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1.
Mol Metab ; 24: 108-119, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30940487

RESUMEN

OBJECTIVE: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease-gene interactions during adipocyte differentiation. METHODS: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene. RESULTS: Over 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein-protein and gene regulatory interactions. CONCLUSIONS: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.


Asunto(s)
Adipocitos/citología , Adipogénesis , Diabetes Mellitus/genética , Redes Reguladoras de Genes , Mapas de Interacción de Proteínas , Aciltransferasas/genética , Aciltransferasas/metabolismo , Adipocitos/metabolismo , Adipocitos/patología , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , Diabetes Mellitus/patología , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Humanos , Resistencia a la Insulina , Perilipina-1/genética , Perilipina-1/metabolismo , Fenotipo , Transcriptoma
2.
Diabetologia ; 56(11): 2498-506, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23989774

RESUMEN

AIMS/HYPOTHESIS: In humans, disruption of the gene BSCL2, encoding the protein seipin, causes congenital generalised lipodystrophy (CGL) with severe insulin resistance and dyslipidaemia. While the causative gene has been known for over a decade, the molecular functions of seipin are only now being uncovered. Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts. However, several more subtle mutations have been reported that cause premature stop codons or single amino acid substitutions. Here we have examined these mutant forms of seipin to gain insight into how they may cause CGL. METHODS: We generated constructs expressing mutant seipin proteins and determined their expression and localisation. We also assessed their capacity to recruit the key adipogenic phosphatidic acid phosphatase lipin 1, a recently identified molecular role of seipin in developing adipocytes. Finally, we used atomic force microscopy to define the oligomeric structure of seipin and to determine whether this is affected by the mutations. RESULTS: We show that the R275X mutant of seipin is not expressed in pre-adipocytes. While the other premature stop mutant forms fail to bind lipin 1 appropriately, the point mutants T78A, L91P and A212P all retain this capacity. We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not. CONCLUSIONS/INTERPRETATION: Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.


Asunto(s)
Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Lipodistrofia/genética , Lipodistrofia/metabolismo , Adipocitos/metabolismo , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Subunidades gamma de la Proteína de Unión al GTP/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Humanos , Immunoblotting , Inmunoprecipitación , Lipodistrofia/patología , Ratones , Microscopía de Fuerza Atómica , Mutación
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