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1.
Cell Stem Cell ; 25(2): 273-289.e5, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374199

RESUMO

Human monogenic diabetes, caused by mutations in genes involved in beta cell development and function, has been a challenge to study because multiple mouse models have not fully recapitulated the human disease. Here, we use genome edited human embryonic stem cells to understand the most common form of monogenic diabetes, MODY3, caused by mutations in the transcription factor HNF1A. We found that HNF1A is necessary to repress an alpha cell gene expression signature, maintain endocrine cell function, and regulate cellular metabolism. In addition, we identified the human-specific long non-coding RNA, LINKA, as an HNF1A target necessary for normal mitochondrial respiration. These findings provide a possible explanation for the species difference in disease phenotypes observed with HNF1A mutations and offer mechanistic insights into how the HNF1A gene may also influence type 2 diabetes.

2.
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.

3.
Diabetes ; 68(5): 988-1001, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30833470

RESUMO

Type 1 diabetes studies consistently generate data showing islet ß-cell dysfunction and T cell-mediated anti-ß-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the ß-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single ß-cells. Consistent with immunohistological studies, ß-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These ß-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin+CD68- ß-cells, specifically found in islets with lymphocytic infiltrates. ß-Cell surface expression of HLA Class II was detected on a portion of CD45-insulin+ ß-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic ß-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. ß-Cell expression of Class II molecules suggests that ß-cells may interact directly with islet-infiltrating CD4+ T cells and may play an immunopathogenic role.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Apresentação do Antígeno/imunologia , Autoimunidade/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Humanos , Insulina/metabolismo
4.
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.

5.
Diabetes ; 2018 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425060

RESUMO

The sustained expression of the MAFB transcription factor in human islet ß cells represents a distinct difference to mouse. Moreover, mRNA expression of closely related and islet ß cell-enriched MAFA does not peak in humans until after nine years in age. Here we show that the MAFA protein is also weakly produced within the juvenile human islet ß cell population, and that MafB expression is postnatally restricted in mouse ß cells by de novo DNA methylation. To obtain insight into how MAFB affects human ß cells, we developed a mouse model to ectopically express MafB in adult mouse ß cells using MafA transcriptional control sequences. Co-expression of MafB with MafA had no overt impact on mouse ß cells, suggesting that the human adult ß cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet ß cell defects in a mouse mutant lacking MafA in ß cells. Interestingly, transgenic production of MafB in ß cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives serotonin biosynthesis critical for adaptive maternal ß cell responses. Together, these studies provide novel insight into the role of MAFB in human islet ß cells.

6.
Cell Rep ; 22(10): 2667-2676, 2018 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-29514095

RESUMO

Many patients with type 1 diabetes (T1D) have residual ß cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual ß cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant ß cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and ß cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-ß cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.

9.
Nat Med ; 22(12): 1482-1487, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27798614

RESUMO

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood-derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.


Assuntos
Autoantígenos/imunologia , Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígeno HLA-A2/imunologia , Antígenos HLA-DQ/imunologia , Ilhotas Pancreáticas/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Criança , Feminino , Humanos , Masculino , Adulto Jovem
10.
Med Educ Online ; 21: 31534, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27499363

RESUMO

Leadership skills are essential for a successful career as a physician-scientist, yet many MD-PhD training programs do not offer formal training in leadership. The Vanderbilt Medical Scientist Training Program (MSTP) previously established a 2-day leadership workshop that has been held biennially since 2006 for students in the first and second years of the graduate school portion of combined MD and PhD training (G1/G2 students). Workshop attendees have consistently rated this workshop as a highly effective experience. However, opportunities for structured training in leadership competencies during the subsequent 3-5 years of MD-PhD training are limited. Given the success of the G1/G2 leadership workshop and the need for continuity in this model of leadership training, we developed a half-day workshop for MSTP students in the clinical years of medical school (M3/M4 students) to foster continued training in leadership. Our workshop curriculum, based in part on original cases drafted by Vanderbilt MSTP students, provides concrete strategies to manage conflict and navigate leadership transitions in the physician-scientist career path. The curriculum emphasizes both short-term competencies, such as effective participation as a member of a clinical team, and long-term competencies, such as leadership of a research team, division, or department. Our inaugural senior leadership workshop, held in August, 2015, was judged by student participants to be well organized and highly relevant to leadership concepts and skills. It will be offered biennially in our training curriculum for M3 and M4 MSTP students.


Assuntos
Pesquisa Biomédica/educação , Educação de Pós-Graduação em Medicina/organização & administração , Liderança , Escolha da Profissão , Currículo , Humanos , Negociação
11.
J Neurosci ; 33(7): 2761-72, 2013 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-23407936

RESUMO

Brain sexual differentiation in rodents results from the perinatal testicular androgen surge. In the preoptic area (POA), estradiol aromatized from testosterone upregulates the production of the proinflammatory molecule, prostaglandin E(2) (PGE(2)) to produce sex-specific brain development. PGE(2) produces a two-fold greater density of dendritic spines in males than in females and masculinizes adult copulatory behavior. One neonatal dose of PGE(2) masculinizes the POA and behavior, and simultaneous treatment with an inhibitor of additional prostaglandin synthesis prevents this masculinization, indicating a positive feedforward process that leads to sustained increases in PGE(2). The mechanisms underlying this feedforward process were unknown. Microglia, the primary immunocompetent cells in the brain, are active neonatally, contribute to normal brain development, and both produce and respond to prostaglandins. We investigated whether there are sex differences in microglia in the POA and whether they influence developmental masculinization. Neonatal males had twice as many ameboid microglia as females and a more activated morphological profile, and both estradiol and PGE(2) masculinized microglial number and morphology in females. Microglial inhibition during the critical period for sexual differentiation prevented sex differences in microglia, estradiol-induced masculinization of dendritic spine density, and adult copulatory behavior. Microglial inhibition also prevented the estradiol-induced upregulation of PGE(2), indicating that microglia are essential to the feedforward process through which estradiol upregulates prostaglandin production. These studies demonstrate that immune cells in the brain interact with the nervous and endocrine systems during development, and are crucial for sexual differentiation of brain and behavior.


Assuntos
Comportamento Animal/fisiologia , Encéfalo/citologia , Encéfalo/fisiologia , Microglia/fisiologia , Diferenciação Sexual/fisiologia , Animais , Western Blotting , Química Encefálica/fisiologia , Contagem de Células , Células Cultivadas , Espinhas Dendríticas/fisiologia , Dinoprostona/metabolismo , Dinoprostona/fisiologia , Estradiol/farmacologia , Estradiol/fisiologia , Feminino , Imunofluorescência , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Minociclina/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Área Pré-Óptica/crescimento & desenvolvimento , Área Pré-Óptica/metabolismo , Área Pré-Óptica/fisiologia , Ratos , Comportamento Sexual Animal/fisiologia , Maturidade Sexual
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