígado/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Ativação Enzimática , Gluconeogênese , Glucose/farmacologia , Técnica Clamp de Glucose , Glucose-6-Fosfatase/fisiologia , Homeostase , Insulina/administração & dosagem , Insulina/sangue , Insulina/farmacologia , Resistência à Insulina , Interleucina-6/análise , Interleucina-6/fisiologia , Células de Kupffer/química , Células de Kupffer/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfoenolpiruvato Carboxilase/fisiologia , Fosforilação , Receptor de Insulina/fisiologia , Transdução de SinaisRESUMO
Krüppel-like zinc finger transcription factors (KLFs) play diverse roles during cell differentiation and development in mammals. We have now shown by microarray analysis that expression of the KLF15 gene is markedly up-regulated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Inhibition of the function of KLF15, either by expression of a dominant negative mutant or by RNA interference, both reduced the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and blocked adipogenesis in 3T3-L1 preadipocytes exposed to inducers of adipocyte differentiation. However, the dominant negative mutant of KLF15 did not affect the expression of CCAAT/enhancer-binding protein beta (C/EBPbeta) elicited by inducers of differentiation in 3T3-L1 preadipocytes. In addition, ectopic expression of KLF15 in NIH 3T3 or C2C12 cells triggered both lipid accumulation and the expression of PPARgamma in the presence of inducers of adipocyte differentiation. Ectopic expression of C/EBPbeta, C/EBPdelta, or C/EBPalpha in NIH 3T3 cells also elicited the expression of KLF15 in the presence of inducers of adipocyte differentiation. Moreover, KLF15 and C/EBPalpha acted synergistically to increase the activity of the PPARgamma2 gene promoter in 3T3-L1 adipocytes. Our observations thus demonstrate that KLF15 plays an essential role in adipogenesis in 3T3-L1 cells through its regulation of PPAR gamma expression.
Assuntos
Adipócitos/citologia , PPAR gama/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Northern Blotting , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA , Fibroblastos/metabolismo , Genes Dominantes , Vetores Genéticos , Fatores de Transcrição Kruppel-Like , Luciferases/metabolismo , Camundongos , Modelos Biológicos , Mutação , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Retroviridae/genética , Fatores de Tempo , Regulação para CimaRESUMO
Regulation of hepatic gene expression is important for energy homeostasis. We now show that hepatic expression of the gene for the transcription factor Kruppel-like factor 15 (KLF15) is increased by food deprivation and reduced by feeding in mice. Expression of the KLF15 gene in mouse liver was also down-regulated by a euglycemic-hyperinsulinemic clamp and was increased by inhibition of phosphatidylinositol 3-kinase. In cultured rat hepatocytes, KLF15 gene expression was induced by dexamethasone and a non-hydrolyzing analog of cAMP, and this effect was inhibited by insulin in a manner dependent on phosphatidylinositol 3-kinase signaling. Forced expression of KLF15 in cultured hepatocytes increased both the expression and the promoter activity of the gene for phosphoenolpyruvate carboxykinase (PEPCK). These results suggest that insulin and its counteracting hormones regulate the hepatic expression of KLF15, and that this transcription factor contributes to the regulation of PEPCK gene expression in the liver.