RESUMEN
Glucagon-like peptide-1 receptor (GLP1R) agonists and dipeptidyl peptidase 4 inhibitors are widely prescribed diabetes drugs due to their ability to stimulate insulin secretion from remaining ß cells and to reduce caloric intake. Unfortunately, they fail to increase human ß cell proliferation. Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) are able to induce adult human ß cell proliferation, but rates are modest (~2%), and their specificity to ß cells is limited. Here, we provide evidence that combining any member of the GLP1R agonist class with any member of the DYRK1A inhibitor class induces a synergistic increase in human ß cell replication (5 to 6%) accompanied by an actual increase in numbers of human ß cells. GLP1R agonist-DYRK1A inhibitor synergy required combined inhibition of DYRK1A and an increase in cAMP and did not lead to ß cell dedifferentiation. These beneficial effects on proliferation were seen in both normal human ß cells and ß cells derived from individuals with type 2 diabetes. The ability of the GLP1R agonist-DYRK1A inhibitor combination to enhance human ß cell proliferation, human insulin secretion, and blood glucose control extended in vivo to studies of human islets transplanted into euglycemic and streptozotocin-diabetic immunodeficient mice. No adverse events were observed in the mouse studies during a 1-week period. Because of the relative ß cell specificity of GLP1R agonists, the combination provides an improved, although not complete, degree of human ß cell specificity.
Asunto(s)
Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón/agonistas , Células Secretoras de Insulina , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adulto , Animales , Humanos , Ratones , Regeneración , Quinasas DyrKRESUMEN
Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human ß cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human ß cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater ß cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human ß cell proliferation, and we provide clarity for future efforts in structure-based drug design for human ß cell regenerative drugs.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/efectos de los fármacos , Adolescente , Adulto , Anciano , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Harmina/metabolismo , Harmina/farmacología , Humanos , Insulinoma , Masculino , Persona de Mediana Edad , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Adulto Joven , Quinasas DyrKRESUMEN
Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFßSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers. This synergy reflects activation of cyclins and cdks by DYRK1A inhibition, accompanied by simultaneous reductions in key cell-cycle inhibitors (CDKN1C and CDKN1A). The latter results from interference with the basal Trithorax- and SMAD-mediated transactivation of CDKN1C and CDKN1A. Notably, combined DYRK1A and TGFß inhibition allows preservation of beta cell differentiated function. These beneficial effects extend from normal human beta cells and stem cell-derived human beta cells to those from people with type 2 diabetes, and occur both in vitro and in vivo.
Asunto(s)
Diabetes Mellitus Tipo 2 , Harmina/farmacología , Células Secretoras de Insulina , Inhibidores de la Monoaminooxidasa/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Adolescente , Adulto , Anciano , Animales , Línea Celular , Proliferación Celular , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Femenino , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Smad/antagonistas & inhibidores , Células Madre , Adulto Joven , Quinasas DyrKRESUMEN
The numbers of insulin-secreting pancreatic beta cells are reduced in people with type 1 and type 2 diabetes. Driving beta cell regeneration in the pancreases of people with diabetes would be an attractive approach to reversing diabetes. While adult human beta cells have long been believed to be terminally differentiated and, therefore, irreversibly quiescent, it has become clear over recent years that this is not true. More specifically, both candidate and unbiased high-throughput screen approaches have revealed several classes of molecules that are clearly able to induce human beta cell proliferation. Here, we review recent approaches and accomplishments in human beta cell regenerative drug discovery. We also list the challenges that this rapidly moving field must confront to translate beta cell regenerative therapy from the laboratory to the clinic.
Asunto(s)
Descubrimiento de Drogas/métodos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Páncreas/citología , Páncreas/metabolismo , Regeneración/fisiologíaRESUMEN
Diabetes is the result of the insufficiency or dysfunction of pancreatic beta cells alone or in combination with insulin resistance. The replacement or regeneration of beta cells can effectively reverse diabetes in humans and rodents. Therefore, the identification of novel small molecules that promote pancreatic beta-cell proliferation is an attractive approach for diabetic therapy. While numerous hormones, small molecules, and growth factors are able to drive rodent beta cells to replicate, only a few small molecules have demonstrated the ability to stimulate human beta-cell proliferation. Hence, there is an urgent need for therapeutic agents that induce regeneration and expansion of adult human beta cells. Here, we describe a detailed protocol for coating chamber slides, culturing primary islets, performing islet cell disassociation, seeding cells on chamber slides, treating islet cells with compounds or infecting them with adenovirus, immunostaining of proliferation markers and imaging, and data analysis.
Asunto(s)
Descubrimiento de Drogas , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Biomarcadores , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/metabolismo , Descubrimiento de Drogas/métodos , Técnica del Anticuerpo Fluorescente , Humanos , Hipoglucemiantes/farmacología , Inmunohistoquímica , Transducción GenéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease, characterized by motor neuron (MN) death, for which there are no truly effective treatments. Here, we describe a new small molecule survival screen carried out using MNs from both wild-type and mutant SOD1 mouse embryonic stem cells. Among the hits we found, kenpaullone had a particularly impressive ability to prolong the healthy survival of both types of MNs that can be attributed to its dual inhibition of GSK-3 and HGK kinases. Furthermore, kenpaullone also strongly improved the survival of human MNs derived from ALS-patient-induced pluripotent stem cells and was more active than either of two compounds, olesoxime and dexpramipexole, that recently failed in ALS clinical trials. Our studies demonstrate the value of a stem cell approach to drug discovery and point to a new paradigm for identification and preclinical testing of future ALS therapeutics.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Células Madre Embrionarias/citología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Células Madre Pluripotentes Inducidas/citología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Inhibidores de Proteínas Quinasas/análisis , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/patología , Animales , Benzazepinas/química , Benzazepinas/farmacología , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colestenonas/química , Colestenonas/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Indoles/química , Indoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Transgénicos , Neuronas Motoras/enzimología , Mutación , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Relación Estructura-Actividad , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
BACKGROUND/AIMS: The steroid hormones, including estradiol (E) and progesterone, act in the brain to regulate female reproductive behavior and physiology. These hormones mediate many of their biological effects by binding to their respective intracellular receptors. The receptors for estrogens (ER) and progestins (PR) interact with nuclear receptor coactivators to initiate transcription of steroid-responsive genes. Work from our laboratory and others reveals that nuclear receptor coactivators, including steroid receptor coactivator-1 (SRC-1) and SRC-2, function in brain to modulate ER-mediated induction of the PR gene and hormone-dependent behaviors. In order for steroid receptors and coactivators to function together, both must be expressed in the same cells. METHODS: Triple-label immunofluorescence was used to determine if E-induced PR cells also express SRC-1 or SRC-2 in reproductively relevant brain regions of the female mouse. RESULTS: The majority of E-induced PR cells in the medial preoptic area (61%), ventromedial nucleus of the hypothalamus (63%) and arcuate nucleus (76%) coexpressed both SRC-1 and SRC-2. A smaller proportion of PR cells expressed either SRC-1 or SRC-2, while a few PR cells expressed neither coactivator. In addition, compared to control animals, 17ß-estradiol benzoate (EB) treatment increased SRC-1 levels in the arcuate nucleus, but not the medial preoptic area or the ventromedial nucleus of the hypothalamus. EB did not alter SRC-2 expression in any of the three brain regions analyzed. CONCLUSIONS: Taken together, the present findings identify a population of cells in which steroid receptors and nuclear receptor coactivators may interact to modulate steroid sensitivity in brain and regulate hormone-dependent behaviors in female mice. Given that cell culture studies reveal that SRC-1 and SRC-2 can mediate distinct steroid-signaling pathways, the present findings suggest that steroids can produce a variety of complex responses in these specialized brain cells.