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1.
Proc Natl Acad Sci U S A ; 113(26): E3706-15, 2016 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-27303042

RESUMEN

Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Linfocitos B/citología , Cardiomiopatías/metabolismo , Proteínas Portadoras/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Linfocitos B/enzimología , Linfocitos B/metabolismo , Cardiomiopatías/genética , Proteínas Portadoras/metabolismo , Recuento de Células , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genética
2.
Am J Physiol Endocrinol Metab ; 311(4): E706-E719, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27577855

RESUMEN

AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory ß (ß1 and ß2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 µM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Bencimidazoles/farmacología , Benzoatos/farmacología , Activadores de Enzimas/farmacología , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Músculo Esquelético/efectos de los fármacos , Ribonucleótidos/farmacología , Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Aminoimidazol Carboxamida/farmacología , Animales , Anticuerpos Bloqueadores/farmacología , Humanos , Técnicas In Vitro , Isoenzimas , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos
3.
Stem Cells ; 33(10): 3077-86, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26175344

RESUMEN

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1-null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self-renewal capacity of MOSPD1-null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)-like cells expressing CD73, CD90, and CD105 were generated from MOSPD1-null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1-null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA-sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre-MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation.


Asunto(s)
Diferenciación Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Proteínas de la Membrana/genética , Células Madre Mesenquimatosas , Adipocitos/metabolismo , Médula Ósea/metabolismo , Linaje de la Célula/genética , Células Madre Embrionarias/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Osteoblastos/metabolismo , ARN Mensajero/biosíntesis
4.
Nat Commun ; 14(1): 853, 2023 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-36792598

RESUMEN

Following the diagnosis of a paediatric disorder caused by an apparently de novo mutation, a recurrence risk of 1-2% is frequently quoted due to the possibility of parental germline mosaicism; but for any specific couple, this figure is usually incorrect. We present a systematic approach to providing individualized recurrence risk. By combining locus-specific sequencing of multiple tissues to detect occult mosaicism with long-read sequencing to determine the parent-of-origin of the mutation, we show that we can stratify the majority of couples into one of seven discrete categories associated with substantially different risks to future offspring. Among 58 families with a single affected offspring (representing 59 de novo mutations in 49 genes), the recurrence risk for 35 (59%) was decreased below 0.1%, but increased owing to parental mixed mosaicism for 5 (9%)-that could be quantified in semen for paternal cases (recurrence risks of 5.6-12.1%). Implementation of this strategy offers the prospect of driving a major transformation in the practice of genetic counselling.


Asunto(s)
Padre , Parto , Masculino , Embarazo , Femenino , Humanos , Niño , Mutación , Medición de Riesgo , Células Germinativas , Mosaicismo , Linaje , Mutación de Línea Germinal
6.
Nat Commun ; 8(1): 1258, 2017 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-29097735

RESUMEN

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.


Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Bradicardia/genética , Calcio/metabolismo , Frecuencia Cardíaca/genética , Sarcolema/metabolismo , Nodo Sinoatrial/metabolismo , Adulto , Animales , Bradicardia/metabolismo , Electrocardiografía Ambulatoria , Ejercicio Físico , Corazón/diagnóstico por imagen , Humanos , Imagen por Resonancia Cinemagnética , Espectroscopía de Resonancia Magnética , Ratones , Microscopía Electrónica de Transmisión , Mutación , Miocardio/metabolismo , Miocardio/patología , Miocardio/ultraestructura , Condicionamiento Físico Animal , Resistencia Física , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Nodo Sinoatrial/patología
7.
Cell Metab ; 23(5): 821-36, 2016 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-27133129

RESUMEN

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/patología , Obesidad/enzimología , Adiposidad/genética , Adulto , Envejecimiento/patología , Proteína Relacionada con Agouti/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Metabolismo Energético/genética , Activación Enzimática , Conducta Alimentaria , Femenino , Heterocigoto , Humanos , Hiperfagia/complicaciones , Hiperfagia/enzimología , Hiperfagia/genética , Hiperfagia/patología , Hipotálamo/metabolismo , Insulina/metabolismo , Masculino , Ratones , Mitocondrias/metabolismo , Mutación/genética , Neuronas/metabolismo , Obesidad/sangre , Obesidad/complicaciones , Obesidad/patología , Fosforilación Oxidativa , Receptores de Ghrelina/metabolismo , Ribosomas/metabolismo , Transducción de Señal/genética , Transcriptoma/genética , Regulación hacia Arriba/genética
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