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OBJECTIVES: Referencing literature and professional experiences of multiple academic affairs deans in Doctor of Pharmacy degree granting programs, this commentary examines ways offices of academic affairs (OAA) play an integral part in the professional identity formation (PIF) of students, faculty, preceptors, and staff. Overlapping roles of academic affairs and other program and university offices are examined with a focus on approaches that impact student PIF. FINDINGS: While size, organizational structure, and roles or responsibilities can vary among OAA within pharmacy education, five focus areas are identified as opportunities where most, if not all, OAA can positively impact PIF for students. These five areas include 1) curriculum design, delivery, and operations, 2) leadership, 3) professional development, 4) student support and progression, and 5) accreditation. SUMMARY: OAA can help to shape PIF within pharmacy education in a variety of ways. Identifying and reflecting upon the five focus areas described in this paper may help individuals within OAA units recognize where PIF efforts may be developed, refined, or assessed within their program. By intentionally layering PIF strategies across these five focus areas, OAA personnel and those from other units within and outside of the university system may thoughtfully work together to develop graduates who have increased self-awareness, confidence, and ability to lead within the profession of pharmacy.
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Vertical sleeve gastrectomy (VSG) is one of the most effective and durable therapies for morbid obesity and its related complications. Although bile acids (BAs) have been implicated as downstream mediators of VSG, the specific mechanisms through which BA changes contribute to the metabolic effects of VSG remain poorly understood. Here, we confirm that high fat diet-fed global farnesoid X receptor (Fxr) knockout mice are resistant to the beneficial metabolic effects of VSG. However, the beneficial effects of VSG were retained in high fat diet-fed intestine- or liver-specific Fxr knockouts, and VSG did not result in Fxr activation in the liver or intestine of control mice. Instead, VSG decreased expression of positive hepatic Fxr target genes, including the bile salt export pump (Bsep) that delivers BAs to the biliary pathway. This reduced small intestine BA levels in mice, leading to lower intestinal fat absorption. These findings were verified in sterol 27-hydroxylase (Cyp27a1) knockout mice, which exhibited low intestinal BAs and fat absorption and did not show metabolic improvements following VSG. In addition, restoring small intestinal BA levels by dietary supplementation with taurocholic acid (TCA) partially blocked the beneficial effects of VSG. Altogether, these findings suggest that reductions in intestinal BAs and lipid absorption contribute to the metabolic benefits of VSG.
Assuntos
Colestanotriol 26-Mono-Oxigenase/genética , Gastrectomia/métodos , Obesidade Mórbida/cirurgia , Receptores Citoplasmáticos e Nucleares/genética , Animais , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/metabolismo , Dieta Hiperlipídica/efeitos adversos , Humanos , Metabolismo dos Lipídeos/genética , Lipídeos/genética , Camundongos , Camundongos Knockout , Obesidade Mórbida/metabolismo , Obesidade Mórbida/fisiopatologia , Redução de Peso/genéticaRESUMO
The 2018-2019 Student Affairs Standing Committee addressed charges related to examining the institutional leadership models and professional development needs of faculty and staff to optimize achievement of Strategic Priority #1 on the applicant pipeline. The report provides five recommendations to AACP and twelve suggestions for colleges and schools of pharmacy. The committee focused on the need to develop tailored leadership training and mentoring programs for admissions personnel on relevant topics, including change management, holistic thinking, leadership, problem solving, technical knowledge, professional development, paths for promotion, conflict resolution, networking, persuasive communication, and strategic planning. Rather than develop new resources, the committee identified existing professional competencies and assessment resources developed by other organizations for student affairs and admissions personnel that could spur enhanced strategic marketing and professional development opportunities in pharmacy education. It also reaffirmed the need for student diversity and the use of data to drive strategic decisions in recruitment. To identify gaps in knowledge among AACP member institutions, the committee analyzed the results of its fall 2018 survey on the current depth and breadth of student recruitment activities and their perceived effectiveness. The committee also recommended ways institutions can encourage faculty and others outside of the admissions office to participate in student recruitment activities. Finally, the committee concluded that it will be necessary for colleges and schools to collaborate across the academy to promote the benefits of pharmacy profession to prospective students, rather than individual colleges and schools of pharmacy, and be more responsive to the expectations of Gen Z students before the tide in applications will shift in a positive direction.
Assuntos
Faculdades de Farmácia/organização & administração , Educação em Farmácia/organização & administração , Docentes/organização & administração , Humanos , Liderança , Assistência Farmacêutica/organização & administração , Farmácia/organização & administração , Estudantes de FarmáciaRESUMO
The 2016-2017 AACP Student Affairs Standing Committee addressed charges related to recruitment to the profession of pharmacy and a national awareness campaign for pharmacy careers, as well as promotion of student wellness and stress management. The Committee report provides six recommendations to the American Association of Colleges of Pharmacy (AACP) and one proposed policy statement for the AACP House of Delegates related to recruitment to the pharmacy profession. The Committee report also provides three recommendations to AACP and one proposed policy statement for the AACP House of Delegates related to student wellness and stress management. In addition, this report provides recommendations for future AACP Student Affairs Standing Committee work.
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Educação em Farmácia , Faculdades de Farmácia , Sociedades Farmacêuticas , Estudantes de Farmácia , Comitês Consultivos , Relatórios Anuais como Assunto , Humanos , Política Organizacional , Estados UnidosRESUMO
BACKGROUND: Obesity and inflammatory bowel disease (IBD) represent chronic inflammatory conditions. Bariatric surgery improves some obesity-related co-morbidities, but the effects of bariatric surgery on IBD have not been well studied. OBJECTIVES: To examine if bariatric surgery may attenuate colitis in an obese murine model of IBD and study the mechanisms underlying the postsurgical amelioration of intestinal inflammation. SETTING: University of California Irvine, Department of Surgery and Microbiology laboratories. METHODS: Obese mice were assigned to one of 2 bariatric procedures [Duodenojejunal Bypass (DJB n = 6), Sleeve Gastrectomy (SG n = 8)]. Sham-operated mice were (Sham n = 8) were used as a control. After recovering from surgery, IBD was induced by administration of 2% dextran sodium sulfate. Fecal samples were collected before and after IBD induction for microbiome analysis. Pathologic analyses and immunohistochemical staining were performed on colon. RESULTS: Survival after DJB and SG was higher relative to Sham mice. Histologically, DJB mice had significantly less intestinal inflammation. The observed improvements were not related to a difference in weight among the groups. Farnesoid X receptor staining in the colon was observed quantitatively more in DJB than in SG and sham mice. A statistically significant increase in the number of Lactobacillales was observed in the stool of mice after DJB. CONCLUSION: These results suggest that bariatric surgery, in particular DJB, reduces the severity of colitis in a chemically-induced IBD murine model. The anticolitis effects of DJB may be associated with Farnesoid X receptor regulation and gut microbiome rearrangements.
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Cirurgia Bariátrica , Colite Ulcerativa/complicações , Colo/patologia , Obesidade/cirurgia , Animais , Colite Ulcerativa/diagnóstico , Modelos Animais de Doenças , Progressão da Doença , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/complicaçõesRESUMO
UNLABELLED: Vertical sleeve gastrectomy (VSG) is one of the most commonly performed clinical bariatric surgeries used for the remission of obesity and diabetes. However, the precise molecular mechanism by which VSG exerts its beneficial effects remains elusive. We report that the membrane-bound G protein-coupled bile acid receptor, GPBAR-1 (also known as TGR5), is required to mediate the effects of anti-obesity, anti-hyperglycemia, and improvements of fatty liver of VSG in mice. In the absence of TGR5, the beneficial metabolic effects of VSG in mice are lost. Moreover, we found that the expression of TGR5 increased significantly after VSG, and VSG alters both BA levels and composition in mice, resulting in enhancement of TGR5 signaling in the ileum and brown adipose tissues, concomitant with improved glucose control and increased energy expenditure. CONCLUSION: Our study elucidates a novel underlying mechanism by which VSG achieves its postoperative therapeutic effects through enhanced TGR5 signaling. (Hepatology 2016;64:760-773).
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Gastrectomia , Receptores Acoplados a Proteínas G/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Ácidos e Sais Biliares/sangue , Metabolismo Energético , Fígado Gorduroso/cirurgia , Íleo/metabolismo , Resistência à Insulina , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética , Redução de PesoRESUMO
GP-BAR1 (also known as TGR5), a novel G-protein coupled receptor regulating various non-genomic functions via bile acid signaling, has emerged as a promising target for metabolic disorders, including obesity and type II diabetes. However, given that many bile acids (BAs) are poorly tolerated for systemic therapeutic use, there is significant need to develop GP-BAR1 agonists with improved potency and specificity and there also is significant impetus to develop a stereoselective synthetic methodology for GP-BAR1 agonists. Here, we report the development of highly stereo-controlled strategies to investigate a series of naturally occurring bile acid derivatives with markedly enhanced GP-BAR1 activity. These novel GP-BAR1 agonists are evaluated in vitro using luciferase-based reporter and cAMP assays to elucidate their biological properties. In vivo studies revealed that the GP-BAR1 agonist 23(S)-m-LCA increased intestinal GLP-1 transcripts by 26-fold. Additionally, computational modeling studies of selected ligands that exhibit enhanced potency and specificity for GP-BAR1 provide information on potential binding sites for these ligands in GP-BAR1.
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Ácidos e Sais Biliares/síntese química , Modelos Moleculares , Receptores Acoplados a Proteínas G/agonistas , Sequência de Aminoácidos , Animais , Ácidos e Sais Biliares/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , EstereoisomerismoRESUMO
Liver X receptors (Lxrα and Lxrß) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinson's disease.
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Mesencéfalo/metabolismo , Neurogênese , Receptores Nucleares Órfãos/metabolismo , Animais , Mapeamento Encefálico/métodos , Diferenciação Celular , Núcleo Celular/metabolismo , Colesterol/análogos & derivados , Colesterol/metabolismo , Ácido Cólico/metabolismo , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/citologia , Ligantes , Receptores X do Fígado , Camundongos , Modelos Biológicos , Fatores de Tempo , Transfecção , Peixe-ZebraRESUMO
A three-layer microfluidic device was developed that combined perfusion of cultured cells with on-line chemical analysis for near real-time monitoring of cellular secretions. Two layers were reversibly sealed to form a cell chamber that allowed cells grown on coverslips to be loaded directly into the chip. The outlet of the chamber was in fluidic contact with a third layer that was permanently bonded. Perfusate from the cell chamber flowed into this third layer where a fluorescence enzyme assay for non-esterified fatty acid (NEFA) was performed on-line. The device was used to monitor efflux of NEFAs from approximately 6,200 cultured adipocytes with 83 s temporal resolution. Perfusion of murine 3T3-L1 cultured adipocytes resulted in an average basal concentration of 24.2 +/- 2.4 microM NEFA (SEM, n = 6) detected in the effluent corresponding to 3.31 x 10(-5) nmol cell(-1) min(-1). Upon pharmacological treatment with a beta-adrenergic agonist to stimulate lipolysis, a 6.9 +/- 0.7-fold (SEM, n = 6) sustained increase in NEFA secretion was observed. This multilayer device provides a versatile platform that could be adapted for use with other cell types to study corresponding cellular secretions in near real-time.
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Adipócitos/química , Adipócitos/metabolismo , Ácidos Graxos não Esterificados/análise , Técnicas Analíticas Microfluídicas/métodos , Células 3T3-L1 , Animais , Técnicas de Cultura de Células , Ácidos Graxos não Esterificados/metabolismo , Lipólise , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Sistemas On-Line/instrumentaçãoRESUMO
In this study, we explored the roles of microRNAs in adipocyte differentiation and metabolism. We first knocked down Argonaute2 (Ago2), a key enzyme in the processing of micro-RNAs (miRNAs), to investigate a potential role for miRNAs in adipocyte differentiation and/or metabolism. Although we did not observe dramatic differences in adipogenesis between Ago2 knock-down and control 3T3-L1 cells, incorporation of [(14)C]glucose or acetate into triacylglycerol, and steady-state levels of triacyglycerol were all reduced, suggesting a role for miRNAs in adipocyte metabolism. To study roles of specific miRNAs in adipocyte biology, we screened for miRNAs that are differentially expressed between preadipocytes and adipocytes for the 3T3-L1 and ST2 cell lines. Distinct subsets of miRNAs decline or increase during adipocyte conversion, whereas most miRNAs are not regulated. One locus encoding two miRNAs, 378/378*, contained within the intron of PGC-1beta is highly induced during adipogenesis. When overexpressed in ST2 mesenchymal precursor cells, miRNA378/378* increases the size of lipid droplets and incorporation of [(14)C]acetate into triacylglycerol. Although protein and mRNA expression levels of C/EBPalpha, C/EBPbeta, C/EBPdelta, and PPARgamma1 are unchanged, microarray and quantitative RT-PCR analyses indicate that a set of lipogenic genes are upregulated, perhaps due to increased expression of PPARgamma2. Knock-down of miRNA378 and/or miRNA378* decreases accumulation of triacylglycerol. Interestingly, we made the unexpected finding that miRNA378/378* specifically increases transcriptional activity of C/EBPalpha and C/EBPbeta on adipocyte gene promoters.
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Adipócitos/metabolismo , Expressão Gênica/fisiologia , Lipogênese/fisiologia , MicroRNAs/genética , Células 3T3-L1 , Animais , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/fisiologia , Expressão Gênica/genética , Lipídeos/biossíntese , Lipogênese/genética , Luciferases/genética , Camundongos , MicroRNAs/isolamento & purificação , Análise em Microsséries , PPAR gama/biossíntese , PPAR gama/genética , Plasmídeos , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/fisiologia , Transfecção , Triglicerídeos/metabolismoRESUMO
Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that functions as a master transcriptional regulator of adipocyte conversion. During PPARgamma transactivation, multiple signaling pathways interact with one another, leading to the differentiation of both white and brown adipose tissue. Ligand activation of the PPARgamma-RXR heterodimer complex also enhances insulin sensitivity, and this property has been heavily exploited to develop effective pharmacotherapies for the treatment of type 2 diabetes mellitus. PPARgamma is also expressed in stem cells and plays a critical role in mesenchymal stromal cell differentiation and lineage determination events. The many facets of PPARgamma activity within the bone marrow niche where adipocytes, osteoblasts, and hematopoietic cells reside make this molecule an attractive target for pharmacological investigation. Additional findings that osteoblasts can alter energy metabolism by influencing adiposity and insulin sensitivity, and observations of decreased bone turnover in diabetic subjects, underscore the contribution of the skeleton to systemic energy requirements. Studies into the role of PPARgamma in skeletal acquisition and maintenance may lead to a better understanding of the molecular mechanisms governing stromal cell differentiation in the mesenchyme compartment and whether PPARgamma activity can be manipulated to benefit skeletal remodeling events and energy metabolism.
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Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/fisiologia , Osso e Ossos/fisiologia , PPAR gama/fisiologia , Adipogenia/fisiologia , Animais , Remodelação Óssea/fisiologia , Osso e Ossos/citologia , Humanos , Células-Tronco Mesenquimais/fisiologia , EsqueletoRESUMO
Wnts are secreted, lipidated proteins that regulate multiple aspects of brain development, including dopaminergic neuron development. In this study, we perform the first purification and signaling analysis of Wnt2 and define the function of Wnt2 in ventral midbrain precursor cultures, as well as in Wnt2-null mice in vivo. We found that purified Wnt2 induces the phosphorylation of both Lrp5/6 and Dvl-2/3, and activates beta-catenin in SN4741 dopaminergic cells. Moreover, purified Wnt2 increases progenitor proliferation, and the number of dopaminergic neurons in ventral midbrain precursor cultures. In agreement with these findings, analysis of the ventral midbrain of developing Wnt2-null mice revealed a decrease in progenitor proliferation and neurogenesis that lead to a decrease in the number of postmitotic precursors and dopaminergic neurons. Collectively, our observations identify Wnt2 as a novel regulator of dopaminergic progenitors and dopaminergic neuron development.
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Proliferação de Células , Mesencéfalo , Células-Tronco/fisiologia , Proteína Wnt2/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Dopamina/metabolismo , Feminino , Mesencéfalo/citologia , Mesencéfalo/embriologia , Camundongos , Camundongos Knockout , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Gravidez , Processamento de Proteína Pós-Traducional , Células-Tronco/citologia , Proteína Wnt2/genética , Proteína Wnt2/isolamento & purificação , beta Catenina/metabolismoRESUMO
Wnts are known to bind and activate multiple membrane receptors/coreceptors and to regulate dopaminergic (DA) neuron development and ventral midbrain (VM) morphogenesis. The low density lipoprotein receptor-related protein (Lrp6) is a Wnt co-receptor, yet it remains unclear whether Lrp6 is required for DA neuron development or VM morphogenesis. Lrp6 is expressed ubiquitously in the developing VM. In this study, we show that Lrp6(-/-) mice exhibit normal patterning, proliferation and cell death in the VM, but display a delay in the onset of DA precursor differentiation. A transient 50% reduction in tyrosine hydroxylase-positive DA neurons and in the expression of DA markers such as Nurr1 and Pitx3, as well as a defect in midbrain morphogenesis was detected in the mutant embryos at embryonic day 11.5. Our results, therefore, suggest a role for Lrp6 in the onset of DA neuron development in the VM as well as a role in midbrain morphogenesis.
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Diferenciação Celular/genética , Dopamina/metabolismo , Mesencéfalo/embriologia , Morfogênese/genética , Neurônios/citologia , Animais , Bromodesoxiuridina , Diferenciação Celular/fisiologia , Genótipo , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Microscopia Confocal , Neurônios/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Control over progenitor proliferation and neurogenesis remains a key challenge for stem cell neurobiology and a prerequisite for successful stem cell replacement therapies for neurodegenerative diseases like Parkinson's disease (PD). Here, we examined the function of two nuclear receptors, liver X receptors (Lxralpha and beta) and their ligands, oxysterols, as regulators of cell division, ventral midbrain (VM) neurogenesis, and dopaminergic (DA) neuron development. Deletion of Lxrs reduced cell cycle progression and VM neurogenesis, resulting in decreased DA neurons at birth. Activation of Lxrs with oxysterol ligands increased the number of DA neurons in mouse embryonic stem cells (ESCs) and in wild-type but not Lxralphabeta(-/-) VM progenitor cultures. Likewise, oxysterol treatment of human ESCs (hESCs) during DA differentiation increased neurogenesis and the number of mature DA neurons, while reducing proliferating progenitors. Thus, Lxr ligands may improve current hESC replacement strategies for PD by selectively augmenting the generation of DA neurons.
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Colesterol/análogos & derivados , Colesterol/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Mesencéfalo/citologia , Neurogênese/efeitos dos fármacos , Receptores Nucleares Órfãos/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Dopamina/metabolismo , Células-Tronco Embrionárias/citologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Receptores X do Fígado , Mesencéfalo/efeitos dos fármacos , Camundongos , Neurogênese/genética , Receptores Nucleares Órfãos/genética , Reação em Cadeia da PolimeraseRESUMO
In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC)-mass spectrometry (MS) and tandem mass spectrometry (MS(n)) low levels of oxysterols (estimated 2-165 ng g(-1) wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7 alpha-, 7 beta-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 microg g(-1) in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng g(-1) in Ctx and 13 ng g(-1) in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng g(-1) in Ctx and 91 ng g(-1) in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg g(-1) in Ctx and 1.15 mg g(-1) in Sc) and its precursors were determined by gas chromatography-mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain development.
Assuntos
Sistema Nervoso Central/embriologia , Esteróis/análise , Animais , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Colesterol/análise , Colesterol/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , Medula Espinal/metabolismo , Esteróis/metabolismoRESUMO
A dual-chip microfluidic platform that coupled perfusion of cultured adipocytes with on-line fluorescence-based enzyme assay was developed to monitor glycerol secretions in real time from cultured adipocytes. The perfusion cell chip, which could be reversibly sealed to allow reloading of cells and reuse of the chip, was shown by modeling to generate low shear stress on the cells under study. Effluent from the perfusion chip was pumped into an enzyme assay chip for monitoring of secretion from the cells. The on-line enzyme assay had a limit of detection (LOD) of 4 microM glycerol. The temporal resolution of the combined system for detecting changes in glycerol concentration was 90 s. The microfluidic device was used to continuously monitor glycerol secretion from murine 3T3-L1 adipocytes, grown and differentiated on glass coverslips, for at least 2 h. An average basal glycerol concentration of 28 microM was detected in the effluent. Pharmacological treatment with a beta-adrenergic agonist to stimulate lipolysis evoked a 3-fold increase in glycerol secretion followed by sustained release that was 40% higher than basal concentration. The ability to monitor changes in cellular secretion over time may provide insight into adipocyte metabolism and the dysregulation that occurs with obesity-related disorders.
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Adipócitos/metabolismo , Ensaios Enzimáticos/métodos , Glicerol/análise , Células 3T3-L1 , Agonistas Adrenérgicos beta/farmacologia , Animais , Ensaios Enzimáticos/instrumentação , Corantes Fluorescentes/química , Camundongos , Técnicas Analíticas Microfluídicas , Fatores de TempoRESUMO
UNLABELLED: Overexpression of Wnt10b from the osteocalcin promoter in transgenic mice increases postnatal bone mass. Increases in osteoblast perimeter, mineralizing surface, and bone formation rate without detectable changes in pre-osteoblast proliferation, osteoblast apoptosis, or osteoclast number and activity suggest that, in this animal model, Wnt10b primarily increases bone mass by stimulating osteoblastogenesis. INTRODUCTION: Wnt signaling regulates many aspects of development including postnatal accrual of bone. Potential mechanisms for how Wnt signaling increases bone mass include regulation of osteoblast and/or osteoclast number and activity. To help differentiate between these possibilities, we studied mice in which Wnt10b is expressed specifically in osteoblast lineage cells or in mice devoid of Wnt10b. MATERIALS AND METHODS: Transgenic mice, in which mouse Wnt10b is expressed from the human osteocalcin promoter (Oc-Wnt10b), were generated in C57BL/6 mice. Transgene expression was evaluated by RNase protection assay. Quantitative assessment of bone variables was done by radiography, muCT, and static and dynamic histomorphometry. Mechanisms of bone homeostasis were evaluated with assays for BrdU, TUNEL, and TRACP5b activity, as well as serum levels of C-terminal telopeptide of type I collagen (CTX). The endogenous role of Wnt10b in bone was assessed by dynamic histomorphometry in Wnt10b(-/-) mice. RESULTS: Oc-Wnt10b mice have increased mandibular bone and impaired eruption of incisors during postnatal development. Analyses of femoral distal metaphyses show significantly higher BMD, bone volume fraction, and trabecular number. Increased bone formation is caused by increases in number of osteoblasts per bone surface, rate of mineral apposition, and percent mineralizing surface. Although number of osteoclasts per bone surface is not altered, Oc-Wnt10b mice have increased total osteoclast activity because of higher bone mass. In Wnt10b(-/-) mice, changes in mineralizing variables and osteoblast perimeter in femoral distal metaphyses were not observed; however, bone formation rate is reduced because of decreased total bone volume and trabecular number. CONCLUSIONS: High bone mass in Oc-Wnt10b mice is primarily caused by increased osteoblastogenesis, with a minor contribution from elevated mineralizing activity of osteoblasts.
Assuntos
Diferenciação Celular , Osteoblastos/metabolismo , Osteocalcina , Osteogênese , Células-Tronco/metabolismo , Proteínas Wnt/biossíntese , Fosfatase Ácida/biossíntese , Fosfatase Ácida/genética , Animais , Animais Recém-Nascidos , Apoptose/genética , Densidade Óssea/genética , Diferenciação Celular/genética , Proliferação de Células , Homeostase/genética , Humanos , Incisivo/crescimento & desenvolvimento , Incisivo/metabolismo , Incisivo/patologia , Isoenzimas/biossíntese , Isoenzimas/genética , Mandíbula/crescimento & desenvolvimento , Mandíbula/metabolismo , Mandíbula/patologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Tamanho do Órgão/genética , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Células-Tronco/patologia , Fosfatase Ácida Resistente a Tartarato , Transgenes , Proteínas Wnt/genéticaRESUMO
Nurr1 is an orphan nuclear receptor required for the development of midbrain dopaminergic neurons. To better understand the molecular consequences of Nurr1 expression, we compared the transcriptomes of two independent control and Nurr1-expressing NSC lines using Affymetrix cDNA microarrays. These data reveal the regulation of genes involved in promoting cell survival (trophic/growth factors and stress response genes) and in preventing cell death (decreased caspase-3 and caspase-11 expression). We found that conditioned medium from Nurr1-expressing NSC lines enhanced the survival of midbrain dopaminergic neurons in primary cultures and that Nurr1-expressing NSC lines themselves were more resistant to oxidative stress. These findings are accompanied by a dynamic pattern of gene regulation that is consistent with a role for Nurr1 in promoting both the acquisition of brain-region-specific identity (Engrailed-1) and neuronal differentiation (tubulin beta III). Interestingly, our gene expression profiles suggested that tenascin-C was regulated by Nurr1 in developing dopaminergic neurons. This was further confirmed in vitro and in Nurr1 knockout mice where low levels of tenascin-C mRNA were observed. Analysis of tenascin-C-null mice revealed an increase in the number of Nurr1(+) cells that become tyrosine hydroxylase-positive (TH(+)) dopaminergic neurons at embryonic day 11.5, suggesting that tenascin-C normally delays the acquisition of TH by Nurr1(+) precursors. Thus, our results confirm the presence of both secreted and cell-intrinsic survival signals modulated by Nurr1 and suggest that Nurr1 is a key regulator of both survival and dopaminergic differentiation.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Neurônios/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Dopamina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Estresse Oxidativo/efeitos dos fármacos , Reprodutibilidade dos Testes , Células-Tronco/efeitos dos fármacos , Tenascina/deficiência , Tenascina/metabolismo , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Cognitive decline in Alzheimer's disease (AD) stems from the progressive dysfunction of synaptic connections within cortical neuronal microcircuits. Recently, soluble amyloid beta protein oligomers (Abeta(ol)s) have been identified as critical triggers for early synaptic disorganization. However, it remains unknown whether a deficit of Hebbian-related synaptic plasticity occurs during the early phase of AD. Therefore, we studied whether age-dependent Abeta accumulation affects the induction of spike-timing-dependent synaptic potentiation at excitatory synapses on neocortical layer 2/3 (L2/3) pyramidal cells in the APPswe/PS1dE9 transgenic mouse model of AD. Synaptic potentiation at excitatory synapses onto L2/3 pyramidal cells was significantly reduced at the onset of Abeta pathology and was virtually absent in mice with advanced Abeta burden. A decreased alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/N-methyl-D-aspartate (NMDA) receptor-mediated current ratio implicated postsynaptic mechanisms underlying Abeta synaptotoxicity. The integral role of Abeta(ol)s in these processes was verified by showing that pretreatment of cortical slices with Abeta((25-35)ol)s disrupted spike-timing-dependent synaptic potentiation at unitary connections between L2/3 pyramidal cells, and reduced the amplitude of miniature excitatory postsynaptic currents therein. A robust decrement of AMPA, but not NMDA, receptor-mediated currents in nucleated patches from L2/3 pyramidal cells confirmed that Abeta(ol)s perturb basal glutamatergic synaptic transmission by affecting postsynaptic AMPA receptors. Inhibition of AMPA receptor desensitization by cyclothiazide significantly increased the amplitude of excitatory postsynaptic potentials evoked by afferent stimulation, and rescued synaptic plasticity even in mice with pronounced Abeta pathology. We propose that soluble Abeta(ol)s trigger the diminution of synaptic plasticity in neocortical pyramidal cell networks during early stages of AD pathogenesis by preferentially targeting postsynaptic AMPA receptors.