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1.
Kidney Int ; 101(2): 256-273, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34774555

RESUMO

Chronic kidney disease (CKD) triggers the risk of developing uremic cardiomyopathy as characterized by cardiac hypertrophy, fibrosis and functional impairment. Traditionally, animal studies are used to reveal the underlying pathological mechanism, although variable CKD models, mouse strains and readouts may reveal diverse results. Here, we systematically reviewed 88 studies and performed meta-analyses of 52 to support finding suitable animal models for future experimental studies on pathological kidney-heart crosstalk during uremic cardiomyopathy. We compared different mouse strains and the direct effect of CKD on cardiac hypertrophy, fibrosis and cardiac function in "single hit" strategies as well as cardiac effects of kidney injury combined with additional cardiovascular risk factors in "multifactorial hit" strategies. In C57BL/6 mice, CKD was associated with a mild increase in cardiac hypertrophy and fibrosis and marginal systolic dysfunction. Studies revealed high variability in results, especially regarding hypertrophy and systolic function. Cardiac hypertrophy in CKD was more consistently observed in 129/Sv mice, which express two instead of one renin gene and more consistently develop increased blood pressure upon CKD induction. Overall, "multifactorial hit" models more consistently induced cardiac hypertrophy and fibrosis compared to "single hit" kidney injury models. Thus, genetic factors and additional cardiovascular risk factors can "prime" for susceptibility to organ damage, with increased blood pressure, cardiac hypertrophy and early cardiac fibrosis more consistently observed in 129/Sv compared to C57BL/6 strains.


Assuntos
Cardiomiopatias , Insuficiência Renal Crônica , Animais , Cardiomiopatias/genética , Modelos Animais de Doenças , Fibrose , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/complicações
2.
Mol Biol Rep ; 40(7): 4521-8, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23652999

RESUMO

Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG ß-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K(+) (P < 0.05). The MSG group also released less insulin in response to 100 µM carbachol, 10 µM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of ß-cell dysfunction.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ilhotas Pancreáticas/metabolismo , Obesidade/metabolismo , Proteína Quinase C/metabolismo , Receptor Muscarínico M3/metabolismo , Transdução de Sinais , Animais , Glicemia , Modelos Animais de Doenças , Quinases do Centro Germinativo , Glucose/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Insulina/metabolismo , Secreção de Insulina , Masculino , Obesidade/induzido quimicamente , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Glutamato de Sódio/administração & dosagem , Glutamato de Sódio/efeitos adversos
3.
Mol Vis ; 18: 194-202, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22312187

RESUMO

PURPOSE: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers. METHODS: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin). RESULTS: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased. CONCLUSIONS: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.


Assuntos
Envelhecimento/metabolismo , Expressão Gênica , Aparelho Lacrimal/metabolismo , Envelhecimento/genética , Animais , Biomarcadores/metabolismo , Western Blotting , Córnea/citologia , Córnea/metabolismo , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Aparelho Lacrimal/citologia , Peroxidação de Lipídeos , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Lágrimas/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Vitamina E/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismo
4.
Redox Biol ; 55: 102419, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35933903

RESUMO

Islet transplantation is a promising treatment strategy for type 1 diabetes mellitus (T1DM) patients. However, oxidative stress-induced graft failure due to an insufficient revascularization is a major problem of this therapeutic approach. NADPH oxidase (NOX)2 is an important producer of reactive oxygen species (ROS) and several studies have already reported that this enzyme plays a crucial role in the endocrine function and viability of ß-cells. Therefore, we hypothesized that targeting islet NOX2 improves the outcome of islet transplantation. To test this, we analyzed the cellular composition and viability of isolated wild-type (WT) and Nox2-/- islets by immunohistochemistry as well as different viability assays. Ex vivo, the effect of Nox2 deficiency on superoxide production, endocrine function and anti-oxidant protein expression was studied under hypoxic conditions. In vivo, we transplanted WT and Nox2-/- islets into mouse dorsal skinfold chambers and under the kidney capsule of diabetic mice to assess their revascularization and endocrine function, respectively. We found that the loss of NOX2 does not affect the cellular composition and viability of isolated islets. However, decreased superoxide production, higher glucose-stimulated insulin secretion as well as expression of nuclear factor erythroid 2-related factor (Nrf)2, heme oxygenase (HO)-1 and superoxide dismutase 1 (SOD1) was detected in hypoxic Nox2-/- islets when compared to WT islets. Moreover, we detected an early revascularization, a higher take rate and restoration of normoglycemia in diabetic mice transplanted with Nox2-/- islets. These findings indicate that the suppression of NOX2 activity represents a promising therapeutic strategy to improve engraftment and function of isolated islets.

5.
Front Immunol ; 13: 831680, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35265081

RESUMO

TNF-related apoptosis inducing ligand (TRAIL) is expressed on cytotoxic T lymphocytes (CTLs) and TRAIL is linked to progression of diabetes. However, the impact of high glucose on TRAIL expression and its related killing function in CTLs still remains largely elusive. Here, we report that TRAIL is substantially up-regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. Non-mitochondrial reactive oxygen species, NFκB and PI3K/Akt are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Treatment with metformin and vitamin D reduces HG-enhanced expression of TRAIL in CTLs and coherently protects 1.4E7 cells from TRAIL-mediated apoptosis. Our work suggests that HG-induced TRAILhigh CTLs might contribute to the destruction of pancreatic beta cells in a hyperglycemia condition.


Assuntos
Linfócitos T Citotóxicos , Ligante Indutor de Apoptose Relacionado a TNF , Glucose/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Linfócitos T Citotóxicos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
6.
Cells ; 10(12)2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34943836

RESUMO

A high caloric intake, rich in saturated fats, greatly contributes to the development of obesity, which is the leading risk factor for type 2 diabetes (T2D). A persistent caloric surplus increases plasma levels of fatty acids (FAs), especially saturated ones, which were shown to negatively impact pancreatic ß-cell function and survival in a process called lipotoxicity. Lipotoxicity in ß-cells activates different stress pathways, culminating in ß-cells dysfunction and death. Among all stresses, endoplasmic reticulum (ER) stress and oxidative stress have been shown to be strongly correlated. One main source of oxidative stress in pancreatic ß-cells appears to be the reactive oxygen species producer NADPH oxidase (NOX) enzyme, which has a role in the glucose-stimulated insulin secretion and in the ß-cell demise during both T1 and T2D. In this review, we focus on the acute and chronic effects of FAs and the lipotoxicity-induced ß-cell failure during T2D development, with special emphasis on the oxidative stress induced by NOX, the ER stress, and the crosstalk between NOX and ER stress.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina/patologia , Lipídeos/toxicidade , NADPH Oxidases/metabolismo , Estresse Oxidativo , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Lipídeos/química , Estresse Oxidativo/efeitos dos fármacos
7.
Free Radic Biol Med ; 162: 1-13, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33249137

RESUMO

Modern lifestyles, including lack of physical activity and poor nutritional habits, are driving the rapidly increasing prevalence of obesity and type 2 diabetes. Increased levels of free fatty acids (FFAs), particularly saturated FFAs, in obese individuals have been linked to pancreatic ß-cell failure. This process, termed lipotoxicity, involves activation of several stress responses, including ER stress and oxidative stress. However, the molecular underpinnings and causal relationships between the disparate stress responses remain unclear. Here we employed transgenic mice, expressing a genetically-encoded cytosolic H2O2 sensor, roGFP2-Orp1, to monitor dynamic changes in H2O2 levels in pancreatic islets in response to chronic palmitate exposure. We identified a transient increase in H2O2 levels from 4 to 8 h after palmitate addition, which was mirrored by a concomitant decrease in cellular NAD(P)H levels. Intriguingly, islets isolated from NOX2 knock-out mice displayed no H2O2 transient upon chronic palmitate treatment. Furthermore, NOX2 knockout rescued palmitate-dependent impairment of insulin secretion, calcium homeostasis and viability. Chemical inhibition of NOX activity protected islets from palmitate-induced impairment in insulin secretion, however had no detectable impact upon the induction of ER stress. In summary, our results reveal that transient NOX2-dependent H2O2 production is a likely cause of early palmitate-dependent lipotoxic effects.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Peróxido de Hidrogênio , Insulina , Camundongos , NADPH Oxidase 2/genética , Palmitatos/toxicidade
8.
Redox Rep ; 25(1): 41-50, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32354273

RESUMO

Objective: Investigate the involvement of the fatty acids receptor GPR40 in the assembly and activation of NADPH oxidase and the implications on pancreatic ß-cell function.Methods: BRIN-BD11 ß-cells were exposed to GPR40 agonist (GW9508) or linoleic acid in different glucose concentrations. Superoxide and H2O2 were analyzed, respectively, by DHE fluorescence and by fluorescence of the H2O2 sensor, roGFP2-Orp1. Protein contents of p47phox in plasma membrane and cytosol were analyzed by western blot. NADPH oxidase role was evaluated by p22phox siRNA or by pharmacological inhibition with VAS2870. NOX2 KO islets were used to measure total cytosolic calcium and insulin secretion.Results: GW9508 and linoleic acid increased superoxide and H2O2 contents at 5.6 and 8.3 mM of glucose. In addition, in 5.6 mM, but not at 16.7 mM of glucose, activation of GPR40 led to the translocation of p47phox to the plasma membrane. Knockdown of p22phox abolished the increase in superoxide after GW9508 and linoleic acid. No differences in insulin secretion were found between wild type and NOX2 KO islets treated with GW9508 or linoleic acid.Discussion: We report for the first time that acute activation of GPR40 leads to NADPH oxidase activation in pancreatic ß-cells, without impact on insulin secretion.


Assuntos
Secreção de Insulina , Células Secretoras de Insulina/metabolismo , NADPH Oxidases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Ativação Enzimática , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/genética , Ratos , Receptores Acoplados a Proteínas G/genética
9.
Pharmacol Rep ; 72(6): 1725-1737, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32274767

RESUMO

BACKGROUND: Free fatty acids (FFAs) are known for their dual effects on insulin secretion and pancreatic ß-cell survival. Short-term exposure to FFAs, such as palmitate, increases insulin secretion. On the contrary, long-term exposure to saturated FFAs results in decreased insulin secretion, as well as triggering oxidative stress and endoplasmic reticulum (ER) stress, culminating in cell death. The effects of FFAs can be mediated either via their intracellular oxidation and consequent effects on cellular metabolism or via activation of the membrane receptor GPR40. Both pathways are likely to be activated upon both short- and long-term exposure to FFAs. However, the precise role of GPR40 in ß-cell physiology, especially upon chronic exposure to FFAs, remains unclear. METHODS: We used the GPR40 agonist (GW9508) and antagonist (GW1100) to investigate the impact of chronically modulating GPR40 activity on BRIN-BD11 pancreatic ß-cells physiology and function. RESULTS: We observed that chronic activation of GPR40 did not lead to increased apoptosis, and both proliferation and glucose-induced calcium entry were unchanged compared to control conditions. We also observed no increase in H2O2 or superoxide levels and no increase in the ER stress markers p-eIF2α, CHOP and BIP. As expected, palmitate led to increased H2O2 levels, decreased cell viability and proliferation, as well as decreased metabolism and calcium entry. These changes were not counteracted by the co-treatment of palmitate-exposed cells with the GPR40 antagonist GW1100. CONCLUSIONS: Chronic activation of GPR40 using GW9508 does not negatively impact upon BRIN-BD11 pancreatic ß-cells physiology and function. The GPR40 antagonist GW1100 does not protect against the deleterious effects of chronic palmitate exposure. We conclude that GPR40 is probably not involved in mediating the toxicity associated with chronic palmitate exposure.


Assuntos
Benzoatos/farmacologia , Células Secretoras de Insulina/metabolismo , Metilaminas/farmacologia , Propionatos/farmacologia , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzoatos/administração & dosagem , Cálcio/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Metilaminas/administração & dosagem , Palmitatos/toxicidade , Propionatos/administração & dosagem , Pirimidinas/administração & dosagem , Ratos , Receptores Acoplados a Proteínas G/efeitos dos fármacos
10.
Antioxid Redox Signal ; 30(3): 297-313, 2019 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-29756464

RESUMO

Aims: Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion (GSIS) by pancreatic ß cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 (reduction/oxidation-sensitive enhanced green fluorescent protein fused to oxidant receptor peroxidase 1) and HyPer (hydrogen peroxide sensor) with its pH-control SypHer, to test the acute effects of glucose, monomethyl succinate, leucine with glutamine, and α-ketoisocaproate on ß cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and menadione concentrations on insulin secretion. Results: RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2-5 vs. 10 µM) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15 µM exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase, or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5 mM glucose in the cytosol and 10 mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1-15 µM) did not affect insulin secretion. By contrast, menadione (1-5 µM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20 mM glucose. Innovation: Subcellular changes in ß cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in ß cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. Conclusion: The GSIS occurs independently of a detectable increase in ß cell cytosolic or mitochondrial H2O2 levels.


Assuntos
Citosol/efeitos dos fármacos , Glucose/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Células Secretoras de Insulina/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Citosol/metabolismo , Glucose/metabolismo , Peróxido de Hidrogênio/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Mitocôndrias/metabolismo , Oxirredução , Ratos , Ratos Wistar
11.
Antioxid Redox Signal ; 29(6): 552-568, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-29160083

RESUMO

SIGNIFICANCE: Genetically encoded hydrogen peroxide (H2O2) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available "toolbox" for monitoring cellular H2O2 changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔCR offer considerably improved H2O2 sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous H2O2 levels. CRITICAL ISSUES: The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain. FUTURE DIRECTIONS: The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal H2O2 levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as H2O2 probes, roGFP2-based H2O2 sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552-568.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Sondas Moleculares , Oxirredução , Animais , Técnicas Biossensoriais , Glutationa/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Estresse Oxidativo , Peroxidases/metabolismo , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais , Compostos de Sulfidrila
12.
Redox Biol ; 17: 200-206, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29704824

RESUMO

Disulfide formation in the mitochondrial intermembrane space is an essential process catalyzed by a disulfide relay machinery. In mammalian cells, the key enzyme in this machinery is the oxidoreductase CHCHD4/Mia40. Here, we determined the in vivo CHCHD4 redox state, which is the major determinant of its cellular activity. We found that under basal conditions, endogenous CHCHD4 redox state in cultured cells and mouse tissues was predominantly oxidized, however, degrees of oxidation in different tissues varied from 70% to 90% oxidized. To test whether differences in the ratio between CHCHD4 and ALR might explain tissue-specific differences in the CHCHD4 redox state, we determined the molar ratio of both proteins in different mouse tissues. Surprisingly, ALR is superstoichiometric over CHCHD4 in most tissues. However, the levels of CHCHD4 and the ratio of ALR over CHCHD4 appear to correlate only weakly with the redox state, and although ALR is present in superstoichiometric amounts, it does not lead to fully oxidized CHCHD4.


Assuntos
Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases/genética , Animais , Dissulfetos/química , Camundongos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Especificidade de Órgãos , Oxirredução , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Transporte Proteico/genética
13.
Arq Bras Oftalmol ; 78(3): 158-63, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26222104

RESUMO

PURPOSE: In the lacrimal gland (LG) acinar cells, signaling regulates the release of secretory vesicles through specific Rab and SNARE exocytotic proteins. In diabetes mellitus (DM), the LGs are dysfunctional. The aim of this work was to determine if secretory apparatus changes were associated with any effects on the secretory vesicles (SV) in diabetic rats as well as the expression levels of constituent Rab and members of the SNARE family, and if insulin supplementation reversed those changes. METHODS: DM was induced in male Wistar rats with an intravenous dose of streptozotocin (60 mg/kg). One of the two diabetic groups was then treated every other day with insulin (1 IU). A third control group was injected with vehicle. After 10 weeks, Western blotting and RT-PCR were used to compared the Rab and SNARE secretory factor levels in the LGs. Transmission electron microscopy evaluated acinar cell SV density and integrity. RESULTS: In the diabetes mellitus group, there were fewer and enlarged SV. The Rab 27b, Rab 3d, and syntaxin-1 protein expression declined in the rats with diabetes mellitus. Insulin treatment restored the SV density and the Rab 27b and syntaxin expression to their control protein levels, whereas the Vamp 2 mRNA expression increased above the control levels. CONCLUSIONS: Diabetes mellitus LG changes were associated with the declines in protein expression levels that were involved in supporting exocytosis and vesicular formation. They were partially reversed by insulin replacement therapy. These findings may help to improve therapeutic management of dry eye in diabetes mellitus.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Aparelho Lacrimal/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Acetilcolina/análise , Células Acinares/ultraestrutura , Animais , Western Blotting/métodos , Diabetes Mellitus Experimental/induzido quimicamente , Exocitose/efeitos dos fármacos , Aparelho Lacrimal/metabolismo , Masculino , Modelos Animais , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , RNA Mensageiro/metabolismo , Ratos Wistar , Vesículas Secretórias/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
14.
Diabetes ; 60(10): 2533-45, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21885870

RESUMO

OBJECTIVE: Sarco-endoplasmic reticulum Ca(2+)-ATPase 2b (SERCA2b) and SERCA3 pump Ca(2+) in the endoplasmic reticulum (ER) of pancreatic ß-cells. We studied their role in the control of the free ER Ca(2+) concentration ([Ca(2+)](ER)) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS: ß-Cell [Ca(2+)](ER) of SERCA3(+/+) and SERCA3(-/-) mice was monitored with an adenovirus encoding the low Ca(2+)-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca(2+) concentration ([Ca(2+)](c)) and [Ca(2+)](ER) were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS: Glucose elicited synchronized [Ca(2+)](ER) and [Ca(2+)](c) oscillations. [Ca(2+)](ER) oscillations were smaller in SERCA3(-/-) than in SERCA3(+/+) ß-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca(2+)](ER) rise in SERCA3(+/+) and SERCA3(-/-) ß-cells. In a Ca(2+)-free medium, glucose moderately raised [Ca(2+)](ER) from a highly buffered cytosolic Ca(2+) pool. Increasing [Ca(2+)](c) with high [K] elicited a [Ca(2+)](ER) rise that was larger but more transient in SERCA3(+/+) than SERCA3(-/-) ß-cells because of the activation of a Ca(2+) release from the ER in SERCA3(+/+) ß-cells. Glucose-induced insulin release was larger in SERCA3(-/-) than SERCA3(+/+) islets. SERCA3 ablation did not induce ER stress. CONCLUSIONS: [Ca(2+)](c) and [Ca(2+)](ER) oscillate in phase in response to glucose. Upon [Ca(2+)](c) increase, Ca(2+) is taken up by SERCA2b and SERCA3. Strong Ca(2+) influx triggers a Ca(2+) release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca(2+) uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.


Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Cálcio/farmacologia , Diazóxido/farmacologia , Retículo Endoplasmático/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Engenharia Genética , Glucose/farmacologia , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Vasodilatadores/farmacologia
15.
Arq. bras. oftalmol ; Arq. bras. oftalmol;78(3): 158-163, May-Jun/2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-753015

RESUMO

ABSTRACT Purpose: In the lacrimal gland (LG) acinar cells, signaling regulates the release of secretory vesicles through specific Rab and SNARE exocytotic proteins. In diabetes mellitus (DM), the LGs are dysfunctional. The aim of this work was to determine if secretory apparatus changes were associated with any effects on the secretory vesicles (SV) in diabetic rats as well as the expression levels of constituent Rab and members of the SNARE family, and if insulin supplementation reversed those changes. Methods: DM was induced in male Wistar rats with an intravenous dose of streptozotocin (60 mg/kg). One of the two diabetic groups was then treated every other day with insulin (1 IU). A third control group was injected with vehicle. After 10 weeks, Western blotting and RT-PCR were used to compared the Rab and SNARE secretory factor levels in the LGs. Transmission electron microscopy evaluated acinar cell SV density and integrity. Results: In the diabetes mellitus group, there were fewer and enlarged SV. The Rab 27b, Rab 3d, and syntaxin-1 protein expression declined in the rats with diabetes mellitus. Insulin treatment restored the SV density and the Rab 27b and syntaxin expression to their control protein levels, whereas the Vamp 2 mRNA expression increased above the control levels. Conclusions: Diabetes mellitus LG changes were associated with the declines in protein expression levels that were involved in supporting exocytosis and vesicular formation. They were partially reversed by insulin replacement therapy. These findings may help to improve therapeutic management of dry eye in diabetes mellitus. .


RESUMO Objetivo: Células acinares da glândula lacrimal (GL) sinalizam a regulação da liberação através de vesículas secretórias específicas Rab proteínas exocitóticas SNARE. No diabetes mellitus (DM), as glândulas lacrimais são disfuncionais. O objetivo deste trabalho foi determinar se em ratos diabéticos, alterações dos aparatos secretórios estão associados a efeitos sobre vesículas secretoras (VS) e sobre os níveis de expressão do constituinte Rab, bem como membros da família SNARE, e se a suplementação de insulina reverte as alterações. Métodos: DM foi induzido em ratos Wistar machos com uma dose intravenosa de estreptozotocina (60 mg/kg). Um dos dois grupos diabéticos foi então tratado a cada dois dias com insulina (1 UI). Um terceiro grupo controle foi injetado com o veículo. Após 10 semanas, western blot e RT-PCR comparou níveis de fatores secretórios de Rab e SNARE na glândula lacrimal. Microscopia eletrônica de transmissão (MET) avaliaram a densidade e integridade de VS de célula acinar. Resultados: No grupo diabetes mellitus , houve poucas e alargadas VS. Rab27b, Rab 3d e Sintaxina-1 diminuiu a expressão da proteína em ratos com Diabetes Mellitus. O tratamento com insulina restaurou a densidade das VS e expressão de Rab 27b e Sintaxina para seus níveis de proteína controle, enquanto a expressão de Vamp 2 RNAm aumentou em relação aos controles. Conclusões: Alterações na glândula lacrimal de diabetes mellitus estão associadas a reduções nos níveis de expressão de proteínas envolvidas no apoio a exocitose e formação vesicular. Eles são, em parte, revertida por terapia de reposição de insulina. Estes resultados podem ajudar a melhorar a conduta terapêutica do olho seco no diabetes mellitus. .


Assuntos
Animais , Masculino , Diabetes Mellitus Experimental/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Aparelho Lacrimal/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Acetilcolina/análise , Células Acinares/ultraestrutura , Western Blotting/métodos , Diabetes Mellitus Experimental/induzido quimicamente , Exocitose/efeitos dos fármacos , Aparelho Lacrimal , Modelos Animais , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratos Wistar , RNA Mensageiro/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
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