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1.
Methods Mol Biol ; 2592: 21-36, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36507983

RESUMO

The anterior chamber of the eye is a highly vascularized and innervated location that is also particularly rich in oxygen and immune privileged. This uncommon transplantation site offers unique possibilities for the observation of the transplanted material as well as for local pharmacological intervention. Transplantation of islets and islet organoids to the anterior chamber of the eye of mice and monkeys facilitates a multitude of new approaches for research into islet physiology and pathophysiology and for the treatment of diabetes. We now present a short overview of the experimental possibilities and describe an updated protocol for transplantation of islets and islet organoids into mice and monkeys.


Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Transplante das Ilhotas Pancreáticas/métodos , Haplorrinos , Roedores , Câmara Anterior
2.
Sci Rep ; 11(1): 21539, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34728728

RESUMO

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Oócitos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Sulfonilureias/metabolismo , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico , Oócitos/citologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores de Sulfonilureias/genética , Xenopus laevis
3.
Nutrients ; 13(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209449

RESUMO

Interactions between endocrine α and ß cells are critical to their secretory function in vivo. The interactions are highly regulated, although yet to be fully understood. In this study, we aim to assess the impact of α and ß cell co-culture on hormone secretion. Mouse clonal cell lines α-TC6-1 (α cell line) and MIN-6 (ß cell line) were cultured independently or in combination in a medium containing 5.5, 11.1, or 25 mM glucose, respectively. After 72 h, hormone release was measured using insulin and glucagon secretion assays, the cell distribution was visualized by inverted microscopy and an immunocytochemistry assay, and changes in gene expressions were assessed using the RT-PCR technique. The co-culture of the two cell lines caused a decrease in glucagon secretion from α-TC1-6 cells, while no effect on insulin secretion from MIN-6 cells was revealed. Both types of cells were randomly scattered throughout the culture flask, unlike in mice islets in vivo where ß cells cluster in the core and α cells are localized at the periphery. During the α-ß cell co-culture, the gene expression of glucagon (Gcg) decreased significantly. We conclude that islet ß cells suppress glucagon secretion from α cells, apparently via direct cell-to-cell contact, of which the molecular mechanism needs further verification.


Assuntos
Comunicação Celular , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Glucose/farmacologia , Secreção de Insulina , Camundongos
4.
Metabolites ; 11(6)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198579

RESUMO

The pancreatic islets of Langerhans consist of endocrine cells that secrete peptide hormones into the blood circulation in response to metabolic stimuli. When transplanted into the anterior chamber of the eye (ACE), pancreatic islets engraft and maintain morphological features of native islets as well as islet-specific vascularization and innervation patterns. In sufficient amounts, intraocular islets are able to maintain glucose homeostasis in diabetic mice. Islet organoids (pseudo-islets), which are formed by self-reassembly of islet cells following disaggregation and genetic manipulation, behave similarly to native islets. Here, we tested the hypothesis that genetically engineered intraocular islet organoids can serve as production sites for leptin. To test this hypothesis, we chose the leptin-deficient ob/ob mouse as a model system, which becomes severely obese, hyperinsulinemic, hyperglycemic, and insulin resistant. We generated a Tet-OFF-based beta-cell-specific adenoviral expression construct for mouse leptin, which allowed efficient transduction of native beta-cells, optical monitoring of leptin expression by co-expressed fluorescent proteins, and the possibility to switch-off leptin expression by treatment with doxycycline. Intraocular transplantation of islet organoids formed from transduced islet cells, which lack functional leptin receptors, to ob/ob mice allowed optical monitoring of leptin expression and ameliorated their metabolic phenotype by improving bodyweight, glucose tolerance, serum insulin, and C-peptide levels.

5.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670429

RESUMO

Loss of pancreatic ß-cell function is a critical event in the pathophysiology of type 2 diabetes. However, studies of its underlying mechanisms as well as the discovery of novel targets and therapies have been hindered due to limitations in available experimental models. In this study we exploited the stable viability and function of standardized human islet microtissues to develop a disease-relevant, scalable, and reproducible model of ß-cell dysfunction by exposing them to long-term glucotoxicity and glucolipotoxicity. Moreover, by establishing a method for highly-efficient and homogeneous viral transduction, we were able to monitor the loss of functional ß-cell mass in vivo by transplanting reporter human islet microtissues into the anterior chamber of the eye of immune-deficient mice exposed to a diabetogenic diet for 12 weeks. This newly developed in vitro model as well as the described in vivo methodology represent a new set of tools that will facilitate the study of ß-cell failure in type 2 diabetes and would accelerate the discovery of novel therapeutic agents.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Transplante das Ilhotas Pancreáticas , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Xenoenxertos , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout
6.
J Cell Mol Med ; 25(10): 4800-4813, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33742502

RESUMO

The two insulin receptor (IR) isoforms IR-A and IR-B are responsible for the pleiotropic actions of insulin and insulin-like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR-mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell-type-specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue-dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes.


Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica , Resistência à Insulina , Obesidade/complicações , Receptor de Insulina/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Processamento Alternativo , Animais , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Isoformas de Proteínas , Receptor de Insulina/genética , Transdução de Sinais
7.
Sci Rep ; 10(1): 20145, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33214580

RESUMO

The secretion of glucagon by pancreatic alpha cells is regulated by a number of external and intrinsic factors. While the electrophysiological processes linking a lowering of glucose concentrations to an increased glucagon release are well characterized, the evidence for the identity and function of the glucose sensor is still incomplete. In the present study we aimed to address two unsolved problems: (1) do individual alpha cells have the intrinsic capability to regulate glucagon secretion by glucose, and (2) is glucokinase the alpha cell glucose sensor in this scenario. Single cell RT-PCR was used to confirm that glucokinase is the main glucose-phosphorylating enzyme expressed in rat pancreatic alpha cells. Modulation of glucokinase activity by pharmacological activators and inhibitors led to a lowering or an increase of the glucose threshold of glucagon release from single alpha cells, measured by TIRF microscopy, respectively. Knockdown of glucokinase expression resulted in a loss of glucose control of glucagon secretion. Taken together this study provides evidence for a crucial role of glucokinase in intrinsic glucose regulation of glucagon release in rat alpha cells.


Assuntos
Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Glucoquinase/metabolismo , Glucose/metabolismo , Animais , Técnicas Biossensoriais , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Glucagon/genética , Células Secretoras de Glucagon/efeitos dos fármacos , Glucoquinase/genética , Glucose/farmacologia , Isoenzimas/metabolismo , Manoeptulose/farmacologia , Microscopia de Fluorescência , Ratos Wistar , Análise de Célula Única/métodos , Sulfonas/farmacologia , Tiazóis/farmacologia
8.
Diabetologia ; 63(10): 2064-2075, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32894317

RESUMO

The islet of Langerhans is a complex endocrine micro-organ consisting of a multitude of endocrine and non-endocrine cell types. The two most abundant and prominent endocrine cell types, the beta and the alpha cells, are essential for the maintenance of blood glucose homeostasis. While the beta cell produces insulin, the only blood glucose-lowering hormone of the body, the alpha cell releases glucagon, which elevates blood glucose. Under physiological conditions, these two cell types affect each other in a paracrine manner. While the release products of the beta cell inhibit alpha cell function, the alpha cell releases factors that are stimulatory for beta cell function and increase glucose-stimulated insulin secretion. The aim of this review is to provide a comprehensive overview of recent research into the regulation of beta cell function by alpha cells, focusing on the effect of alpha cell-secreted factors, such as glucagon and acetylcholine. The consequences of differences in islet architecture between species on the interplay between alpha and beta cells is also discussed. Finally, we give a perspective on the possibility of using an in vivo imaging approach to study the interactions between human alpha and beta cells under in vivo conditions. Graphical abstract.


Assuntos
Acetilcolina/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Animais , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/metabolismo , Humanos , Ilhotas Pancreáticas/anatomia & histologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Comunicação Parácrina
9.
FASEB J ; 34(1): 945-959, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914664

RESUMO

The dynamics of cytoplasmic free Ca2+ concentration ([Ca2+]i) in pancreatic ß cells is central to our understanding of ß-cell physiology and pathology. In this context, there are numerous in vitro studies available but existing in vivo data are scarce. We now critically evaluate the anterior chamber of the eye as an in vivo, non-invasive, imaging site for measuring [Ca2+]i dynamics longitudinally in three dimensions and at single-cell resolution. By applying a fluorescently labeled glucose analogue 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose in vivo, we followed how glucose almost simultaneously distributes to all cells within the islet volume, resulting in [Ca2+]i changes. We found that almost all ß cells in healthy mice responded to a glucose challenge, while in hyperinsulinemic, hyperglycemic mice about 80% of the ß cells could not be further stimulated from fasting basal conditions. This finding indicates that our imaging modality can resolve functional heterogeneity within the ß-cell population in terms of glucose responsiveness. Importantly, we demonstrate that glucose homeostasis is markedly affected using isoflurane compared to hypnorm/midazolam anesthetics, which has major implications for [Ca2+]i measurements. In summary, this setup offers a powerful tool to further investigate in vivo pancreatic ß-cell [Ca2+]i response patterns at single-cell resolution in health and disease.


Assuntos
Cálcio/química , Células Secretoras de Insulina/metabolismo , Anestésicos/farmacologia , Animais , Câmara Anterior/cirurgia , Cálcio/metabolismo , Cruzamentos Genéticos , Feminino , Glucose/farmacologia , Teste de Tolerância a Glucose , Heterozigoto , Homeostase , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas , Isoflurano/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Midazolam/farmacologia , Fenótipo
10.
FASEB J ; 33(1): 204-218, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29957055

RESUMO

Although convincing in genetic models, the relevance of ß-cell insulin resistance in diet-induced type 2 diabetes (T2DM) remains unclear. Exemplified by diabetes-prone, male, C57B1/6J mice being fed different combinations of Western-style diet, we show that ß-cell insulin resistance occurs early during T2DM progression and is due to a combination of lipotoxicity and increased ß-cell workload. Within 8 wk of being fed a high-fat, high-sucrose diet, mice became obese, developed impaired insulin and glucose tolerances, and displayed noncompensatory insulin release, due, at least in part, to reduced expression of syntaxin-1A. Through reporter islets transplanted to the anterior chamber of the eye, we demonstrated a concomitant loss of functional ß-cell mass. When mice were changed from diabetogenic diet to normal chow diet, the diabetes phenotype was reversed, suggesting a remarkable plasticity of functional ß-cell mass in the early phase of T2DM development. Our data reinforce the relevance of diet composition as an environmental factor determining different routes of diabetes progression in a given genetic background. Employing the in vivo reporter islet-monitoring approach will allow researchers to define key times in the dynamics of reversible loss of functional ß-cell mass and, thus, to investigate the underlying, molecular mechanisms involved in the progression toward T2DM manifestation.-Paschen, M., Moede, T., Valladolid-Acebes, I., Leibiger, B., Moruzzi, N., Jacob, S., García-Prieto, C. F., Brismar, K., Leibiger, I. B., Berggren, P.-O. Diet-induced ß-cell insulin resistance results in reversible loss of functional ß-cell mass.


Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Sacarose Alimentar/efeitos adversos , Resistência à Insulina , Células Secretoras de Insulina/patologia , Insulina/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
11.
Sci Rep ; 6: 21448, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26899548

RESUMO

Insulin resistance contributes to the development of cardio-vascular disease and diabetes. An important but unresolved task is to study the dynamics of insulin resistance in selective cell types of insulin target tissues in vivo. Here we present a novel technique to monitor insulin resistance dynamics non-invasively and longitudinally in vivo in a cell type-specific manner, exemplified by the pancreatic ß-cell situated within the micro-organ the islet of Langerhans. We utilize the anterior chamber of the eye (ACE) as a transplantation site and the cornea as a natural body-window to study the development and reversibility of insulin resistance. Engrafted islets in the ACE that express a FoxO1-GFP-based biosensor in their ß-cells, report on insulin resistance measured by fluorescence microscopy at single-cell resolution in the living mouse. This technique allows monitoring of cell type specific insulin sensitivity/resistance in real-time in the context of whole body insulin resistance during progression and intervention of disease.


Assuntos
Linhagem da Célula/genética , Rastreamento de Células/métodos , Resistência à Insulina/genética , Células Secretoras de Insulina/metabolismo , Animais , Córnea/metabolismo , Córnea/patologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/transplante , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Microscopia de Fluorescência , Análise de Célula Única
12.
Cell Rep ; 13(1): 15-22, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26387957

RESUMO

Insulin resistance is a syndrome that affects multiple insulin target tissues, each having different biological functions regulated by insulin. A remaining question is to mechanistically explain how an insulin target cell/tissue can be insulin resistant in one biological function and insulin sensitive in another at the same time. Here, we provide evidence that in pancreatic ß cells, knockdown of PI3K-C2α expression results in rerouting of the insulin signal from insulin receptor (IR)-B/PI3K-C2α/PKB-mediated metabolic signaling to IR-B/Shc/ERK-mediated mitogenic signaling, which allows the ß cell to switch from a highly glucose-responsive, differentiated state to a proliferative state. Our data suggest the existence of IR-cascade-selective insulin resistance, which allows rerouting of the insulin signal within the same target cell. Hence, factors involved in the rerouting of the insulin signal represent tentative therapeutic targets in the treatment of insulin resistance.


Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/genética , Receptor de Insulina/genética , Transdução de Sinais , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Obesos , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/metabolismo
13.
Proc Natl Acad Sci U S A ; 112(20): E2611-9, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25941406

RESUMO

Insulin resistance and ß-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and ß-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of ß-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of ß-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.


Assuntos
Apolipoproteína C-III/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/patologia , Análise de Variância , Animais , Apolipoproteína C-III/genética , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
14.
Nat Commun ; 5: 5308, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25374274

RESUMO

Type 2 diabetes mellitus is affecting more than 382 million people worldwide. Although much progress has been made, a comprehensive understanding of the underlying disease mechanism is still lacking. Here we report a role for the ß-cell primary cilium in type 2 diabetes susceptibility. We find impaired glucose handling in young Bbs4(-/-) mice before the onset of obesity. Basal body/ciliary perturbation in murine pancreatic islets leads to impaired first phase insulin release ex and in vivo. Insulin receptor is recruited to the cilium of stimulated ß-cells and ciliary/basal body integrity is required for activation of downstream targets of insulin signalling. We also observe a reduction in the number of ciliated ß-cells along with misregulated ciliary/basal body gene expression in pancreatic islets in a diabetic rat model. We suggest that ciliary function is implicated in insulin secretion and insulin signalling in the ß-cell and that ciliary dysfunction could contribute to type 2 diabetes susceptibility.


Assuntos
Cílios/fisiologia , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Suscetibilidade a Doenças/etiologia , Suscetibilidade a Doenças/fisiopatologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Glucose/metabolismo , Homeostase/fisiologia , Secreção de Insulina , Ilhotas Pancreáticas/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Obesidade/complicações , Obesidade/fisiopatologia , Fenótipo , Transdução de Sinais/fisiologia
15.
Cell ; 158(1): 41-53, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24995977

RESUMO

A hallmark of type 2 diabetes mellitus (T2DM) is the development of pancreatic ß cell failure, which results in insulinopenia and hyperglycemia. We show that the adipokine adipsin has a beneficial role in maintaining ß cell function. Animals genetically lacking adipsin have glucose intolerance due to insulinopenia; isolated islets from these mice have reduced glucose-stimulated insulin secretion. Replenishment of adipsin to diabetic mice treated hyperglycemia by boosting insulin secretion. We identify C3a, a peptide generated by adipsin, as a potent insulin secretagogue and show that the C3a receptor is required for these beneficial effects of adipsin. C3a acts on islets by augmenting ATP levels, respiration, and cytosolic free Ca(2+). Finally, we demonstrate that T2DM patients with ß cell failure are deficient in adipsin. These findings indicate that the adipsin/C3a pathway connects adipocyte function to ß cell physiology, and manipulation of this molecular switch may serve as a therapy in T2DM.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Tecido Adiposo/metabolismo , Animais , Complemento C3a/metabolismo , Fator D do Complemento/genética , Fator D do Complemento/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta Hiperlipídica , Glucose/metabolismo , Humanos , Inflamação/metabolismo , Insulina/metabolismo , Secreção de Insulina , Camundongos
16.
Proc Natl Acad Sci U S A ; 109(51): 20925-30, 2012 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-23213228

RESUMO

Peptide hormones are powerful regulators of various biological processes. To guarantee continuous availability and function, peptide hormone secretion must be tightly coupled to its biosynthesis. A simple but efficient way to provide such regulation is through an autocrine feedback mechanism in which the secreted hormone is "sensed" by its respective receptor and initiates synthesis at the level of transcription and/or translation. Such a secretion-biosynthesis coupling has been demonstrated for insulin; however, because of insulin's unique role as the sole blood glucose-decreasing peptide hormone, this coupling is considered an exception rather than a more generally used mechanism. Here we provide evidence of a secretion-biosynthesis coupling for glucagon, one of several peptide hormones that increase blood glucose levels. We show that glucagon, secreted by the pancreatic α cell, up-regulates the expression of its own gene by signaling through the glucagon receptor, PKC, and PKA, supporting the more general applicability of an autocrine feedback mechanism in regulation of peptide hormone synthesis.


Assuntos
Comunicação Autócrina , Glucagon/biossíntese , Glucagon/química , Transdução de Sinais , Animais , Glicemia/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Hormônios/química , Humanos , Insulina/metabolismo , Camundongos , Peptídeos/química , Ligação Proteica , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glucagon/química , Receptores de Glucagon/metabolismo
17.
J Biol Chem ; 287(34): 28932-42, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22718751

RESUMO

We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR). We now hypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YF-Nephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin-Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfate-induced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function.


Assuntos
Dinaminas/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Substituição de Aminoácidos , Dinaminas/genética , Inativação Gênica , Glucose/farmacologia , Células HEK293 , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Edulcorantes/metabolismo , Edulcorantes/farmacologia , Vanadatos/farmacologia
18.
Integr Biol (Camb) ; 4(2): 209-19, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22267247

RESUMO

Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 ± 1.4%, n = 3 for rat; 95.4 ± 1.7%, n = 4 for human, mean ± SEM). These cell fractions showed ~5-fold and ~4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca(2+)](i) upon stimulation with L-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 µM), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat alpha cells, as well as established a novel approach to separate human alpha cells using neither antibodies nor dyes possibly interfering with cellular functions.


Assuntos
Citometria de Fluxo/métodos , Células Secretoras de Glucagon/citologia , Ilhotas Pancreáticas/citologia , Adulto , Idoso , Animais , Cálcio/análise , Sobrevivência Celular , Feminino , Células Secretoras de Glucagon/química , Humanos , Ilhotas Pancreáticas/química , Masculino , Pessoa de Meia-Idade , RNA/química , RNA/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
19.
FASEB J ; 24(6): 1824-37, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20061534

RESUMO

Phosphatidylinositide 3-kinases (PI3Ks) play central roles in insulin signal transduction. While the contribution of class Ia PI3K members has been extensively studied, the role of class II members remains poorly understood. The diverse actions of class II PI3K-C2alpha have been attributed to its lipid product PI(3)P. By applying pharmacological inhibitors, transient overexpression and small-interfering RNA-based knockdown of PI3K and PKB/Akt isoforms, together with PI-lipid profiling and live-cell confocal and total internal reflection fluorescence microscopy, we now demonstrate that in response to insulin, PI3K-C2alpha generates PI(3,4)P(2), which allows the selective activation of PKBalpha/Akt1. Knockdown of PI3K-C2alpha expression and subsequent reduction of PKBalpha/Akt1 activity in the pancreatic beta-cell impaired glucose-stimulated insulin release, at least in part, due to reduced glucokinase expression and increased AS160 activity. Hence, our results identify signal transduction via PI3K-C2alpha as a novel pathway whereby insulin activates PKB/Akt and thus discloses PI3K-C2alpha as a potential drugable target in type 2 diabetes. The high degree of codistribution of PI3K-C2alpha and PKBalpha/Akt1 with insulin receptor B type, but not A type, in the same plasma membrane microdomains lends further support to the concept that selectivity in insulin signaling is achieved by the spatial segregation of signaling events.


Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Edulcorantes/farmacologia , Androstadienos/farmacologia , Animais , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Classe II de Fosfatidilinositol 3-Quinases , Imunofluorescência , Glucoquinase/metabolismo , Imunoprecipitação , Antagonistas da Insulina/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Lipídeos , Camundongos , Camundongos Obesos , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptor de Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Wortmanina
20.
Nat Med ; 14(5): 574-8, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18327249

RESUMO

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.


Assuntos
Diagnóstico por Imagem/métodos , Ilhotas Pancreáticas/ultraestrutura , Análise de Variância , Animais , Fluorescência , Iris/cirurgia , Ilhotas Pancreáticas/cirurgia , Transplante das Ilhotas Pancreáticas , Camundongos , Microscopia Confocal
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