RESUMEN
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
Although diabetes results in part from a deficiency of normal pancreatic beta cells, inducing human beta cells to regenerate is difficult. Reasoning that insulinomas hold the "genomic recipe" for beta cell expansion, we surveyed 38 human insulinomas to obtain insights into therapeutic pathways for beta cell regeneration. An integrative analysis of whole-exome and RNA-sequencing data was employed to extensively characterize the genomic and molecular landscape of insulinomas relative to normal beta cells. Here, we show at the pathway level that the majority of the insulinomas display mutations, copy number variants and/or dysregulation of epigenetic modifying genes, most prominently in the polycomb and trithorax families. Importantly, these processes are coupled to co-expression network modules associated with cell proliferation, revealing candidates for inducing beta cell regeneration. Validation of key computational predictions supports the concept that understanding the molecular complexity of insulinoma may be a valuable approach to diabetes drug discovery.Diabetes results in part from a deficiency of functional pancreatic beta cells. Here, the authors study the genomic and epigenetic landscapes of human insulinomas to gain insight into possible pathways for therapeutic beta cell regeneration, highlighting epigenetic genes and pathways.
Asunto(s)
Proliferación Celular/genética , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Neoplasias Pancreáticas/genética , Regeneración/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Diabetes Mellitus Tipo 1/metabolismo , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Insulinoma/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismoRESUMEN
Pregnancy in rodents is associated with a two- to threefold increase in ß-cell mass, which is attributable to large increases in ß-cell proliferation, complimented by increases in ß-cell size, survival, and function and mediated mainly by the lactogenic hormones prolactin (PRL) and placental lactogens. In humans, however, ß-cell mass does not increase as dramatically during pregnancy, and PRL fails to activate proliferation in human islets in vitro. To determine why, we explored the human PRL-prolactin receptor (hPRLR)-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5)-cyclin-cdk signaling cascade in human ß-cells. Surprisingly, adult human ß-cells express little or no PRLR. As expected, restoration of the hPRLR in human ß-cells rescued JAK2-STAT5 signaling in response to PRL. However, rescuing hPRLR-STAT5 signaling nevertheless failed to confer proliferative ability on adult human ß-cells in response to PRL. Surprisingly, mouse (but not human) Stat5a overexpression led to upregulation of cyclins D1-3 and cdk4, as well as their nuclear translocation, all of which are associated with ß-cell cycle entry. Collectively, the findings show that human ß-cells fail to proliferate in response to PRL for multiple reasons, one of which is a paucity of functional PRL receptors, and that murine Stat5 overexpression is able to bypass these impediments.
Asunto(s)
Proliferación Celular/fisiología , Células Secretoras de Insulina/metabolismo , Prolactina/farmacología , Receptores de Prolactina/metabolismo , Factor de Transcripción STAT5/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Ciclina D/genética , Ciclina D/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Receptores de Prolactina/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia ArribaRESUMEN
ß-Cell regeneration is a key goal of diabetes research. Progression through the cell cycle is associated with retinoblastoma protein (pRb) inactivation via sequential phosphorylation by the "early" cyclins and cyclin-dependent kinases (cdks) (d-cyclins cdk4/6) and the "late" cyclins and cdks (cyclin A/E and cdk1/2). In ß-cells, activation of either early or late G1/S cyclins and/or cdks is an efficient approach to induce cycle entry, but it is unknown whether the combined expression of early and late cyclins and cdks might have synergistic or additive effects. Thus, we explored whether a combination of both early and late cyclins and cdks might more effectively drive human ß-cell cell cycle entry than either group alone. We also sought to determine whether authentic replication with the expansion of adult human ß-cells could be demonstrated. Late cyclins and cdks do not traffic in response to the induction of replication by early cyclins and cdks in human ß-cells but are capable of nuclear translocation when overexpressed. Early plus late cyclins and cdks, acting via pRb phosphorylation on distinct residues, complementarily induce greater proliferation in human ß-cells than either group alone. Importantly, the combination of early and late cyclins and cdks clearly increased human ß-cell numbers in vitro. These findings provide additional insight into human ß-cell expansion. They also provide a novel tool for assessing ß-cell expansion in vitro.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Células Secretoras de Insulina/metabolismo , Envejecimiento , Animales , Proliferación Celular/fisiología , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Regulación de la Expresión Génica/fisiología , Glucosa/farmacología , Humanos , Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Fosforilación , Ratas , Ratas Sprague-Dawley , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismoRESUMEN
Expansion of pancreatic ß-cells is a key goal of diabetes research, yet induction of adult human ß-cell replication has proven frustratingly difficult. In part, this reflects a lack of understanding of cell cycle control in the human ß-cell. Here, we provide a comprehensive immunocytochemical "atlas" of G1/S control molecules in the human ß-cell. This atlas reveals that the majority of these molecules, previously known to be present in islets, are actually present in the ß-cell. More importantly, and in contrast to anticipated results, the human ß-cell G1/S atlas reveals that almost all of the critical G1/S cell cycle control molecules are located in the cytoplasm of the quiescent human ß-cell. Indeed, the only nuclear G1/S molecules are the cell cycle inhibitors, pRb, p57, and variably, p21: none of the cyclins or cdks necessary to drive human ß-cell proliferation are present in the nuclear compartment. This observation may provide an explanation for the refractoriness of human ß-cells to proliferation. Thus, in addition to known obstacles to human ß-cell proliferation, restriction of G1/S molecules to the cytoplasm of the human ß-cell represents an unanticipated obstacle to therapeutic human ß-cell expansion.
Asunto(s)
Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Proliferación Celular , Células Secretoras de Insulina/fisiología , Adolescente , Adulto , Niño , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Persona de Mediana Edad , Fracciones SubcelularesRESUMEN
Harnessing control of human ß-cell proliferation has proven frustratingly difficult. Most G1/S control molecules, generally presumed to be nuclear proteins in the human ß-cell, are in fact constrained to the cytoplasm. Here, we asked whether G1/S molecules might traffic into and out of the cytoplasmic compartment in association with activation of cell cycle progression. Cdk6 and cyclin D3 were used to drive human ß-cell proliferation and promptly translocated into the nucleus in association with proliferation. In contrast, the cell cycle inhibitors p15, p18, and p19 did not alter their location, remaining cytoplasmic. Conversely, p16, p21, and p27 increased their nuclear frequency. In contrast once again, p57 decreased its nuclear frequency. Whereas proliferating ß-cells contained nuclear cyclin D3 and cdk6, proliferation generally did not occur in ß-cells that contained nuclear cell cycle inhibitors, except p21. Dynamic cytoplasmic-nuclear trafficking of cdk6 was confirmed using green fluorescent protein-tagged cdk6 and live cell imaging. Thus, we provide novel working models describing the control of cell cycle progression in the human ß-cell. In addition to known obstacles to ß-cell proliferation, cytoplasmic-to-nuclear trafficking of G1/S molecules may represent an obstacle as well as a therapeutic opportunity for human ß-cell expansion.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Fase G1/fisiología , Células Secretoras de Insulina/metabolismo , Fase S/fisiología , Adolescente , Adulto , Animales , Proteínas de Ciclo Celular/genética , División Celular , Línea Celular Tumoral , Núcleo Celular/genética , Proliferación Celular , Niño , Citoplasma/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Transporte de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
The transcription factor HNF4α (hepatocyte nuclear factor-4α) is required for increased ß-cell proliferation during metabolic stress in vivo. We hypothesized that HNF4α could induce proliferation of human ß-cells. We employed adenoviral-mediated overexpression of an isoform of HNF4α (HNF4α8) alone, or in combination with cyclin-dependent kinase (Cdk)6 and Cyclin D3, in human islets. Heightened HNF4α8 expression led to a 300-fold increase in the number of ß-cells in early S-phase. When we overexpressed HNF4α8 together with Cdk6 and Cyclin D3, ß-cell cycle entry was increased even further. However, the punctate manner of bromodeoxyuridine incorporation into HNF4α(High) ß-cells indicated an uncoupling of the mechanisms that control the concise timing and execution of each cell cycle phase. Indeed, in HNF4α8-induced bromodeoxyuridine(+,punctate) ß-cells we observed signs of dysregulated DNA synthesis, cell cycle arrest, and activation of a double stranded DNA damage-associated cell cycle checkpoint mechanism, leading to the initiation of loss of ß-cell lineage fidelity. However, a substantial proportion of ß-cells stimulated to enter the cell cycle by Cdk6 and Cyclin D3 alone also exhibited a DNA damage response. HNF4α8 is a mitogenic signal in the human ß-cell but is not sufficient for completion of the cell cycle. The DNA damage response is a barrier to efficient ß-cell proliferation in vitro, and we suggest its evaluation in all attempts to stimulate ß-cell replication as an approach to diabetes treatment.
Asunto(s)
Factor Nuclear 4 del Hepatocito/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Ciclo Celular , División Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Glucosa/farmacología , Factor Nuclear 4 del Hepatocito/genética , Humanos , Etiquetado Corte-Fin in Situ , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Glucose stimulates rodent and human ß-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic ß-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated ß-cell proliferation. The relative expression of ChREBP was determined in liver and ß-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human ß-cells. Proliferation was measured by 5-bromo-2'-deoxyuridine incorporation, [(3)H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in ß-cells isolated from ChREBP(-/-) mice, in INS-1-derived 832/13 cells, and in primary rat and human ß-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human ß-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic ß-cells.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Proteínas de Ciclo Celular/fisiología , Proliferación Celular/efectos de los fármacos , Humanos , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Ratones , RatasRESUMEN
Induction of proliferation in adult human ß-cells is challenging. It can be accomplished by introduction of cell cycle molecules such as cyclin-dependent kinase 6 (cdk6) and cyclin D1, but their continuous overexpression raises oncogenic concerns. We attempted to mimic normal, transient, perinatal human ß-cell proliferation by delivering these molecules in a regulated and reversible manner. Adult cadaveric islets were transduced with doxycycline (Dox)-inducible adenoviruses expressing cdk6 or cyclin D1. End points were cdk6/cyclin D1 expression and human ß-cell proliferation, survival, and function. Increasing doses of Dox led to marked dose- and time-related increases in cdk6 and cyclin D1, accompanied by a 20-fold increase in ß-cell proliferation. Notably, Dox withdrawal resulted in a reversal of both cdk6 and cyclin D1 expression as well as ß-cell proliferation. Re-exposure to Dox reinduced both cdk/cyclin expression and proliferation. ß-Cell function and survival were not adversely affected. The adenoviral tetracycline (tet)-on system has not been used previously to drive human ß-cell proliferation. Human ß-cells can be induced to proliferate or arrest in a regulated, reversible manner, temporally and quantitatively mimicking the transient perinatal physiological proliferation that occurs in human ß-cells.
Asunto(s)
Células Secretoras de Insulina/fisiología , Adenoviridae/genética , Adulto , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/fisiología , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/fisiología , Doxiciclina/farmacología , HumanosRESUMEN
Parathyroid hormone-related protein (PTHrP) contains a classical bipartite nuclear localization signal. Nuclear PTHrP induces proliferation of arterial vascular smooth muscle cells (VSMC). In the arterial wall, PTHrP is markedly up-regulated in response to angioplasty and promotes arterial restenosis. PTHrP overexpression exacerbates arterial restenosis, and knockout of the PTHrP gene results in decreased VSMC proliferation in vivo. In arterial VSMC, expression of the cell cycle inhibitor, p27, rapidly decreases after angioplasty, and replacement of p27 markedly reduces neointima development. We have shown that PTHrP overexpression in VSMC leads to p27 down-regulation, mostly through increased proteosomal degradation. Here, we determined the molecular mechanisms through which PTHrP targets p27 for degradation. S-phase kinase-associated protein 2 (skp2) and c-myc, two critical regulators of p27 expression and stability, and neointima formation were up-regulated in PTHrP overexpression in VSMC. Normalization of skp2 or c-myc using small interfering RNA restores normal cell cycle and p27 expression in PTHrP overexpression in VSMC. These data indicate that skp2 and c-myc mediate p27 loss and proliferation induced by PTHrP. c-myc promoter activity was increased, and c-myc target genes involved in p27 stability were up-regulated in PTHrP overexpression in VSMC. In primary VSMC, PTHrP overexpression led to increased c-myc and decreased p27. Conversely, knockdown of PTHrP in primary VSMC from PTHrP(flox/flox) mice led to cell cycle arrest, p27 up-regulation, with c-myc and skp2 down-regulation. Collectively, these data describe for the first time the role of PTHrP in the regulation of skp2 and c-myc in VSMC. This novel PTHrP-c-myc-skp2 pathway is a potential target for therapeutic manipulation of the arterial response to injury.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Músculo Liso Vascular/citología , Neointima/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Regulación de la Expresión Génica , Ratones , Mutación , Neointima/patología , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Quinasas Asociadas a Fase-S/genéticaRESUMEN
A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Islotes Pancreáticos/citología , Cultivo Primario de Células/métodos , Línea Celular , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Reproducibilidad de los Resultados , Bibliotecas de Moléculas PequeñasRESUMEN
Adult human ß-cells replicate slowly. Also, despite the abundance of rodent ß-cell lines, there are no human ß-cell lines for diabetes research or therapy. Prior studies in four commonly studied rodent ß-cell lines revealed that all four lines displayed an unusual, but strongly reproducible, cell cycle signature: an increase in seven G(1)/S molecules, i.e. cyclins A, D3, and E, and cdk1, -2, -4, and -6. Here, we explore the upstream mechanism(s) that drive these cell cycle changes. Using biochemical, pharmacological and molecular approaches, we surveyed potential upstream mitogenic signaling pathways in Ins 1 and RIN cells. We used both underexpression and overexpression to assess effects on rat and human ß-cell proliferation, survival and cell cycle control. Our results indicate that cMyc is: 1) uniquely up-regulated among other candidates; 2) principally responsible for the increase in the seven G(1)/S molecules; and, 3) largely responsible for proliferation in rat ß-cell lines. Importantly, cMyc expression in ß-cell lines, although some 5- to 7-fold higher than normal rat ß-cells, is far below the levels (75- to 150-fold) previously associated with ß-cell death and dedifferentiation. Notably, modest overexpression of cMyc is able to drive proliferation without cell death in normal rat and human ß-cells. We conclude that cMyc is an important driver of replication in the two most commonly employed rat ß-cell lines. These studies reverse the current paradigm in which cMyc overexpression is inevitably associated with ß-cell death and dedifferentiation. The cMyc pathway provides potential approaches, targets, and tools for driving and sustaining human ß-cell replication.
Asunto(s)
Células Secretoras de Insulina/patología , Insulinoma/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Fase G1 , Regulación Neoplásica de la Expresión Génica , Humanos , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Fase S , Transducción de Señal , Regulación hacia ArribaRESUMEN
OBJECTIVE: PKC-ζ activation is a key signaling event for growth factor-induced ß-cell replication in vitro. However, the effect of direct PKC-ζ activation in the ß-cell in vivo is unknown. In this study, we examined the effects of PKC-ζ activation in ß-cell expansion and function in vivo in mice and the mechanisms associated with these effects. RESEARCH DESIGN AND METHODS: We characterized glucose homeostasis and ß-cell phenotype of transgenic (TG) mice with constitutive activation of PKC-ζ in the ß-cell. We also analyzed the expression and regulation of signaling pathways, G1/S cell cycle molecules, and ß-cell functional markers in TG and wild-type mouse islets. RESULTS: TG mice displayed increased plasma insulin, improved glucose tolerance, and enhanced insulin secretion with concomitant upregulation of islet insulin and glucokinase expression. In addition, TG mice displayed increased ß-cell proliferation, size, and mass compared with wild-type littermates. The increase in ß-cell proliferation was associated with upregulation of cyclins D1, D2, D3, and A and downregulation of p21. Phosphorylation of D-cyclins, known to initiate their rapid degradation, was reduced in TG mouse islets. Phosphorylation/inactivation of GSK-3ß and phosphorylation/activation of mTOR, critical regulators of D-cyclin expression and ß-cell proliferation, were enhanced in TG mouse islets, without changes in Akt phosphorylation status. Rapamycin treatment in vivo eliminated the increases in ß-cell proliferation, size, and mass; the upregulation of cyclins Ds and A in TG mice; and the improvement in glucose tolerance-identifying mTOR as a novel downstream mediator of PKC-ζ-induced ß-cell replication and expansion in vivo. CONCLUSIONS PKC:-ζ, through mTOR activation, modifies the expression pattern of ß-cell cycle molecules leading to increased ß-cell replication and mass with a concomitant enhancement in ß-cell function. Approaches to enhance PKC-ζ activity may be of value as a therapeutic strategy for the treatment of diabetes.
Asunto(s)
Intolerancia a la Glucosa/metabolismo , Células Secretoras de Insulina/enzimología , Proteína Quinasa C/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Antibacterianos/farmacología , Glucemia , Regulación de la Expresión Génica/fisiología , Intolerancia a la Glucosa/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Insulina/sangre , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Transgénicos , Fosforilación , Proteína Quinasa C/genética , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genéticaRESUMEN
OBJECTIVE: Inducing human ß-cell growth while enhancing function is a major goal in the treatment of diabetes. Parathyroid hormone-related protein (PTHrP) enhances rodent ß-cell growth and function through the parathyroid hormone-1 receptor (PTH1R). Based on this, we hypothesized that PTH1R is expressed in human ß-cells and that PTHrP has the potential to enhance human ß-cell proliferation and/or function. RESEARCH DESIGN AND METHODS: PTH1R expression, ß-cell proliferation, glucose-stimulated insulin secretion (GSIS), and expression of differentiation and cell-cycle genes were analyzed in human islets transduced with adenoviral PTHrP constructs or treated with PTHrP peptides. The effect of overexpression of late G1/S cell cycle molecules was also assessed on human ß-cell proliferation. RESULTS: We found that human ß-cells express PTH1R. More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human ß-cell proliferation. Furthermore, the amino terminus PTHrP(1-36) peptide is sufficient to increase replication as well as expression of the late G1/S cell-cycle proteins cyclin E and cyclin-dependent kinase 2 (cdk2) in human islets. Notably, PTHrP(1-36) also enhances GSIS. Finally, overexpression of cyclin E alone, but not cdk2, augments human ß-cell proliferation, and when both molecules are expressed simultaneously there is a further marked synergistic increase in replication. CONCLUSIONS: PTHrP(1-36) peptide enhances human ß-cell proliferation as well as function, with associated upregulation of two specific cell-cycle activators that together can induce human ß-cell proliferation several fold. The future therapeutic potential of PTHrP(1-36) for the treatment of diabetes is especially relevant given the complementary therapeutic efficacy of PTHrP(1-36) in postmenopausal osteoporosis.
Asunto(s)
Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/genética , Células Secretoras de Insulina/fisiología , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Receptor de Hormona Paratiroídea Tipo 1/genética , Adolescente , Adulto , Anciano , Ciclo Celular/genética , Diferenciación Celular/genética , División Celular , Niño , Preescolar , Femenino , Regulación de la Expresión Génica , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Persona de Mediana Edad , Osteoporosis Posmenopáusica/tratamiento farmacológico , Proteína Relacionada con la Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Proteína Relacionada con la Hormona Paratiroidea/uso terapéutico , Fragmentos de Péptidos/farmacologíaRESUMEN
OBJECTIVE: Most knowledge on human beta-cell cycle control derives from immunoblots of whole human islets, mixtures of beta-cells and non-beta-cells. We explored the presence, subcellular localization, and function of five early G1/S phase molecules-cyclins D1-3 and cdk 4 and 6-in the adult human beta-cell. RESEARCH DESIGN AND METHODS: Immunocytochemistry for the five molecules and their relative abilities to drive human beta-cell replication were examined. Human beta-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse. RESULTS: Human beta-cells contain easily detectable cdks 4 and 6 and cyclin D3 but variable cyclin D1. Cyclin D2 was only marginally detectable. All five were principally cytoplasmic, not nuclear. Overexpression of the five, alone or in combination, led to variable increases in human beta-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control beta-cells). A single molecule, cdk6, proved to be capable of driving human beta-cell replication in vitro and enhancing human islet engraftment/proliferation in vivo, superior to normal islets and as effectively as the combination of cdk6 plus a D-cyclin. CONCLUSIONS: Human beta-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent beta-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal beta-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human beta-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human beta-cell proliferation and function in vivo.
Asunto(s)
Ciclina D/fisiología , Quinasa 6 Dependiente de la Ciclina/genética , Células Secretoras de Insulina/fisiología , Células Secretoras de Insulina/trasplante , Adulto , Animales , Western Blotting , División Celular , Ciclina D1/fisiología , Ciclina D2/fisiología , Ciclina D3/fisiología , Quinasa 6 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/fisiología , Fase G1/fisiología , Humanos , Células Secretoras de Insulina/citología , Islotes Pancreáticos/citología , Ratones , Fase S , Especificidad de la EspecieRESUMEN
Increasing evidence suggests that elevation of plasma fatty acids that often accompanies insulin resistance contributes to beta-cell insufficiency in obesity-related type 2 diabetes. Circulating levels of hepatocyte growth factor (HGF) are increased in humans with metabolic syndrome and obesity. HGF is known to protect beta-cells against streptozotocin and during islet engraftment. However, whether HGF is a beta-cell prosurvival factor in situations of excessive lipid supply has not been deciphered. Mice overexpressing HGF in the beta-cell [rat insulin type II promoter (RIP)-HGF transgenic mice] fed with standard chow display improved glucose homeostasis and increased beta-cell mass and proliferation compared with normal littermates. However, after 15 wk of high-fat feeding, glucose homeostasis and beta-cell expansion and proliferation are indistinguishable between normal and transgenic mice. Interestingly, RIP-HGF transgenic mouse beta-cells and normal beta-cells treated with HGF display increased sensitivity to palmitate-mediated apoptosis in vitro. Palmitate completely eliminates Akt and Bad phosphorylation in RIP-HGF transgenic mouse islets. HGF-overexpressing islets also show significantly decreased AMP-activated protein kinase-alpha and acetyl-coenzyme A carboxylase phosphorylation, diminished fatty acid oxidation, increased serine palmitoyltransferase expression, and enhanced ceramide formation compared with normal islets. Importantly, human islets overexpressing HGF also display increased beta-cell apoptosis in the presence of palmitate. Treatment of both mouse and human islet cells with the de novo ceramide synthesis inhibitors myriocin and fumonisin B1 abrogates beta-cell apoptosis induced by HGF and palmitate. Collectively, these studies indicate that HGF can be detrimental for beta-cell survival in an environment with excessive fatty acid supply.
Asunto(s)
Apoptosis/fisiología , Ácidos Grasos/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Células Secretoras de Insulina/patología , Ácido Palmítico/metabolismo , Páncreas/patología , Análisis de Varianza , Animales , Glucemia/metabolismo , Western Blotting , Proliferación Celular , Tamaño de la Célula , Células Cultivadas , Ceramidas/análisis , Grasas de la Dieta/administración & dosificación , Factor de Crecimiento de Hepatocito/genética , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Células Secretoras de Insulina/química , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Transgénicos , Ácido Palmítico/farmacología , Páncreas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Letal Asociada a bcl/metabolismoRESUMEN
OBJECTIVE: Ceramide is now recognized as a negative regulator of insulin signaling by impairing protein kinase B (PKB)/Akt activation. In different cells, two distinct mechanisms have been proposed to mediate ceramide inhibition of PKB/Akt: one involving atypical protein kinase C zeta (PKCzeta) and the other the protein phosphatase-2 (PP2A). We hypothesized that ceramide action through PKCzeta or PP2A might depend on plasma membrane (PM) structural organization and especially on caveolin-enriched domain (CEM) abundance. RESEARCH DESIGN AND METHODS: We have used different PKCzeta mutant constructs or the PP2A inhibitor, okadaic acid (OKA), to selectively inhibit PKCzeta- and PP2A-dependent pathways in cells expressing different caveolin-1 levels and evaluated the impact of insulin and ceramide on PKB/Akt activity in different PM subdomains. RESULTS: Although the PKCzeta-mediated negative effect of ceramide on insulin-stimulated PKB/Akt was dominant in adipocytes, a ceramide action through PP2A outside CEMs, prevented by OKA, was also unraveled. To test the importance of CEM to direct ceramide action through the PKCzeta pathway, we treated 3T3-L1 preadipocytes devoid of CEMs with ceramide and we saw a shift of the lipid-negative action on PKB/Akt to a PP2A-mediated mechanism. In fibroblasts with low CEM abundance, the ceramide-activated PP2A pathway dominated, but could be shifted to a ceramide-activated PKCzeta pathway after caveolin-1 overexpression. CONCLUSIONS: Our results show that ceramide can switch from a PKCzeta-dependent mechanism to a PP2A pathway, acting negatively on PKB/Akt, and hence revealing a critical role of CEMs of the PM in this process.
Asunto(s)
Adipocitos/metabolismo , Membrana Celular/enzimología , Ceramidas/farmacología , Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Caveolinas/metabolismo , Compartimento Celular/fisiología , Fibroblastos/citología , Humanos , Resistencia a la Insulina/fisiología , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/fisiología , Ratones , Palmitatos/metabolismo , Palmitatos/farmacología , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/fisiología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVES: To comprehensively inventory the proteins that control the G1/S cell cycle checkpoint in the human islet and compare them with those in the murine islet, to determine whether these might therapeutically enhance human beta-cell replication, to determine whether human beta-cell replication can be demonstrated in an in vivo model, and to enhance human beta-cell function in vivo. RESEARCH DESIGN AND METHODS: Thirty-four G1/S regulatory proteins were examined in human islets. Effects of adenoviruses expressing cdk-6, cdk-4, and cyclin D1 on proliferation in human beta-cells were studied in both in vitro and in vivo models. RESULTS: Multiple differences between murine and human islets occur, most strikingly the presence of cdk-6 in human beta-cells versus its low abundance in the murine islet. Cdk-6 and cyclin D1 in vitro led to marked activation of retinoblastoma protein phosphorylation and cell cycle progression with no induction of cell death. Human islets transduced with cdk-6 and cyclin D1 were transplanted into diabetic NOD-SCID mice and markedly outperformed native human islets in vivo, maintaining glucose control for the entire 6 weeks of the study. CONCLUSIONS: The human G1/S proteome is described for the first time. Human islets are unlike their rodent counterparts in that they contain easily measurable cdk-6. Cdk-6 overexpression, alone or in combination with cyclin D1, strikingly stimulates human beta-cell replication, both in vitro as well as in vivo, without inducing cell death or loss of function. Using this model, human beta-cell replication can be induced and studied in vivo.
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Ciclina D1/fisiología , Quinasa 6 Dependiente de la Ciclina/fisiología , Fase G1/genética , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/fisiología , Proteoma , Fase S/genética , Animales , Ciclo Celular , División Celular , Ciclina D1/genética , Quinasa 6 Dependiente de la Ciclina/genética , Cartilla de ADN , Regulación de la Expresión Génica , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/trasplante , Cinética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la EspecieRESUMEN
Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (DeltaNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro. This is of clinical relevance because adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which DeltaNLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, DeltaNLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G(0)/G(1) arrest. This pRb underphosphorylation was associated with stable levels of cdks 2, 4, and 6, the D and E cyclins, p16, p18, p19, and p21, but was associated with a dramatic decrease in cdk-2 and cdk4 kinase activities. Cyclin A was reduced, but restoring cyclin A adenovirally to normal did not promote cell cycle progression in DeltaNLS-PTH-related protein VSMC. More importantly, p15(INK4) and p27(kip1), two critical inhibitors of the G(1/S) progression, were markedly increased. Normalization of both p15(INK4b) and p27(kip1) by small interfering RNA knockdown normalized cell cycle progression. These data indicate that the changes in p15(INK4b) and p27(kip1) fully account for the marked cell cycle slowing induced by DeltaNLS-PTH-related protein in VSMCs. Finally, DeltaNLS-PTH-related protein is able to induce p15(INK4) and p27(kip1) expression when delivered adenovirally to primary murine VSMCs. These studies provide a mechanistic understanding of DeltaNLS-PTH-related protein actions, and suggest that DeltaNLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Músculo Liso Vascular/efectos de los fármacos , Señales de Localización Nuclear , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Animales , Arterias/efectos de los fármacos , Arterias/metabolismo , Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Reestenosis Coronaria/prevención & control , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Señales de Localización Nuclear/fisiología , Proteína Relacionada con la Hormona Paratiroidea/química , Proteína Relacionada con la Hormona Paratiroidea/genética , ARN Interferente Pequeño/farmacología , Ratas , Túnica Íntima/efectos de los fármacos , Túnica Íntima/crecimiento & desarrollo , Túnica Íntima/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: Adenoviral delivery of hepatocyte growth factor (HGF) to rodent islets improves islet graft survival and function, markedly reducing the number of islets required to achieve glucose control. Here, we asked whether these prior observations in rodent models extend to nonhuman primate (NHP) islets. RESEARCH DESIGN AND METHODS: NHP islets were transduced with murine (Ad.mHGF) or human (Ad.hHGF) adenoviral HGF (Ad.HGF) at low multiplicity of infection and studied in vitro. To study the function of Ad.HGF-transduced NHP islets in vivo, a renal subcapsular marginal mass islet transplant model was developed in streptozotocin-induced diabetic NOD-SCID mice. RESULTS: Baseline glucose values were 454.7 +/- 11.3 mg/dl (n = 7). Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 +/- 9.7 mg/dl, n = 5). In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0 +/- 6.2 mg/dl, n = 7) for the 6-week duration of the experiment. Unilateral nephrectomy resulted in an immediate return of glucose to baseline diabetic levels. Interestingly, adenoviral DNA, as well as mouse HGF (mHGF) mRNA derived from the adenovirus, were present for 42 days posttransplantation. Surprisingly, transplant of 500 IE with Ad.hHGF, as compared with Ad.mHGF, resulted in only marginal correction of blood glucose, suggesting that human HGF is less efficient than mHGF in this system. CONCLUSIONS: These studies demonstrate that mHGF markedly improves islet transplant outcomes in the highest preclinical species examined to date. HGF has promise as an agent that can improve islet mass and function in transplant models and likely in other models of types 1 and 2 diabetes.