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1.
Nat Commun ; 13(1): 735, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35136059

RESUMO

Insulin receptor (Insr) protein is present at higher levels in pancreatic ß-cells than in most other tissues, but the consequences of ß-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in ß-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined ß-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout ß-cells from female, but not male mice, whereas only male ßInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female ßInsrKO and ßInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter ß-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include ß-cell insulin resistance, which predicts that ß-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female ßInsrKO and ßInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of ß-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that ß-cell insulin resistance in the form of reduced ß-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.


Assuntos
Diabetes Mellitus Tipo 2/genética , Hiperinsulinismo/genética , Resistência à Insulina/genética , Células Secretoras de Insulina/metabolismo , Receptor de Insulina/genética , Animais , Conjuntos de Dados como Assunto , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Glucose/metabolismo , Humanos , Hiperinsulinismo/sangue , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patologia , Insulina/sangue , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Receptor de Insulina/deficiência , Fatores Sexuais
2.
Diabetes ; 68(5): 975-987, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30833468

RESUMO

Pancreatic ß-cell regeneration, the therapeutic expansion of ß-cell number to reverse diabetes, is an important goal. Replication of differentiated insulin-producing cells is the major source of new ß-cells in adult mice and juvenile humans. Nucleoside analogs such as BrdU, which are incorporated into DNA during S-phase, have been widely used to quantify ß-cell proliferation. However, reports of ß-cell nuclei labeling with both BrdU and γ-phosphorylated H2A histone family member X (γH2AX), a DNA damage marker, have raised questions about the fidelity of BrdU to label S-phase, especially during conditions when DNA damage is present. We performed experiments to clarify the causes of BrdU-γH2AX double labeling in mouse and human ß-cells. BrdU-γH2AX colabeling is neither an age-related phenomenon nor limited to human ß-cells. DNA damage suppressed BrdU labeling and BrdU-γH2AX colabeling. In dispersed islet cells, but not in intact islets or in vivo, pro-proliferative conditions promoted both BrdU and γH2AX labeling, which could indicate DNA damage, DNA replication stress, or cell cycle-related intrinsic H2AX phosphorylation. Strategies to increase ß-cell number must not only tackle the difficult challenge of enticing a quiescent cell to enter the cell cycle, but also achieve safe completion of the cell division process.


Assuntos
Bromodesoxiuridina/química , Bromodesoxiuridina/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos
3.
Lab Chip ; 13(15): 3090-7, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23753064

RESUMO

Intercellular electromechanical transduction in adult cardiac myocytes plays an important role in regulating heart function. The efficiency of intercellular electromechanical transduction has so far been investigated only to a limited extent, which is largely due to the lack of appropriate tools that can quantitatively assess the contractile performance of interconnected adult cardiac myocytes. In this paper we report a microengineered device that is capable of applying electrical stimulation to the selected adult cardiac myocyte in a longitudinally connected cell doublet and quantifying the intercellular electromechanical transduction by measuring the contractile performance of stimulated and un-stimulated cells in the same doublet. The capability of applying selective electrical stimulation on only one cell in the doublet is validated by examining cell contractile performance while blocking the intercellular communication. Quantitative assessment of cell contractile performance in isolated adult cardiac myocytes is performed by measuring the change in cell length. The proof-of-concept assessment of gap junction performance shows that the device is useful in studying the efficiency of gap junctions in adult cardiac myocytes, which is most relevant to the synchronized pumping performance of native myocardium. Collectively, this work provides a quantitative tool for studying intercellular electromechanical transduction and is expected to develop a comprehensive understanding of the role of intercellular communication in various heart diseases.


Assuntos
Estimulação Elétrica/instrumentação , Dispositivos Lab-On-A-Chip , Miócitos Cardíacos/fisiologia , Animais , Comunicação Celular , Células Cultivadas , Desenho de Equipamento , Junções Comunicantes/fisiologia , Ratos , Ratos Sprague-Dawley
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