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1.
Diabetologia ; 62(6): 1036-1047, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30955045

RESUMO

AIMS/HYPOTHESIS: The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases. METHODS: We developed matrix-assisted laser desorption/ionisation (MALDI) IMS protocols to study metabolite, lipid and protein distributions in mouse (wild-type and ob/ob mouse models) and human pancreases. IMS allows for the facile discrimination of chemically similar lipid and metabolite isoforms that cannot be distinguished using standard immunohistochemical techniques. Co-registration of MS images with immunofluorescence images acquired from serial tissue sections allowed accurate cross-registration of cell types. By acquiring immunofluorescence images first, this serial section approach guides targeted high spatial resolution IMS analyses (down to 15 µm) of regions of interest and leads to reduced time requirements for data acquisition. RESULTS: MALDI IMS enabled the molecular identification of specific phospholipid and glycolipid isoforms in pancreatic islets with intra-islet spatial resolution. This technology shows that subtle differences in the chemical structure of phospholipids can dramatically affect their distribution patterns and, presumably, cellular function within the islet and exocrine compartments of the pancreas (e.g. 18:1 vs 18:2 fatty acyl groups in phosphatidylcholine lipids). We also observed the localisation of specific GM3 ganglioside lipids [GM3(d34:1), GM3(d36:1), GM3(d38:1) and GM3(d40:1)] within murine islet cells that were correlated with a higher level of GM3 synthase as verified by immunostaining. However, in human pancreas, GM3 gangliosides were equally distributed in both the endocrine and exocrine tissue, with only one GM3 isoform showing islet-specific localisation. CONCLUSIONS/INTERPRETATION: The development of more complete molecular profiles of pancreatic tissue will provide important insight into the molecular state of the pancreas during islet development, normal function, and diseased states. For example, this study demonstrates that these results can provide novel insight into the potential signalling mechanisms involving phospholipids and glycolipids that would be difficult to detect by targeted methods, and can help raise new hypotheses about the types of physiological control exerted on endocrine hormone-producing cells in islets. Importantly, the in situ measurements afforded by IMS do not require a priori knowledge of molecules of interest and are not susceptible to the limitations of immunohistochemistry, providing the opportunity for novel biomarker discovery. Notably, the presence of multiple GM3 isoforms in mouse islets and the differential localisation of lipids in human tissue underscore the important role these molecules play in regulating insulin modulation and suggest species, organ, and cell specificity. This approach demonstrates the importance of both high spatial resolution and high molecular specificity to accurately survey the molecular composition of complex, multi-functional tissues such as the pancreas.

2.
JCI Insight ; 52019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30964447

RESUMO

Imatinib (Gleevec) reverses type 1 diabetes (T1D) in NOD mice and is currently in clinical trials in individuals with recent-onset disease. While research has demonstrated that imatinib protects islet ß cells from the harmful effects of ER stress, the role the immune system plays in its reversal of T1D has been less well understood, and specific cellular immune targets have not been identified. In this study, we demonstrate that B lymphocytes, an immune subset that normally drives diabetes pathology, are unexpectedly required for reversal of hyperglycemia in NOD mice treated with imatinib. In the presence of B lymphocytes, reversal was linked to an increase in serum insulin concentration, but not an increase in islet ß cell mass or proliferation. However, improved ß cell function was reflected by a partial recovery of MafA transcription factor expression, a sensitive marker of islet ß cell stress that is important to adult ß cell function. Imatinib treatment was found to increase the antioxidant capacity of B lymphocytes, improving reactive oxygen species (ROS) handling in NOD islets. This study reveals a novel mechanism through which imatinib enables B lymphocytes to orchestrate functional recovery of T1D ß cells.

3.
J Clin Invest ; 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30507613

RESUMO

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained ß cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.

4.
Front Genet ; 8: 35, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28424732

RESUMO

The two main types of diabetes mellitus have distinct etiologies, yet a similar outcome: loss of islet ß-cell function that is solely responsible for the secretion of the insulin hormone to reduce elevated plasma glucose toward euglycemic levels. Type 1 diabetes (T1D) has traditionally been characterized by autoimmune-mediated ß-cell death leading to insulin-dependence, whereas type 2 diabetes (T2D) has hallmarks of peripheral insulin resistance, ß-cell dysfunction, and cell death. However, a growing body of evidence suggests that, especially during T2D, key components of ß-cell failure involves: (1) loss of cell identity, specifically proteins associated with mature cell function (e.g., insulin and transcription factors like MAFA, PDX1, and NKX6.1), as well as (2) de-differentiation, defined by regression to a progenitor or stem cell-like state. New technologies have allowed the field to compare islet cell characteristics from normal human donors to those under pathophysiological conditions by single cell RNA-Sequencing and through epigenetic analysis. This has revealed a remarkable level of heterogeneity among histologically defined "insulin-positive" ß-cells. These results not only suggest that these ß-cell subsets have different responses to insulin secretagogues, but that defining their unique gene expression and epigenetic modification profiles will offer opportunities to develop cellular therapeutics to enrich/maintain certain subsets for correcting pathological glucose levels. In this review, we will summarize the recent literature describing how ß-cell heterogeneity and plasticity may be influenced in T2D, and various possible avenues of therapeutic intervention.

5.
Diabetes ; 65(8): 2331-41, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27217483

RESUMO

ß-Cell proliferation and expansion during pregnancy are crucial for maintaining euglycemia in response to increased metabolic demands placed on the mother. Prolactin and placental lactogen signal through the prolactin receptor (PRLR) and contribute to adaptive ß-cell responses in pregnancy; however, the in vivo requirement for PRLR signaling specifically in maternal ß-cell adaptations remains unknown. We generated a floxed allele of Prlr, allowing conditional loss of PRLR in ß-cells. In this study, we show that loss of PRLR signaling in ß-cells results in gestational diabetes mellitus (GDM), reduced ß-cell proliferation, and failure to expand ß-cell mass during pregnancy. Targeted PRLR loss in maternal ß-cells in vivo impaired expression of the transcription factor Foxm1, both G1/S and G2/M cyclins, tryptophan hydroxylase 1 (Tph1), and islet serotonin production, for which synthesis requires Tph1. This conditional system also revealed that PRLR signaling is required for the transient gestational expression of the transcription factor MafB within a subset of ß-cells during pregnancy. MafB deletion in maternal ß-cells also produced GDM, with inadequate ß-cell expansion accompanied by failure to induce PRLR-dependent target genes regulating ß-cell proliferation. These results unveil molecular roles for PRLR signaling in orchestrating the physiologic expansion of maternal ß-cells during pregnancy.


Assuntos
Diabetes Gestacional/metabolismo , Células Secretoras de Insulina/metabolismo , Fator de Transcrição MafB/metabolismo , Receptores da Prolactina/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Ciclina A2/genética , Ciclina B1/genética , Ciclina B2/genética , Ciclina D1/genética , Ciclina D2/genética , Diabetes Gestacional/fisiopatologia , Feminino , Proteína Forkhead Box M1/genética , Insulina/metabolismo , Fator de Transcrição MafB/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Receptores da Prolactina/genética , Serotonina/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo
6.
Dev Cell ; 34(6): 656-68, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26418295

RESUMO

For glucose-stimulated insulin secretion (GSIS), insulin granules have to be localized close to the plasma membrane. The role of microtubule-dependent transport in granule positioning and GSIS has been debated. Here, we report that microtubules, counterintuitively, restrict granule availability for secretion. In ß cells, microtubules originate at the Golgi and form a dense non-radial meshwork. Non-directional transport along these microtubules limits granule dwelling at the cell periphery, restricting granule availability for secretion. High glucose destabilizes microtubules, decreasing their density; such local microtubule depolymerization is necessary for GSIS, likely because granule withdrawal from the cell periphery becomes inefficient. Consistently, microtubule depolymerization by nocodazole blocks granule withdrawal, increases their concentration at exocytic sites, and dramatically enhances GSIS in vitro and in mice. Furthermore, glucose-driven MT destabilization is balanced by new microtubule formation, which likely prevents over-secretion. Importantly, microtubule density is greater in dysfunctional ß cells of diabetic mice.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Microtúbulos/fisiologia , Animais , Células Cultivadas , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Feminino , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Edulcorantes/farmacologia
7.
Islets ; 1(3): 269-72, 2009 Nov-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21099283

RESUMO

Histone tail acetylation and methylation are known to enhance accessibility of islet genes to transcription factors and the basal transcriptional machinery.  In this brief report, we follow up on a recent study in which we identified the islet enriched factor Set7/9 as a potentially important histone methyltransferase in ß-cells (Deering, et al. Diabetes 2009; 58:185-93).  We had suggested that the methylation of H3-Lys4 by Set7/9 enhances accessibility of the insulin gene to the basal transcriptional machinery.  Consistent with this hypothesis, we show here that RNA polymerase II occupancy at the insulin and IAPP genes is considerably enhanced in ß-cells compared to α cells (or NIH3T3 cells), and that the converse is true for RNA polymerase II occupancy at the glucagon gene. The enrichment of Set7/9 in ß-cells appears to be dependent upon Pdx1, as knockdown of Pdx1 in INS-1 ß-cells using small hairpin RNAs almost completely abolishes Set7/9 expression.  A LacZ expression vector driven by the -6.5 kilobase pair Set7/9 promoter that contains putative Pdx1 binding sites shows ß-cell-line-specific expression.  Taken together, our data support further the hypothesis that Pdx1-dependent Set7/9 expression may be crucial to enhancing chromatin accessibility and transcription of ß-cell genes.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/fisiologia , Ilhotas Pancreáticas/metabolismo , Animais , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Ilhotas Pancreáticas/fisiologia , Camundongos , Células NIH 3T3 , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologia , Transcrição Genética/genética
8.
Mol Cell Biol ; 27(11): 4093-104, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17403901

RESUMO

The critical pancreatic transcription factor Pdx1 is expressed throughout the pancreas early but enriched in insulin-producing beta cells postnatally. Previous studies showed that the 5' conserved promoter regions areas I and II (Pdx1(PB)) direct endocrine cell expression, while an adjacent region (Pdx1(XB)) containing conserved area III directs transient beta-cell expression. In this study, we used Cre-mediated lineage tracing to track cells that activated these regions. Pdx1(PB)Cre mediated only endocrine cell recombination, while Pdx1(XB)Cre directed broad and early recombination in the developing pancreas. Also, a reporter transgene containing areas I, II, and III was expressed throughout the embryonic day 10.5 (E10.5) pancreas and gradually became beta cell enriched, similar to endogenous Pdx1. These data suggested that sequences within area III mediate early pancreas-wide Pdx1 expression. Area III contains a binding site for PTF1, a transcription factor complex essential for pancreas development. This site contributed to area III-dependent reporter gene expression in the acinar AR42J cell line, while PTF1 specifically trans-activated area III-containing reporter expression in a nonpancreatic cell line. Importantly, Ptf1a occupied sequences spanning the endogenous PTF1 site in area III of E11.5 pancreatic buds. These data strongly suggest that PTF1 is an important early activator of Pdx1 in acinar and endocrine progenitor cells during pancreas development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Pâncreas/fisiologia , Regiões Promotoras Genéticas , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Feminino , Genes Reporter , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Transgênicos , Pâncreas/citologia , Pâncreas/embriologia , Transativadores/genética , Fatores de Transcrição/genética , Transgenes
9.
Dev Biol ; 298(2): 616-31, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16962573

RESUMO

The unique, well-demarcated expression domain of Pdx1 within the posterior foregut suggests that investigating its transcriptional regulation will provide insight into mechanisms that regionally pattern the endoderm. Previous phylogenetic comparison identified conserved noncoding regions that stimulate transcriptional activity selectively in cultured pancreatic beta cells. Characterization of these regulatory elements is helping to dissect the transcription factor networks that operate within beta cells, which is important for understanding the etiology of beta cell dysfunction and diabetes, as well as for developing methods to produce beta cells in vitro for cell-based therapies. We recently reported that deletion of three proximally located conserved areas (Area I-II-III) from the endogenous Pdx1 locus resulted in severely reduced expression of Pdx1 in the pancreas, and a milder decrease in other foregut tissues. Here, we report transgene-based complementation experiments on Pdx1 null mice, which reveal that the proximal promoter/enhancer region, including Area I-II-III, rescues the pancreatic defects caused by Pdx1 deficiency, but only weakly promotes expression of Pdx1 in the postnatal stomach and duodenum. These results reveal a role for distal cis-regulatory elements in achieving the correct level of extra-pancreatic Pdx1 expression, which is necessary for the production of duodenal GIP cells and stomach gastrin cells.


Assuntos
Duodeno/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Pâncreas/metabolismo , Elementos Reguladores de Transcrição , Transativadores/genética , Transativadores/fisiologia , Animais , Diferenciação Celular , Duodeno/citologia , Células Enteroendócrinas/metabolismo , Mucosa Gástrica/metabolismo , Dosagem de Genes , Expressão Gênica , Teste de Complementação Genética , Intolerância à Glucose , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Pâncreas/fisiologia , Fenótipo , Transativadores/metabolismo
10.
Genes Dev ; 20(2): 253-66, 2006 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16418487

RESUMO

Pdx1 (IPF-1 in humans, which is altered in MODY-4) is essential for pancreas development and mature beta-cell function. Pdx1 is expressed dynamically within the developing foregut, but how its expression characteristics are linked to the various steps of organ specification, differentiation, and function is unknown. Deletion of a conserved enhancer region (Area I-II-III) from Pdx1 produced a hypomorphic allele (Pdx1(DeltaI-II-III)) with altered timing and level of expression, which was studied in combination with wild-type and protein-null alleles. Lineage labeling in homozygous Area I-II-III deletion mutants (Pdx1(DeltaI-II-III/DeltaI-II-III)) revealed lack of ventral pancreatic bud specification and early-onset hypoplasia in the dorsal bud. Acinar tissue formed in the hypoplastic dorsal bud, but endocrine maturation was greatly impaired. While Pdx1(-/-) (protein-null) mice have nonpancreatic abnormalities (e.g., distorted pylorus, absent Brunner's glands), these structures formed normally in Pdx1(DeltaI-II-III/DeltaI-II-III) and Pdx1(DeltaI-II-III/-) mice. Surprisingly, heterozygous (Pdx1(+/DeltaI-II-III)) mice had abnormal islets and a more severe prediabetic condition than Pdx1(+/-) mice. These findings provide in vivo evidence of the differential requirements for the level of Pdx1 gene activity in the specification and differentiation of the various organs of the posterior foregut, as well as in pancreas and gut endocrine cell differentiation.


Assuntos
Proteínas de Homeodomínio/genética , Mucosa Intestinal/metabolismo , Pâncreas/embriologia , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/genética , Animais , Diferenciação Celular/genética , Sequência Conservada , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Enteroendócrinas/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Organogênese/genética , Pâncreas/metabolismo , Sequências Reguladoras de Ácido Nucleico/fisiologia , Transativadores/metabolismo
11.
Gene ; 313: 179-88, 2003 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-12957389

RESUMO

The selective expression of the Xenopus TFIIIA gene in immature oocytes is principally regulated by a single 5'-flanking DNA sequence element, termed element 3 (i.e. E3). We describe the isolation and characterization of a cDNA for a protein present in immature Xenopus ooctyes, termed B3.65, which appears to bind to and activate E3-mediated expression. The approximate molecular weight of the E3 binding protein(s) was determined by ultraviolet light cross-linking analysis. B3.65, a protein of the appropriate molecular weight, was purified biochemically from immature Xenopus ooctye extracts by affinity chromatography. Antiserum to purified B3.65 super-shifted the E3 activator complex. In addition, B3.65 mRNA was found to be highly enriched in immature oocytes. All of these data are consistent with B3.65 either being the E3 activator, or antigenically related to the specific activator required for XenopusTFIIIA gene transcription. B3.65 is a member of the K-homologous (KH) domain family of proteins, with almost absolute identity to Xenopus Vg1 RBP/VERA (97%) and significant similarity to human koc (82%). The koc mRNA is over-expressed in human pancreatic cancer tissues, and B3.65 mRNA was detected in Xenopus pancreas and kidney. Interestingly, KH proteins, like Vg1RBP/VERA, are most commonly associated with RNA metabolism, in their capacity to regulate RNA localization, stability, and translation. Our results suggest that B3.65 is a key regulator of both RNA- and DNA metabolism.


Assuntos
Fator de Transcrição TFIIIA/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Northern Blotting , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Peso Molecular , Oócitos/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Transcrição Genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis/embriologia , Xenopus laevis/metabolismo
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