RESUMO
Nucleotide metabolism fuels normal DNA replication and is also primarily targeted by the DNA replication checkpoint when replication stalls. To reveal a comprehensive interconnection between genome maintenance and metabolism, we analyzed the metabolomic changes upon replication stress in the budding yeast S. cerevisiae. We found that upon treatment of cells with hydroxyurea, glucose is rapidly diverted to the oxidative pentose phosphate pathway (PPP). This effect is mediated by the AMP-dependent kinase, SNF1, which phosphorylates the transcription factor Mig1, thereby relieving repression of the gene encoding the rate-limiting enzyme of the PPP. Surprisingly, NADPH produced by the PPP is required for efficient recruitment of replication protein A (RPA) to single-stranded DNA, providing the signal for the activation of the Mec1/ATR-Rad53/CHK1 checkpoint signaling kinase cascade. Thus, SNF1, best known as a central energy controller, determines a fast mode of replication checkpoint activation through a redox mechanism. These findings establish that SNF1 provides a hub with direct links to cellular metabolism, redox, and surveillance of DNA replication in eukaryotes.
Assuntos
Replicação do DNA , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Glucose/genética , Glucose/metabolismo , Glicólise/fisiologia , Hidroxiureia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Syntrophins are a family of proteins forming membrane-anchored scaffolds and serving as adaptors for various transmembrane and intracellular signaling molecules. To understand the physiological roles of ß1 syntrophin, one of the least characterized members, we generated mouse models to eliminate ß1 syntrophin specifically in the endocrine or exocrine pancreas. ß1 syntrophin is dispensable for the morphology and function of insulin-producing ß cells. However, mice with ß1 syntrophin deletion in exocrine acinar cells exhibit increased severity of cerulein-induced acute pancreatitis. Reduced expression of cystic fibrosis transmembrane conductance regulator and dilation of acinar lumen are potential predisposition factors. During the disease progression, a relative lack of autophagy is associated with deficiencies in both actin assembly and endoplasmic reticulum nucleation. Our findings reveal, for the first time, that ß1 syntrophin is a critical regulator of actin cytoskeleton and autophagy in pancreatic acinar cells and is potently protective against cerulein-induced acute pancreatitis.
Assuntos
Autofagia , Ceruletídeo/toxicidade , Proteínas Associadas à Distrofina/fisiologia , Pancreatite/prevenção & controle , Substâncias Protetoras , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/metabolismo , Pancreatite/patologiaRESUMO
To investigate the role of GRP78 in adipogenesis and metabolic homeostasis, we knocked down GRP78 in mouse embryonic fibroblasts and 3T3-L1 preadipocytes induced to undergo differentiation into adipocytes. We also created an adipose Grp78-knockout mouse utilizing the aP2 (fatty acid binding protein 4) promoter-driven Cre-recombinase. Adipogenesis was monitored by molecular markers and histology. Tissues were analyzed by micro-CT and electron microscopy. Glucose homeostasis and cytokine analysis were performed. Our results indicate that GRP78 is essential for adipocyte differentiation in vitro. aP2-cre-mediated GRP78 deletion leads to lipoatrophy with â¼90% reduction in gonadal and subcutaneous white adipose tissue and brown adipose tissue, severe growth retardation, and bone defects. Despite severe abnormality in adipose mass and function, adipose Grp78-knockout mice showed normal plasma triglyceride levels, and plasma glucose and insulin levels were reduced by 40-60% compared to wild-type mice, suggesting enhanced insulin sensitivity. The endoplasmic reticulum is grossly expanded in the residual mutant white adipose tissue. Thus, these studies establish that GRP78 is required for adipocyte differentiation, glucose homeostasis, and balanced secretion of adipokines. Unexpectedly, the phenotypes and metabolic parameters of the mutant mice, which showed early postnatal mortality, are uniquely distinct from previously characterized lipodystrophic mouse models.
Assuntos
Adipogenia/fisiologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Glucose/metabolismo , Proteínas de Choque Térmico/metabolismo , Homeostase/fisiologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Adipocinas/genética , Adipocinas/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Branco/citologia , Animais , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Deleção de Genes , Glucose/genética , Proteínas de Choque Térmico/genética , Lipodistrofia/genética , Lipodistrofia/metabolismo , Lipodistrofia/patologia , Camundongos , Camundongos Knockout , Triglicerídeos/sangue , Triglicerídeos/genéticaRESUMO
The disease progression of the metabolic syndrome is associated with prolonged hyperlipidemia and insulin resistance, eventually giving rise to impaired insulin secretion, often concomitant with hypoadiponectinemia. As an adipose tissue derived hormone, adiponectin is beneficial for insulin secretion and ß cell health and differentiation. However, the down-stream pathway of adiponectin in the pancreatic islets has not been studied extensively. Here, along with the overall reduction of endocrine pancreatic function in islets from adiponectin KO mice, we examine PPARα and HNF4α as additional down-regulated transcription factors during a prolonged metabolic challenge. To elucidate the function of ß cell-specific PPARα and HNF4α expression, we developed doxycycline inducible pancreatic ß cell-specific PPARα (ß-PPARα) and HNF4α (ß-HNF4α) overexpression mice. ß-PPARα mice exhibited improved protection from lipotoxicity, but elevated ß-oxidative damage in the islets, and also displayed lowered phospholipid levels and impaired glucose-stimulated insulin secretion. ß-HNF4α mice showed a more severe phenotype when compared to ß-PPARα mice, characterized by lower body weight, small islet mass and impaired insulin secretion. RNA-sequencing of the islets of these models highlights overlapping yet unique roles of ß-PPARα and ß-HNF4α. Given that ß-HNF4α potently induces PPARα expression, we define a novel adiponectin-HNF4α-PPARα cascade. We further analyzed downstream genes consistently regulated by this axis. Among them, the islet amyloid polypeptide (IAPP) gene is an important target and accumulates in adiponectin KO mice. We propose a new mechanism of IAPP aggregation in type 2 diabetes through reduced adiponectin action.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animais , Camundongos , Adiponectina/genética , Adiponectina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismoRESUMO
The endoplasmic reticulum (ER) is abundant in the acinar cells of the exocrine pancreas. To test the role of ER homeostasis in acute pancreatitis, we manipulated GRP78 levels, a major ER chaperone, in mice. Grp78(+/+) and (+/-) littermates were fed either a regular diet (RD) or a high-fat diet. Acinar cells were examined for ER structure by electron microscopy, and ER chaperone levels were assessed by immunoblotting. Pancreatitis was induced by cerulein injection, and multiple pathological parameters were analyzed. Grp78(+/-) mice showed decreased GRP78 expression in acinar cells. Exocrine pancreata of RD-fed Grp78(+/-) mice in an outbred C57BL/6 × 129/sv genetic background exhibited ER lumen dilation, a reduction in chaperones calnexin (CNX) and calreticulin (CRT), and exacerbated pancreatitis associated with high CHOP induction. With the high-fat diet regimen, Grp78 heterozygosity triggered GRP94 up-regulation and restoration of GRP78, CNX, and CRT to wild-type levels, corresponding with mitigated pancreatitis on cerulein insult. Interestingly, after backcrossing into the C57BL/6 background, RD-fed Grp78(+/-) mice exhibited an increase in GRP94 and levels of CNX and CRT equivalent to wild type, associated with decreased experimental pancreatitis severity. Administration of a chemical chaperone, 4-phenolbutyrate, was protective against cerulein-induced death. Thus, in exocrine pancreata, Grp78 heterozygosity regulates ER chaperone balance, in dietary- and genetic background-dependent manners, and improved ER protein folding capacity might be protective against pancreatitis.
Assuntos
Ceruletídeo , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/metabolismo , Pâncreas Exócrino/metabolismo , Pancreatite/genética , Doença Aguda , Animais , Gorduras na Dieta/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Fármacos Gastrointestinais , Proteínas de Choque Térmico/fisiologia , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pâncreas Exócrino/efeitos dos fármacos , Pancreatite/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
GRP78/BiP has recently emerged as a novel biomarker for aggressive prostate cancer. Here, we report that homozygous deletion of Grp78 specifically in mouse prostate epithelium suppresses prostate tumorigenesis without affecting postnatal prostate development and growth. Mouse prostates with double conditional knockout of Grp78 and Pten exhibit normal histology and cytology, in contrast to the invasive adenocarcinoma in mouse prostates with Pten inactivation. AKT activation in Pten null prostate epithelium is inhibited by Grp78 homozygous deletion, corresponding with suppression of AKT phosphorylation by GRP78 knockdown in prostate cancer cell line. Thus, inactivation of GRP78 may represent a previously undescribed approach to stop prostate cancer and potentially other cancers resulting from the loss of PTEN tumor suppression and/or activation of the oncogenic AKT.
Assuntos
Adenocarcinoma/metabolismo , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Animais , Chaperona BiP do Retículo Endoplasmático , Epitélio/patologia , Epitélio/fisiologia , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/metabolismo , PTEN Fosfo-Hidrolase/genética , Próstata/crescimento & desenvolvimento , Próstata/patologia , Próstata/fisiologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologiaRESUMO
Obesity and diabetes are often associated with lipotoxic conditions in multiple tissues. The insulin-producing ß cells are susceptible to elevated lipid levels and the ensuing lipotoxicity. The preservation of ß cell mass and function is one of the main goals of diabetes management under these metabolically stressful conditions. However, the adverse effects from the adaptive signaling pathways that ß cells use to counteract lipotoxic stress have secondary negative effects in their own right. Antilipotoxic signaling cascades in ß cells can contribute to their eventual failure. Such dual roles are seen for many other biological adaptive processes as well.
RESUMO
Adipose tissue in the mammary gland undergoes dramatic remodeling during reproduction. Adipocytes are replaced by mammary alveolar structures during pregnancy and lactation, then reappear upon weaning. The fate of the original adipocytes during lactation and the developmental origin of the re-appearing adipocyte post involution are unclear. Here, we reveal that adipocytes in the mammary gland de-differentiate into Pdgfrα+ preadipocyte- and fibroblast-like cells during pregnancy and remain de-differentiated during lactation. Upon weaning, de-differentiated fibroblasts proliferate and re-differentiate into adipocytes. This cycle occurs over multiple pregnancies. These observations reveal the potential of terminally differentiated adipocytes to undergo repeated cycles of de-differentiation and re-differentiation in a physiological setting.
Assuntos
Adipócitos Brancos/metabolismo , Adipogenia , Tecido Adiposo , Lactação/metabolismo , Glândulas Mamárias Animais , Adipócitos Brancos/citologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , DesmameRESUMO
Adipose tissue is a dynamic organ that makes critical contributions to whole-body metabolic homeostasis. Although recent studies have revealed that different fat depots have distinct molecular signatures, metabolic functions and adipogenic mechanisms, peroxisome proliferator-activated receptor γ (PPARγ) is still widely viewed as the master regulator of adipogenesis and critical for maintaining mature adipocyte function. Using an inducible, adipocyte-specific knockout system, we explored the role of PPARγ in mature adipocytes in vivo Short-term PPARγ deficiency in adipocytes reduces whole-body insulin sensitivity, but adipocytes are viable both in vitro and in vivo However, after exposure to a high-fat diet, even short-term PPARγ deficiency leads to rapid adipocyte death. When mature adipocytes are depleted of both PPARγ and CCAAT-enhancer-binding protein α (C/EBPα), they are rapidly depleted of lipids and undergo adipocyte death, both in vitro and in vivo Surprisingly, although thiazolidinediones (TZDs; PPARγ agonists) are thought to act mainly on PPARγ, PPARγ in adipocytes is not required for the whole-body insulin-sensitizing effect of TZDs. This offers new mechanistic aspects of PPARγ/TZD action and its effect on whole-body metabolic homeostasis.
Assuntos
Adipócitos/metabolismo , Resistência à Insulina/fisiologia , PPAR gama/fisiologia , Adipogenia/fisiologia , Tecido Adiposo/fisiologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Homeostase/fisiologia , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Tiazolidinedionas/farmacologia , Fatores de TranscriçãoRESUMO
The compensatory proliferation of insulin-producing ß cells is critical to maintaining glucose homeostasis at the early stage of type 2 diabetes. Failure of ß cells to proliferate results in hyperglycemia and insulin dependence in patients. To understand the effect of the interplay between ß cell compensation and lipid metabolism upon obesity and peripheral insulin resistance, we eliminated LDL receptor-related protein 1 (LRP1), a pleiotropic mediator of cholesterol, insulin, energy metabolism, and other cellular processes, in ß cells. Upon high-fat diet exposure, LRP1 ablation significantly impaired insulin secretion and proliferation of ß cells. The diminished insulin signaling was partly contributed to by the hypersensitivity to glucose-induced, Ca2+-dependent activation of Erk and the mTORC1 effector p85 S6K1. Surprisingly, in LRP1-deficient islets, lipotoxic sphingolipids were mitigated by improved lipid metabolism, mediated at least in part by the master transcriptional regulator PPARγ2. Acute overexpression of PPARγ2 in ß cells impaired insulin signaling and insulin secretion. Elimination of Apbb2, a functional regulator of LRP1 cytoplasmic domain, also impaired ß cell function in a similar fashion. In summary, our results uncover the double-edged effects of intracellular lipid metabolism on ß cell function and viability in obesity and type 2 diabetes and highlight LRP1 as an essential regulator of these processes.
Assuntos
Dieta , Células Secretoras de Insulina/metabolismo , Metabolismo dos Lipídeos , Obesidade/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Alelos , Animais , Glicemia/metabolismo , Proliferação de Células , Cruzamentos Genéticos , Citoplasma/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glucose/metabolismo , Teste de Tolerância a Glucose , Insulina/sangue , Insulina/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR gama/metabolismo , Esfingolipídeos/metabolismo , Transcrição GênicaRESUMO
Acute neonatal hypoxia, a common stressor, causes a spontaneous decrease in body temperature which may be protective. There is consensus that hypothermia should be prevented during acute hypoxia in the human neonate; however, this may be an additional stress with negative consequences. We hypothesize that maintaining body temperature during hypoxia in the first week of postnatal life alters the subsequent insulin, glucose, and glucagon secretion in adult rats. Rat pups were separated from their dam daily from postnatal days (PD) 2-6 for the following 90 min experimental treatments: (1) normoxic separation (control), (2) hypoxia (8% O2) allowing spontaneous hypothermia, (3) normoxic hypothermia with external cold, and (4) exposure to 8% O2 while maintaining body temperature using external heat. An additional normoxic non-separated control group was performed to determine if separation per se changed the adult phenotype. Plasma insulin, glucose, and glucagon responses to arginine stimulation were evaluated from PD105 to PD133. Maternal separation (compared to non-separated neonates) had more pronounced effects on the adult response to arginine compared to the hypoxic, hypothermic, and hypoxic-isothermic neonatal treatments. Adult males exposed to neonatal maternal separation had augmented insulin and glucose responses to arginine compared to unseparated controls. Additionally, neonatal treatment had a significant effect on body weight gain; adults exposed to neonatal maternal separation were significantly heavier. Female adults had significantly smaller insulin and glucose responses to arginine regardless of neonatal treatment. Neonatal maternal separation during the first week of life significantly altered adult beta-cell function in a sexually dimorphic manner.
RESUMO
BACKGROUND: Evidence hints at the ability of ß-cells to emerge from non-ß-cells upon genetic or pharmacological interventions. However, their quantitative contributions to the process of autonomous ß-cell regeneration without genetic or pharmacological manipulations remain to be determined. METHODS & RESULTS: Using PANIC-ATTAC mice, a model of titratable, acute ß-cell apoptosis capable of autonomous, and effective islet mass regeneration, we demonstrate that an extended washout of residual tamoxifen activity is crucial for ß-cell lineage tracing studies using the tamoxifen-inducible Cre/loxP systems. We further establish a doxycycline-inducible system to label different cell types in the mouse pancreas and pursued a highly quantitative assessment to trace adult ß-cells after various metabolic challenges. Beyond proliferation of pre-existing ß-cells, non-ß-cells contribute significantly to the post-challenge regenerated ß-cell pool. α-cell trans-differentiation is the predominant mechanism upon post-apoptosis regeneration and multiparity. No contributions from exocrine acinar cells were observed. During diet-induced obesity, about 25% of α-cells arise de novo from ß-cells. Ectopic expression of Nkx6.1 promotes α-to-ß conversion and insulin production. CONCLUSIONS: We identify the origins and fates of adult ß-cells upon post-challenge upon autonomous regeneration of islet mass and establish the quantitative contributions of the different cell types using a lineage tracing system with high temporal resolution.
RESUMO
Mitochondrial metabolism plays an integral role in glucose-stimulated insulin secretion (GSIS) in ß-cells. In addition, the diabetogenic role of glucagon released from α-cells plays a major role in the etiology of both type 1 and type 2 diabetes because unopposed hyperglucagonemia is a pertinent contributor to diabetic hyperglycemia. Titrating expression levels of the mitochondrial protein mitoNEET is a powerful approach to fine-tune mitochondrial capacity of cells. Mechanistically, ß-cell-specific mitoNEET induction causes hyperglycemia and glucose intolerance due to activation of a Parkin-dependent mitophagic pathway, leading to the formation of vacuoles and uniquely structured mitophagosomes. Induction of mitoNEET in α-cells leads to fasting-induced hypoglycemia and hypersecretion of insulin during GSIS. MitoNEET-challenged α-cells exert potent antiapoptotic effects on ß-cells and prevent cellular dysfunction associated with mitoNEET overexpression in ß-cells. These observations identify that reduced mitochondrial function in α-cells exerts potently protective effects on ß-cells, preserving ß-cell viability and mass.
Assuntos
Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Sobrevivência Celular , Glucagon/biossíntese , Glucose/metabolismo , Intolerância à Glucose/etiologia , Hiperglicemia/etiologia , Insulina/metabolismo , Secreção de Insulina , Camundongos , Receptor Cross-TalkRESUMO
Pathways that stimulate ß-cell regeneration remain of great clinical interest, yet effective therapeutic avenues that promote survival or reconstitution of ß-cell mass remain elusive. Using a mouse model with inducible ß-cell apoptosis followed by adiponectin-mediated regeneration, we aimed to identify key molecules boosting ß-cell viability. In the regenerating pancreatic islets, we examined changes within the transcriptome and observed an extensive up-regulation of genes encoding proteins involved in lipid transport and metabolism. The most prominent targets were further confirmed by quantitative PCR and immunofluorescence. Among the upstream regulators predicted by pathway analysis of the transcriptome, we detected enhanced levels of 2 key transcription factors, Hepatocyte Nuclear Factor 4α and Peroxisome Proliferator-Activated Receptorα. Our data suggest that improving pancreatic islet lipid metabolism as an important antilipotoxic phenomenon to boost ß-cell regeneration. This is primarily mediated by the adipokine adiponectin that exerts its action on both the beta-cell directly as well as on the adipocyte. Adiponectin induces lipid metabolism gene expression in regenerating islets through Hepatocyte Nuclear Factor 4α and Peroxisome Proliferator-Activated Receptorα. Adiponectin also modulates leptin levels via preserving adipose tissue mass in the insulinopenic state.
Assuntos
Adiponectina/metabolismo , Ilhotas Pancreáticas/metabolismo , Adiponectina/genética , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Imuno-Histoquímica , Células Secretoras de Insulina/metabolismo , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma/genéticaRESUMO
BACKGROUND: The selective estrogen receptor modulator tamoxifen, in combination with the Cre-ER(T2) fusion protein, has been one of the mainstream methods to induce genetic recombination and has found widespread application in lineage tracing studies. METHODS & RESULTS: Here, we report that tamoxifen exposure at widely used concentrations remains detectable by mass-spectrometric analysis in adipose tissue after a washout period of 10 days. Surprisingly, its ability to maintain nuclear translocation of the Cre-ER(T2) protein is preserved beyond 2 months of washout. Tamoxifen treatment acutely leads to transient lipoatrophy, followed by de novo adipogenesis that reconstitutes the original fat mass. In addition, we find a "synthetically lethal" phenotype for adipocytes when tamoxifen treatment is combined with adipocyte-specific loss-of-function mutants, such as an adipocyte-specific PPARγ knockout. This is observed to a lesser extent when alternative inducible approaches are employed. CONCLUSIONS: These findings highlight the potential for tamoxifen-induced adipogenesis, and the associated drawbacks of the use of tamoxifen in lineage tracing studies, explaining the discrepancy in lineage tracing results from different systems with temporal control of gene targeting.
RESUMO
Pathological expansion of adipose tissue contributes to the metabolic syndrome. Distinct depots develop at various times under different physiological conditions. The transcriptional cascade mediating adipogenesis is established in vitro, and centres around a core program involving PPARγ and C/EBPα. We developed an inducible, adipocyte-specific knockout system to probe the requirement of key adipogenic transcription factors at various stages of adipogenesis in vivo. C/EBPα is essential for all white adipogenic conditions in the adult stage, such as adipose tissue regeneration, adipogenesis in muscle and unhealthy expansion of white adipose tissue during high-fat feeding or due to leptin deficiency. Surprisingly, terminal embryonic adipogenesis is fully C/EBPα independent, but does however depend on PPARγ; cold-induced beige adipogenesis is also C/EBPα independent. Moreover, C/EBPα is not vital for adipocyte survival in the adult stage. We reveal a surprising diversity of transcriptional signals required at different stages of adipogenesis in vivo.
Assuntos
Adipócitos/fisiologia , Adipogenia , Tecido Adiposo Branco/citologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Metabolismo dos Carboidratos , Forma Celular , Dieta Hiperlipídica/efeitos adversos , Embrião de Mamíferos/citologia , Feminino , Técnicas de Inativação de Genes , Metabolismo dos Lipídeos , Masculino , Camundongos Obesos , Camundongos Transgênicos , Especificidade de Órgãos , PPAR gama/metabolismo , Transcrição GênicaRESUMO
As an adipokine in circulation, adiponectin has been extensively studied for its beneficial metabolic effects. While many important functions have been attributed to adiponectin under high-fat diet conditions, little is known about its essential role under regular chow. Employing a mouse model with inducible, acute ß-cell ablation, we uncovered an essential role of adiponectin under insulinopenic conditions to maintain minimal lipid homeostasis. When insulin levels are marginal, adiponectin is critical for insulin signaling, endocytosis, and lipid uptake in subcutaneous white adipose tissue. In the absence of both insulin and adiponectin, severe lipoatrophy and hyperlipidemia lead to lethality. In contrast, elevated adiponectin levels improve systemic lipid metabolism in the near absence of insulin. Moreover, adiponectin is sufficient to mitigate local lipotoxicity in pancreatic islets, and it promotes reconstitution of ß-cell mass, eventually reinstating glycemic control. We uncovered an essential new role for adiponectin, with major implications for type 1 diabetes.
Assuntos
Adiponectina/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Insulina/deficiência , Metabolismo dos Lipídeos , Regeneração , Adipócitos/metabolismo , Adipócitos/ultraestrutura , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Tecido Adiposo Branco/ultraestrutura , Animais , Cavéolas/metabolismo , Caveolina 1/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Homeostase/efeitos dos fármacos , Insulina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/toxicidade , Camundongos , Regeneração/efeitos dos fármacos , Estreptozocina , Análise de SobrevidaRESUMO
Elevated levels of TGFß are a negative prognostic indicator for patients diagnosed with pancreatic cancer; as a result, the TGFß pathway is an attractive target for therapy. However, clinical application of pharmacologic inhibition of TGFß remains challenging because TGFß has tumor suppressor functions in many epithelial malignancies, including pancreatic cancer. In fact, direct neutralization of TGFß promotes tumor progression of genetic murine models of pancreatic cancer. Here, we report that neutralizing the activity of murine TGFß receptor 2 using a monoclonal antibody (2G8) has potent antimetastatic activity in orthotopic human tumor xenografts, syngeneic tumors, and a genetic model of pancreatic cancer. 2G8 reduced activated fibroblasts, collagen deposition, microvessel density, and vascular function. These stromal-specific changes resulted in tumor cell epithelial differentiation and a potent reduction in metastases. We conclude that TGFß signaling within stromal cells participates directly in tumor cell phenotype and pancreatic cancer progression. Thus, strategies that inhibit TGFß-dependent effector functions of stromal cells could be efficacious for the therapy of pancreatic tumors. Cancer Res; 74(18); 4996-5007. ©2014 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Antimetabólitos Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Metástase Neoplásica , Neoplasias Pancreáticas/metabolismo , Distribuição Aleatória , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Almost 20 years have passed since the first laboratory evidence emerged that an abundant message encoding a protein with homology to the C1q superfamily is highly specifically expressed in adipocytes. At this stage, we refer to this protein as adiponectin. Despite more than 10,000 reports in the literature since its initial description, we seem to have written only the first chapter in the textbook on adiponectin physiology. With every new aspect we learn about adiponectin, a host of new questions arise with respect to the underlying molecular mechanisms. Here, we aim to summarize recent findings in the field and bring the rodent studies that suggest a causal relationship between adiponectin levels in plasma and systemic insulin sensitivity in perspective with the currently available data on the clinical side.
RESUMO
The inositol 1,4,5-trisphosphate receptors (IP3Rs) as ligand-gated Ca(2)(+) channels are key modulators of cellular processes. Despite advances in understanding their critical role in regulating neuronal function and cell death, how this family of proteins impact cell metabolism is just emerging. Unexpectedly, a transgenic mouse line (D2D) exhibited progressive glucose intolerance as a result of transgene insertion. Inverse PCR was used to identify the gene disruption in the D2D mice. This led to the discovery that Itpr1 is among the ten loci disrupted in chromosome 6. Itpr1 encodes for IP3R1, the most abundant IP3R isoform in mouse brain and also highly expressed in pancreatic ß-cells. To study IP3R1 function in glucose metabolism, we used the Itpr1 heterozygous mutant mice, opt/+. Glucose homeostasis in male mice cohorts was examined by multiple approaches of metabolic phenotyping. Under regular diet, the opt/+ mice developed glucose intolerance but no insulin resistance. Decrease in second-phase glucose-stimulated blood insulin level was observed in opt/+ mice, accompanied by reduced ß-cell mass and insulin content. Strikingly, when fed with high-fat diet, the opt/+ mice were more susceptible to the development of hyperglycemia, glucose intolerance, and insulin resistance. Collectively, our studies identify the gene Itpr1 being interrupted in the D2D mice and uncover a novel role of IP3R1 in regulation of in vivo glucose homeostasis and development of diet-induced diabetes.