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1.
bioRxiv ; 2024 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-38746433

RESUMEN

Islet transplantation can cure type 1 diabetes, but peri-transplant beta cell death limits this procedure to those with low insulin requirements. Improving human beta cell survival or proliferation may make islet transplantation a possibility for more type 1 patients. To identify novel regulators of beta cell survival and proliferation, we conducted a pooled small hairpin RNA (shRNA) screen in primary human beta cells transplanted into immunocompromised mice. shRNAs targeting several cyclin dependent kinase inhibitors were enriched after transplant. Here, we focused on the Gi/o-coupled GPCR, serotonin 1F receptor ( HTR1F, 5-HT 1F ) which our screen identified as a negative regulator of beta cell numbers after transplant. In vitro , 5-HT 1F knockdown induced human beta cell proliferation but only when combined with harmine and exendin-4. In vivo , knockdown of 5-HT 1F reduced beta cell death during transplant. To demonstrate the feasibility of targeting 5-HT 1F in islet transplant, we identified and validated a small molecule 5-HT 1F antagonist. This antagonist increased glucose stimulated insulin secretion from primary human islets and cAMP accumulation in primary human beta cells. Finally, the 5-HT 1F antagonist improved glycemia in marginal mass, human islet transplants into immunocompromised mice. We identify 5-HT 1F as a novel druggable target to improve human beta cell survival in the setting of islet transplantation. One Sentence Summary: Serotonin 1F receptor (5-HT 1F ) negatively regulates insulin secretion and beta cell survival during transplant.

2.
Sci Rep ; 10(1): 5629, 2020 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-32221326

RESUMEN

Gpr27 is a highly conserved, orphan G protein coupled receptor (GPCR) previously implicated in pancreatic beta cell insulin transcription and glucose-stimulated insulin secretion in vitro. Here, we characterize a whole-body mouse knockout of Gpr27. Gpr27 knockout mice were born at expected Mendelian ratios and exhibited no gross abnormalities. Insulin and Pdx1 mRNA in Gpr27 knockout islets were reduced by 30%, but this did not translate to a reduction in islet insulin content or beta cell mass. Gpr27 knockout mice exhibited slightly worsened glucose tolerance with lower plasma insulin levels while maintaining similar insulin tolerance. Unexpectedly, Gpr27 deletion reduced expression of Eif4e3, a neighboring gene, likely by deleting transcription start sites on the anti-sense strand of the Gpr27 coding exon. Our data confirm that loss of Gpr27 reduces insulin mRNA in vivo but has only minor effects on glucose tolerance.


Asunto(s)
Diabetes Mellitus/metabolismo , Insulina/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Glucosa/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Noqueados
3.
Islets ; 12(3): 59-69, 2020 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-32579048

RESUMEN

Virus-mediated gene knockdown in intact pancreatic islets is technically challenging due to poor infection of the center of the islet. Because the cells that do not have knockdown have normal insulin secretion, measuring changes in insulin secretion after gene knockdown is challenging. We describe a method to monitor insulin secretion from only the beta cells with knockdown of a gene of interest in intact islets using a single lentivirus containing a guide RNA, a luciferase insulin secretion reporter and a dCas9-KRAB cassette. This method allows rapid and inexpensive monitoring of insulin secretion from only those beta cells with knockdown, circumventing the problem of incomplete islet infection.


Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Edición Génica , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Secreciones Corporales , Edición Génica/métodos , Técnicas de Silenciamiento del Gen , Ratones , Interferencia de ARN
4.
Endocrinology ; 159(9): 3245-3256, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-30052866

RESUMEN

Mitochondria are dynamic organelles that undergo frequent fission and fusion events. Mitochondrial fission is required for ATP production, the tricarboxylic acid cycle, and processes beyond metabolism in a cell-type specific manner. Ex vivo and cell line studies have demonstrated that Drp1, a central regulator of mitochondrial fission, is required for glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells. Herein, we set out to interrogate the role of Drp1 in ß-cell insulin secretion in vivo. We generated ß-cell-specific Drp1 knockout (KO) mice (Drp1ß-KO) by crossing a conditional allele of Drp1 to Ins1cre mice, in which Cre recombinase replaces the coding region of the Ins1 gene. Drp1ß-KO mice were glucose intolerant due to impaired GSIS but did not progress to fasting hyperglycemia as adults. Despite markedly abnormal mitochondrial morphology, Drp1ß-KO islets exhibited normal oxygen consumption rates and an unchanged glucose threshold for intracellular calcium mobilization. Instead, the most profound consequences of ß-cell Drp1 deletion were impaired second-phase insulin secretion and impaired glucose-stimulated amplification of insulin secretion. Our data establish Drp1 as an important regulator of insulin secretion in vivo and demonstrate a role for Drp1 in metabolic amplification and calcium handling without affecting oxygen consumption.


Asunto(s)
Dinaminas/genética , Secreción de Insulina/genética , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Consumo de Oxígeno/genética , Animales , Calcio/metabolismo , Ayuno/metabolismo , Intolerancia a la Glucosa/genética , Hiperglucemia/genética , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/patología , Dinámicas Mitocondriales
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