RESUMEN
The emergence of drug-resistant viruses is a serious concern in current chemotherapy for human immunodeficiency virus type-1 (HIV-1) infectious diseases. Hence, antiviral drugs aiming at targets that are different from those of approved drugs are still required, and the RNase H activity of HIV-1 reverse transcriptase is a suitable target. In this study, a search of a series of natural compounds was performed to identify the RNase H inhibitors. Three compounds were found to block the RNase H enzymatic activity. A laccaic acid skeleton was observed in all three natural compounds. A hydroxy phenyl group is connected to an anthraquinone backbone in the skeleton. An acetamido-ethyl, amino-carboxy-ethyl, and amino-ethyl are bound to the phenyl in laccaic acids A, C, and E, respectively. Laccaic acid C showed a 50% inhibitory concentration at 8.1 µM. Laccaic acid C also showed inhibitory activity in a cell-based viral proliferation assay. Binding structures of these three laccaic acids were determined by X-ray crystallographic analysis using a recombinant protein composed of the HIV-1 RNase H domain. Two divalent metal ions were located at the catalytic center in which one carbonyl and two hydroxy groups on the anthraquinone backbone chelated two metal ions. Molecular dynamics simulations were performed to examine the stabilities of the binding structures. Laccaic acid C showed the strongest binding to the catalytic site. These findings will be helpful for the design of potent inhibitors with modification of laccaic acids to enhance the binding affinity.
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Infecciones por VIH , Ribonucleasa H , Humanos , Ribonucleasa H/metabolismo , Iones , Antraquinonas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/químicaRESUMEN
The Repeat Expansion Diseases, a large group of human diseases that includes the fragile X-related disorders (FXDs) and Huntington's disease (HD), all result from expansion of a disease-specific microsatellite via a mechanism that is not fully understood. We have previously shown that mismatch repair (MMR) proteins are required for expansion in a mouse model of the FXDs, but that the FANCD2 and FANCI associated nuclease 1 (FAN1), a component of the Fanconi anemia (FA) DNA repair pathway, is protective. FAN1's nuclease activity has been reported to be dispensable for protection against expansion in an HD cell model. However, we show here that in a FXD mouse model a point mutation in the nuclease domain of FAN1 has the same effect on expansion as a null mutation. Furthermore, we show that FAN1 and another nuclease, EXO1, have an additive effect in protecting against MSH3-dependent expansions. Lastly, we show that the loss of FANCD2, a vital component of the Fanconi anemia DNA repair pathway, has no effect on expansions. Thus, FAN1 protects against MSH3-dependent expansions without diverting the expansion intermediates into the canonical FA pathway and this protection depends on FAN1 having an intact nuclease domain.
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Dominio Catalítico , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Enzimas Multifuncionales/metabolismo , Expansión de Repetición de Trinucleótido , Animales , Enzimas Reparadoras del ADN/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Ratones , Ratones Endogámicos C57BL , Enzimas Multifuncionales/química , Enzimas Multifuncionales/genética , Proteína 3 Homóloga de MutS/metabolismo , Mutación PuntualRESUMEN
Chemotherapy of human immunodeficiency virus type-1 (HIV-1) has significantly developed over the last three decades. The emergence of drug-resistant variants is, however, still a severe problem. The RNase H activity of HIV-1 reverse transcriptase is an attractive target for a new class of antiviral drugs because there is no approved inhibitor. The nitro-furan-carbonyl and nitro-thiophene-carbonyl groups are potent scaffolds for the HIV-1 RNase H inhibitor. In this work, the binding structures of six inhibitory compounds were obtained by X-ray crystal analysis in a complex with a recombinant protein of HIV-1 RNase H domain. Every inhibitory compound was found to be bound to the catalytic site with the furan- or thiophene-ring coordinated to two divalent metal ions at the binding pocket. All the atoms in nitro, furan, carbonyl, and two metals were aligned in the nitro-furan derivatives. The straight line connecting nitro and carboxyl groups was parallel to the plane made by two metal ions and a furan O atom. The binding modes of the nitro-thiophene derivatives were slightly different from those of the nitro-furan ones. The nitro and carbonyl groups deviated from the plane made by two metals and a thiophene S atom. Molecular dynamics simulations suggested that the furan O or thiophene S atom and carbonyl O atom were firmly coordinated to the metal ions. The simulations made the planar nitro-furan moiety well aligned to the line connecting the two metal ions. In contrast, the nitro-thiophene derivatives were displaced from the initial positions after the simulations. The computational findings will be a sound basis for developing potent inhibitors for HIV-1 RNase H activity.
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Fármacos Anti-VIH , VIH-1 , Ribonucleasa H , Humanos , Dominio Catalítico , Cristalografía por Rayos X , Furanos/farmacología , Furanos/química , Transcriptasa Inversa del VIH , VIH-1/efectos de los fármacos , VIH-1/enzimología , Metales/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/química , Ribonucleasa H/antagonistas & inhibidores , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacologíaRESUMEN
G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic ß-cells. ß-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of ß-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic ß-cells, knockdown of CK2α expression, or genetic deletion of CK2α in ß-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of ß-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on ß-cell GPCRs may represent novel therapeutic targets.
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Quinasa de la Caseína II/fisiología , Insulina/metabolismo , Receptor Muscarínico M3/fisiología , Animales , Células COS , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Naftiridinas/farmacología , FenazinasRESUMEN
While significant progress has been achieved in utilizing remote sensing technologies for landslide investigation in China, there remains a notable gap in consolidating information on applicable conditions, application stages, and workflows across various remote sensing methodologies. This paper proposes a comprehensive framework for active landslide detection, incorporating multiple stages and data sources, successfully implemented in a vast region of southwestern China. Furthermore, detailed discussions are provided on the effects of the geometric distortion, land cover type, and various InSAR methods on the accuracy of active landslide identification results. Additionally, the paper delves into the advantages of integrated remote sensing technology in active landslide investigation, encompassing the assessment of current landslide activity status, precise delineation of boundaries, identification of different deformation stages, and determination of damage patterns. Through comprehensive analysis of multisource data, it enhances understanding of the active landslide process, ultimately contributing to the mitigation of casualties and property damage.
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Ghrelin is a gastric peptide hormone that controls appetite and energy homeostasis. Plasma ghrelin levels rise before a meal and fall quickly thereafter. Elucidation of the regulation of ghrelin secretion has been hampered by the difficulty of directly interrogating ghrelin cells diffusely scattered within the complex gastric mucosa. Therefore, we generated transgenic mice with ghrelin cell expression of green fluorescent protein (GFP) to enable characterization of ghrelin secretion in a pure population of isolated gastric ghrelin-expressing GFP (Ghr-GFP) cells. Using quantitative RT-PCR and immunofluorescence staining, we detected a high level of expression of the long-chain fatty acid (LCFA) receptor GPR120, while the other LCFA receptor, GPR40, was undetectable. In short-term-cultured pure Ghr-GFP cells, the LCFAs docosadienoic acid, linolenic acid, and palmitoleic acid significantly suppressed ghrelin secretion. The physiological mechanism of LCFA inhibition on ghrelin secretion was studied in mice. Serum ghrelin levels were transiently suppressed after gastric gavage of LCFA-rich lipid in mice with pylorus ligation, indicating that the ghrelin cell may directly sense increased gastric LCFA derived from ingested intraluminal lipids. Meal-induced increase in gastric mucosal LCFA was assessed by measuring the transcripts of markers for tissue uptake of LCFA, lipoprotein lipase (LPL), fatty acid translocase (CD36), glycosylphosphatidylinositol-anchored HDL-binding protein 1, and nuclear fatty acid receptor peroxisome proliferator-activated receptor-γ. Quantitative RT-PCR studies indicate significantly increased mRNA levels of lipoprotein lipase, glycosylphosphatidylinositol-anchored HDL-binding protein 1, and peroxisome proliferator-activated receptor-γ in postprandial gastric mucosa. These results suggest that meal-related increases in gastric mucosal LCFA interact with GPR120 on ghrelin cells to inhibit ghrelin secretion.
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Ghrelina/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Animales , Células Cultivadas , Ácidos Grasos/farmacología , Mucosa Gástrica/metabolismo , Ratones , Ratones Transgénicos , Periodo Posprandial , ARN Mensajero/metabolismoRESUMEN
Impaired functioning of pancreatic beta cells is a key hallmark of type 2 diabetes. beta cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific beta cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that beta cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific beta cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in beta cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (G(q/11) versus G(s)). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either beta cell G(q/11) or G(s) G proteins. Here we report the findings that conditional and selective activation of beta cell G(q/11) signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated beta cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of beta cell G(s) triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of beta cell function in vivo.
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Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/fisiología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/anatomía & histología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Animales , Células COS , Chlorocebus aethiops , Clozapina/análogos & derivados , Clozapina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Ratones Transgénicos , Ensayo de Unión Radioligante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Obesity is the major driver of the global epidemic in type 2 diabetes (T2D). In individuals with obesity, impaired insulin action leads to increased lipolysis in adipocytes, resulting in elevated plasma free fatty acid (FFA) levels that promote peripheral insulin resistance, a hallmark of T2D. Here we show, by using a combined genetic/biochemical/pharmacologic approach, that increased adipocyte lipolysis can be prevented by selective activation of adipocyte Gq signaling in vitro and in vivo (in mice). Activation of this pathway by a Gq-coupled designer receptor or by an agonist acting on an endogenous adipocyte Gq-coupled receptor (CysLT2 receptor) greatly improved glucose and lipid homeostasis in obese mice or in mice with adipocyte insulin receptor deficiency. Our findings identify adipocyte Gq signaling as an essential regulator of whole-body glucose and lipid homeostasis and should inform the development of novel classes of GPCR-based antidiabetic drugs.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Adipocitos/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Homeostasis , Lípidos , Lipólisis , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/metabolismoRESUMEN
HIV-associated nephropathy (HIVAN) impairs functions of both glomeruli and tubules. Attention has been previously focused on the HIVAN glomerulopathy. Tubular injury has drawn increased attention because sodium wasting is common in hospitalized HIV/AIDS patients. We used viral protein R (Vpr)-transgenic mice to investigate the mechanisms whereby Vpr contributes to urinary sodium wasting. In phosphoenolpyruvate carboxykinase promoter-driven Vpr-transgenic mice, in situ hybridization showed that Vpr mRNA was expressed in all nephron segments, including the distal convoluted tubule. Vpr-transgenic mice, compared with wild-type littermates, markedly increased urinary sodium excretion, despite similar plasma renin activity and aldosterone levels. Kidneys from Vpr-transgenic mice also markedly reduced protein abundance of the Na+-Cl- cotransporter (NCC), while mineralocorticoid receptor (MR) protein expression level was unchanged. In African green monkey kidney cells, Vpr abrogated the aldosterone-mediated stimulation of MR transcriptional activity. Gene expression of Slc12a3 (NCC) in Vpr-transgenic mice was significantly lower compared with wild-type mice, assessed by both qRT-PCR and RNAScope in situ hybridization analysis. Chromatin immunoprecipitation assays identified multiple MR response elements (MRE), located from 5 kb upstream of the transcription start site and extending to the third exon of the SLC12A3 gene. Mutation of MRE and SP1 sites in the SLC12A3 promoter region abrogated the transcriptional responses to aldosterone and Vpr, indicating that functional MRE and SP1 are required for the SLC12A3 gene suppression in response to Vpr. Thus, Vpr attenuates MR transcriptional activity and inhibits Slc12a3 transcription in the distal convoluted tubule and contributes to salt wasting in Vpr-transgenic mice.
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Productos del Gen vpr , VIH-1 , Aldosterona/metabolismo , Aldosterona/farmacología , Animales , Chlorocebus aethiops , Productos del Gen vpr/metabolismo , VIH-1/genética , Túbulos Renales Distales/metabolismo , Ratones , Ratones Transgénicos , Fosfoenolpiruvato , ARN Mensajero/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Renina/metabolismo , Sodio/metabolismo , Cloruro de Sodio/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , TiazidasRESUMEN
One of the hallmarks of type 2 diabetes is that pancreatic beta cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic beta cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M3 muscarinic acetylcholine receptor subtype in pancreatic beta cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.
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Glucemia/metabolismo , Homeostasis , Células Secretoras de Insulina/fisiología , Insulina/metabolismo , Receptor Muscarínico M3/fisiología , Animales , Dieta , Glucosa/administración & dosificación , Intolerancia a la Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa/métodos , Hiperinsulinismo/metabolismo , Hipoglucemia/metabolismo , Fosfatos de Inositol/biosíntesis , Insulina/administración & dosificación , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Noqueados , Agonistas Muscarínicos/farmacología , Fenotipo , Receptor Muscarínico M3/deficiencia , Receptor Muscarínico M3/metabolismo , Factores de TiempoRESUMEN
CD44(+)/CD24(-) cells have been associated with breast cancer stem/progenitor cell features. However, the status of this phenotype cells in normal, benign and malignant breast tissues has not been studied, and the clinical correlation of this subpopulation in breast cancer is not fully understood. The present study sought to identify these cells in a series of normal, benign, and malignant breast tissues and explore their correlation to the molecular subtypes of breast carcinoma and conventional pathological features. Double-staining immunohistochemistry (DIHC) of CD44 and CD24 was performed on 30 normal breast tissues, 30 breast fibroadenomas (FA), 60 breast invasive ductal carcinomas (IDC), and 3 breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231). In the normal breast tissues and FAs, three phenotypes were observed including CD44(+)/CD24(+), CD44(+)/CD24(-), and CD44(-)/CD24(-) cells. In the IDCs, CD44(-)/CD24(+) cells were detected, in addition to the three aforementioned phenotypes. The strong positive rate (+++, incidence >60%) of CD44(+)/CD24(-) was significantly increased from normal breast tissue, FAs to IDCs (0.0%-->6.7%-->21.7%). However, the CD44(+)/CD24(-) cells didn't correlate with ages of patients, lymph node metastasis, tumor size, molecular subtypes, and the expression of ER, PR, HER-2, PS2, Bcl-2, nm23. The proportion of CD44(+)/CD24(-) cells in MCF-7, MDA-MB-468, and MDA-MB-231 was about 1, 5, and 80%, respectively. The results indicate that the CD44(+)/CD24(-) cells are transit progenitors and have no association with the molecular subtypes and clinicopathological parameters in the IDCs.
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Neoplasias de la Mama/inmunología , Antígeno CD24/metabolismo , Carcinoma Ductal de Mama/inmunología , Fibroadenoma/inmunología , Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/inmunología , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Femenino , Fibroadenoma/patología , Humanos , Inmunohistoquímica , Invasividad Neoplásica , Estadificación de Neoplasias , Células Madre Neoplásicas/patología , FenotipoRESUMEN
PURPOSE: The goal of this study was to analyze the role of somatostatin receptor type 2 (SSTR2) as a molecular target for the imaging and treatment of thyroid cancer through analysis of SSTR2 expression and its epigenetic modulation and testing tumor uptake of different radiolabeled SSTR2 analogues. EXPERIMENTAL DESIGN: We analyzed SSTR2 expression by immunostaining of 92 thyroid cancer tissue samples and quantified standard uptake values (SUVmax) of SSTR2 analogue, 68Ga-DOTA-TATE, by PET/CT imaging in 25 patients with metastatic thyroid cancer. We utilized human thyroid cancer cell lines characterized by differential SSTR2 expression (TT, BCPAP, and FTC133) and rat pancreatic cell line (AR42J) with intrinsically high SSTR2 expression for functional in vitro studies. SSTR2-high (AR42J) and SSTR2-low (FTC133) xenograft mouse models were used to test the uptake of radiolabeled SSTR2 analogues and their therapeutic efficacy in vivo. RESULTS: Thyroid cancer had a higher SSTR2 expression than normal thyroid. Hurthle cell thyroid cancer was characterized by the highest 68Ga-DOTA-TATE uptake [median SUVmax, 16.5 (7.9-29)] than other types of thyroid cancers. In vivo studies demonstrated that radiolabeled DOTA-EB-TATE is characterized by significantly higher tumor uptake than DOTA-TATE (P < 0.001) and DOTA-JR11 (P < 0.001). Treatment with 177Lu-DOTA-EB-TATE extended survival and reduced tumor size in a mouse model characterized by high somatostatin (SST) analogues uptake (SUVmax, 15.16 ± 4.34), but had no effects in a model with low SST analogues uptake (SUVmax, 4.8 ± 0.27). CONCLUSIONS: A novel SST analogue, 177Lu-DOTA-EB-TATE, has the potential to be translated from bench to bedside for the targeted therapy of patients characterized by high uptake of SST analogues in metastatic lesions.
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Radiofármacos/administración & dosificación , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Neoplasias de la Tiroides/tratamiento farmacológico , Adulto , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Persona de Mediana Edad , Metástasis de la Neoplasia , Tomografía Computarizada por Tomografía de Emisión de Positrones , Pronóstico , Radiofármacos/metabolismo , Receptores de Somatostatina/química , Somatostatina/administración & dosificación , Somatostatina/metabolismo , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Alb/TGF-beta(1) transgenic mice overexpress active transforming growth factor-beta(1) (TGF-beta(1)) in the liver, leading to increased circulating levels of the cytokine and progressive renal fibrosis. This study was designed to explore if exogenous all-trans retinoic acid (tRA) prevents renal fibrosis in this animal model. METHODS: The retinoid profile in kidney and liver of wild-type and Alb/TGF-beta(1) transgenic mice was examined by high-performance liquid chromatography and slow-release pellets containing different amounts of tRA were implanted subcutaneously to treat the Alb/TGF-beta(1) transgenic mice, starting at 1 week of age; mice were sacrificed 2 weeks later. RESULTS: Kidneys of 3-week-old wild-type mice had abundant tRA, which was completely absent in kidneys of the transgenic mice. Low doses of tRA (6-10.7 mg/kg/day) failed to affect renal fibrosis although it tended to suppress the mRNA expression of some molecular markers of fibrosis and retinal dehydrogenase 2 (RALDH2), a gene encoding a key tRA-synthesising enzyme. These tendencies disappeared, mortality tended to increase and RALDH2 and connective tissue growth factor (CTGF) mRNAs significantly increased in the medium-dose group (12.7-18.8 mg/kg/day). High doses (20.1-27.4 mg/kg/day) showed even higher toxicity with increased renal fibrosis and significant mortality. CONCLUSIONS: Alb/TGF-beta(1) transgenic mice are characterised by depletion of endogenous renal tRA. Exogenous tRA dose-dependently increases mortality and kidney fibrosis, which is associated with dose-dependent regulation of renal RALDH2 and CTGF mRNA expression.
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Riñón/metabolismo , Tretinoina/metabolismo , Tretinoina/toxicidad , Animales , Factor de Crecimiento del Tejido Conjuntivo/genética , Modelos Animales de Enfermedad , Fibrosis , Riñón/patología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Analysis of individual gametes has a number of applications in the study of the mechanism of repeat expansion in mouse models of the fragile X-related disorders, as well as in mouse models of other repeat expansion diseases. This chapter describes the techniques required to isolate oocytes and male gametes of different stages of maturity, along with the techniques required to accurately determine the repeat number in these gametes.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Oocitos/citología , Espermatocitos/citología , Expansión de Repetición de Trinucleótido , Animales , Separación Celular , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Oocitos/química , Análisis de la Célula Individual , Espermatocitos/químicaRESUMEN
A better understanding of the signaling pathways regulating adipocyte function is required for the development of new classes of antidiabetic/obesity drugs. We here report that mice lacking ß-arrestin-1 (barr1), a cytoplasmic and nuclear signaling protein, selectively in adipocytes showed greatly impaired glucose tolerance and insulin sensitivity when consuming an obesogenic diet. In contrast, transgenic mice overexpressing barr1 in adipocytes were protected against the metabolic deficits caused by a high-calorie diet. Barr1 deficiency led to a myogenic reprogramming of brown adipose tissue (BAT), causing elevated plasma myostatin (Mstn) levels, which in turn led to impaired insulin signaling in multiple peripheral tissues. Additional in vivo studies indicated that barr1-mediated suppression of Mstn expression by BAT is required for maintaining euglycemia. These findings convincingly identify barr1 as a critical regulator of BAT function. Strategies aimed at enhancing barr1 activity in BAT may prove beneficial for the treatment of type 2 diabetes.
RESUMEN
Skeletal muscle (SKM) insulin resistance plays a central role in the pathogenesis of type 2 diabetes. Because G-protein-coupled receptors (GPCRs) represent excellent drug targets, we hypothesized that activation of specific functional classes of SKM GPCRs might lead to improved glucose homeostasis in type 2 diabetes. At present, little is known about the in vivo metabolic roles of the various distinct GPCR signaling pathways operative in SKM. In this study, we tested the hypothesis that selective activation of SKM Gq signaling can improve SKM glucose uptake and whole-body glucose homeostasis under physiological and pathophysiological conditions. Studies with transgenic mice expressing a Gq-linked designer GPCR selectively in SKM cells demonstrated that receptor-mediated activation of SKM Gq signaling greatly promoted glucose uptake into SKM and significantly improved glucose homeostasis in obese, glucose-intolerant mice. These beneficial metabolic effects required the activity of SKM AMPK. In contrast, obese mutant mice that lacked both Gαq and Gα11 selectively in SKM showed severe deficits in glucose homeostasis. Moreover, GPCR-mediated activation of Gq signaling also stimulated glucose uptake in primary human SKM cells. Taken together, these findings strongly suggest that agents capable of enhancing SKM Gq signaling may prove useful as novel antidiabetic drugs.
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Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Intolerancia a la Glucosa/metabolismo , Glucosa/metabolismo , Resistencia a la Insulina , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenilato Quinasa/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Ratones , Ratones Obesos , Ratones Transgénicos , Mioblastos Esqueléticos , Transducción de SeñalRESUMEN
AIMS: Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. METHODS AND RESULTS: We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. CONCLUSION: Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes' Ca2+ handling but also directly alters the heart's electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.
Asunto(s)
Potenciales de Acción , Arritmias Cardíacas/metabolismo , Señalización del Calcio , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Frecuencia Cardíaca , Miocardio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Clozapina/análogos & derivados , Clozapina/farmacología , Conexina 43/metabolismo , Forma MM de la Creatina-Quinasa/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Preparación de Corazón Aislado , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Preadipocytes initiate differentiation into adipocytes through a cascade of events. Mitotic clonal expansion, as one of the earliest events, is essential for adipogenesis. However, the underlying mechanisms that regulate mitotic clonal expansion remain elusive. SIRT6 is a member of the evolutionarily conserved sirtuin family of nicotinamide adenine dinucleotide (NAD)+-dependent protein deacetylases. Here, we show that SIRT6 deficiency in preadipocytes blocks their adipogenesis. Analysis of gene expression during adipogenesis reveals that KIF5C, which belongs to the kinesin family, is negatively regulated by SIRT6. Furthermore, we show that KIF5C is a negative factor for adipogenesis through interacting with CK2α', a catalytic subunit of CK2. This interaction blocks CK2α' nuclear translocation and CK2 kinase activity and inhibits mitotic clonal expansion during adipogenesis. These findings reveal a crucial role of SIRT6 in adipogenesis and provide potential therapeutic targets for obesity.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Diferenciación Celular , Mitosis , Sirtuinas/metabolismo , Células 3T3-L1 , Adipogénesis , Animales , Quinasa de la Caseína II/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular , Células Clonales , Eliminación de Gen , Cinesinas/metabolismo , Ratones , Fenotipo , Transporte de Proteínas , Sirtuinas/deficienciaRESUMEN
ß-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the ß-arrestin-2 gene, barr2, in ß-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in ß-arrestin-2 (barr2) deficient ß-cells. In human ß-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in ß-cells. Importantly, overexpression of barr2 in ß-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of ß-cell function, which may serve as a new target to improve ß-cell function.