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Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate, which controls hormone release and ß-cell viability by acting on glutamate receptors expressed by endocrine cells. We here investigate whether alteration of the excitatory amino acid transporter 2 (EAAT2), the major glutamate clearance system in the islet, may occur in type 2 diabetes mellitus and contribute to ß-cell dysfunction. Increased EAAT2 intracellular localization was evident in islets of Langerhans from T2DM subjects as compared with healthy control subjects, despite similar expression levels. Chronic treatment of islets from healthy donors with high-glucose concentrations led to the transporter internalization in vesicular compartments and reduced [H3]-d-glutamate uptake (65 ± 5% inhibition), phenocopying the findings in T2DM pancreatic sections. The transporter relocalization was associated with decreased Akt phosphorylation protein levels, suggesting an involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the process. In line with this, PI3K inhibition by a 100-µM LY294002 treatment in human and clonal ß-cells caused the transporter relocalization in intracellular compartments and significantly reduced the glutamate uptake compared to control conditions, suggesting that hyperglycemia changes the trafficking of the transporter to the plasma membrane. Upregulation of the glutamate transporter upon treatment with the antibiotic ceftriaxone rescued hyperglycemia-induced ß-cells dysfunction and death. Our data underscore the significance of EAAT2 in regulating islet physiology and provide a rationale for potential therapeutic targeting of this transporter to preserve ß-cell survival and function in diabetes.NEW & NOTEWORTHY The glutamate transporter SLC1A2/excitatory amino acid transporter 2 (EAAT2) is expressed on the plasma membrane of pancreatic ß-cells and controls islet glutamate clearance and ß-cells survival. We found that the EAAT2 membrane expression is lost in the islets of Langerhans from type 2 diabetes mellitus (T2DM) patients due to hyperglycemia-induced downregulation of the phosphoinositide 3-kinase/Akt pathway and modification of its intracellular trafficking. Pharmacological rescue of EAAT2 expression prevents ß-cell dysfunction and death, suggesting EAAT2 as a new potential target of intervention in T2DM.
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Diabetes Mellitus Tipo 2 , Transportador 2 de Aminoácido Excitatório , Ácido Glutâmico , Hiperglicemia , Ilhotas Pancreáticas , Transportador 2 de Aminoácido Excitatório/metabolismo , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Ácido Glutâmico/metabolismo , Hiperglicemia/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Feminino , Transporte Proteico , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Idoso , Adulto , Animais , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Purpose: To evaluate the impact of vitamin D (Vit D) supplementation on systemic biomarkers of collagen degradation, inflammation, oxidative stress, and copper metabolism in adolescent patients with keratoconus (KC). Methods: This was a prospective observational pilot study. Twenty patients (age range, 16-19 years) presenting KC and Vit D insufficiency (<30 ng/mL) were included. Vit D supplementation was prescribed by their general practitioner as per the standard of care. Patients were followed up for 12 months. At each visit, best spectacle-corrected visual acuity (BSCVA), maximal keratometry (Kmax), and thinnest corneal thickness (TCT) were evaluated. The primary outcome of the study was the proportion of patients with Kmax progression of less than 1 D throughout the 12-month follow-up time. Blood samples were collected at different time points to evaluate Vit D levels and systemic markers of collagen degradation, inflammation, oxidative stress, and copper metabolism by ELISA or RT-PCR. Results: Lower Vit D levels in the plasma were correlated with higher levels of systemic biomarkers of collagen degradation. Vit D supplementation increased the cell availability of copper. Moreover, stabilization of KC progression was found in 60% of patients (72% of eyes) after 12 months with Vit D supplementation. BSCVA, Kmax, and TCT rates remained stable during the observation period. Conclusions: Our findings support that Vit D administration could affect ocular and systemic biomarkers in KC and illuminate a possible mechanism that can be used to develop new treatment alternatives. Translational Relevance: Although KC therapy currently relies exclusively on surgical procedures, Vit D supplementation may offer a non-invasive and inexpensive alternative with minimal associated side effects.
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Ceratocone , Fotoquimioterapia , Adolescente , Humanos , Adulto Jovem , Adulto , Ceratocone/tratamento farmacológico , Ceratocone/metabolismo , Fármacos Fotossensibilizantes , Fotoquimioterapia/métodos , Cobre/uso terapêutico , Acuidade Visual , Raios Ultravioleta , Riboflavina , Estudos Prospectivos , Seguimentos , Topografia da Córnea , Colágeno , Inflamação , Vitamina D/uso terapêutico , Suplementos NutricionaisRESUMO
PURPOSE: To assess the accuracy of different corneal powers for intraocular (IOL) power calculation in combined Descemet membrane endothelial keratoplasty (DMEK) and cataract surgery and investigate whether preoperative parameters correlate to the prediction error (PE). METHODS: This prospective case series involved patients with Fuchs endothelial dystrophy receiving combined DMEK and cataract surgery. Preoperatively, patients underwent optical biometry and anterior segment OCT (AS-OCT). AS-OCT measurements were repeated 6 months postoperatively, when final refraction was assessed. The PE was calculated using the preoperative average keratometry (Kave) measured by the optical biometer and User Group for Laser Interference Biometry (ULIB) constants. It was also calculated, after constant optimization, using the preoperative Kave from both devices and the total corneal power (TCP) measured by AS-OCT, as well as the postoperative Kave and TCP measured by AS-OCT. RESULTS: ULIB constants resulted in the highest hyperopic PE (P < .0001). Constant optimization improved the results, because the PE was zeroed out and the absolute PEs decreased. No significant difference was found among the Barrett Universal II, Emmetropia Verifying Optical 2.0, Haigis, Hoffer Q, Holladay 1, Kane, and SRK/T formulas. Further improvement was achieved with the postoperative Kave and TCP, although the accuracy remained moderate. The PE based on preoperative corneal measurements was correlated to the amount of corneal flattening; the latter could be predicted by multiple linear regression accounting for anterior and posterior corneal radii (P = .0002) and was correlated to the preoperative anterior/posterior ratio. CONCLUSIONS: Constant optimization is beneficial for combined DMEK and phacoemulsification. Predicting postoperative corneal flattening may improve the results of IOL power accuracy. [J Refract Surg. 2022;38(7):435-442.].
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Catarata , Transplante de Córnea , Lentes Intraoculares , Facoemulsificação , Biometria/métodos , Lâmina Limitante Posterior , Humanos , Óptica e Fotônica , Facoemulsificação/métodos , Refração Ocular , Estudos RetrospectivosRESUMO
Substantial epidemiological evidence indicates that a diet rich in polyphenols protects against developing type 2 diabetes. The phenylethanoid glycoside verbascoside/acteoside, a widespread polyphenolic plant compound, has several biological properties including strong antioxidant, anti-inflammatory and neuroprotective activities. The aim of this research was to test the possible effects of verbascoside on pancreatic ß-cells, a target never tested before. Mouse and human ß-cells were incubated with verbascoside (0.8-16 µM) for up to five days and a combination of biochemical and imaging techniques were used to assess the ß-cell survival and function under normal or endoplasmic reticulum (ER)-stress inducing conditions. We found a dose-dependent protective effect of verbascoside against oxidative stress in clonal and human ß-cells. Mechanistic studies revealed that the polyphenol protects ß-cells against ER-stress mediated dysfunctions, modulating the activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response and promoting mitochondrial dynamics. As a result, increased viability, mitochondrial function and insulin content were detected in these cells. These studies provide the evidence that verbascoside boosts the ability of ß-cells to cope with ER-stress, an important contributor of ß-cell dysfunction and failure in diabetic conditions and support the therapeutic potential of verbascoside in diabetes.
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AIMS: Despite the very clear association between polycystic ovary syndrome (PCOS) and dysglycemia, few studies have explored the continuum of glycemic alterations leading from minor glucose abnormalities to overt diabetes. The purpose of this review is to trace the natural history of glycemic alteration in women with PCOS. METHODS: We performed a literature review without time limit until August 2019. Inclusion criteria were studies addressing the association between impaired glucose tolerance or impaired fasting glucose or type 2 diabetes (T2D) and PCOS with at least an English abstract. The exclusion criteria were no PCOS or impaired glucose tolerance or impaired fasting glucose or T2D as outcome. The outcomes of interest were the onset of impaired glucose tolerance, impaired fasting glucose, T2D, and the progression from impaired glucose tolerance or impaired fasting glucose to T2D. RESULTS: Healthy diet and physical activity are the first-line therapy for PCOS. Treatment with metformin was associated with significant lower 2-hour postload glucose levels and with reduction in fasting glucose when compared to placebo. Thiazolidinediones were more effective in reducing fasting glucose levels compared to placebo. Metformin and pioglitazone treatments showed similar effects on fasting glucose levels. The sodium-glucose cotransporter-2 inhibitor empagliflozin did not show differences in metabolic parameters when compared to metformin. The combination therapy with metformin plus the glucagon-like peptide-1 receptor agonist liraglutide was associated with significant improvements in basal and postload glucose levels compared with only liraglutide. Likewise, a combination therapy with the dipeptidyl peptidase-4 inhibitor saxagliptin and metformin demonstrated superiority versus metformin in fasting glucose and oral glucose tolerance test normalization. Myo-inositol supplementation was associated with lower insulin levels, glucose levels, and insulin resistance when compared with placebo, metformin, or estrogen treatments. CONCLUSIONS: The use of insulin-sensitizing agents, such as metformin and inositols, along with lifestyle interventions may improve the metabolic profile in PCOS women.
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Extracellular ATP (eATP) activates T cells by engaging the P2X7R receptor. We identified two loss-of-function P2X7R mutations that are protective against type 1 diabetes (T1D) and thus hypothesized that eATP/P2X7R signaling may represent an early step in T1D onset. Specifically, we observed that in patients with newly diagnosed T1D, P2X7R is upregulated on CD8+ effector T cells in comparison with healthy control subjects. eATP is released at high levels by human/murine islets in vitro in high-glucose/inflammatory conditions, thus upregulating P2X7R on CD8+ T cells in vitro. P2X7R blockade with oxidized ATP reduces the CD8+ T cell-mediated autoimmune response in vitro and delays diabetes onset in NOD mice. Autoreactive CD8+ T-cell activation is highly dependent upon eATP/P2X7R-mediated priming, while a novel sP2X7R recombinant protein abrogates changes in metabolism and the autoimmune response associated with CD8+ T cells. eATP/P2X7R signaling facilitates the onset of autoimmune T1D by fueling autoreactive CD8+ cells and therefore represents a novel targeted therapeutic for the disorder.
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Trifosfato de Adenosina/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Autoimunidade/genética , Autoimunidade/fisiologia , Diabetes Mellitus Tipo 1/genética , Feminino , Citometria de Fluxo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Mutação/genética , Receptores Purinérgicos P2X7/genéticaAssuntos
Diabetes Mellitus Tipo 1/cirurgia , Everolimo/uso terapêutico , Hipoglicemiantes/uso terapêutico , Imunossupressores/uso terapêutico , Transplante das Ilhotas Pancreáticas , Peptídeos/uso terapêutico , Peçonhas/uso terapêutico , Aloenxertos , Peptídeo C/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/diagnóstico , Everolimo/efeitos adversos , Exenatida , Hemoglobinas Glicadas/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Hipoglicemiantes/efeitos adversos , Imunossupressores/efeitos adversos , Peptídeos/efeitos adversos , Projetos Piloto , Fatores de Tempo , Resultado do Tratamento , Peçonhas/efeitos adversosRESUMO
AIMS/HYPOTHESIS: The cellular composition of the islet of Langerhans is essential to ensure its physiological function. Morphophysiological islet abnormalities are present in type 2 diabetes but the relationship between fasting plasma glucose (FPG) and islet cell composition, particularly the role of delta cells, is unknown. We explored these questions in pancreases from baboons (Papio hamadryas) with FPG ranging from normal to type 2 diabetic values. METHODS: We measured the volumes of alpha, beta and delta cells and amyloid in pancreatic islets of 40 baboons (Group 1 [G1]: FPG < 4.44 mmol/l [n = 10]; G2: FPG = 4.44-5.26 mmol/l [n = 9]; G3: FPG = 5.27-6.94 mmol/l [n = 9]; G4: FPG > 6.94 mmol/l [n = 12]) and correlated islet composition with metabolic and hormonal variables. We also performed confocal microscopy including TUNEL, caspase-3, and anti-caspase cleavage product of cytokeratin 18 (M30) immunostaining, electron microscopy, and immuno-electron microscopy with anti-somatostatin antibodies in baboon pancreases. RESULTS: Amyloidosis preceded the decrease in beta cell volume. Alpha cell volume increased â¼ 50% in G3 and G4 (p < 0.05), while delta cell volume decreased in these groups by 31% and 39%, respectively (p < 0.05). In G4, glucagon levels were higher, while insulin and HOMA index of beta cell function were lower than in the other groups. Immunostaining of G4 pancreatic sections with TUNEL, caspase-3 and M30 showed apoptosis of beta and delta cells, which was also confirmed by immuno-electron microscopy with anti-somatostatin antibodies. CONCLUSIONS/INTERPRETATION: In diabetic baboons, changes in islet composition correlate with amyloid deposition, with increased alpha cell and decreased beta and delta cell volume and number due to apoptosis. These data argue for an important role of delta cells in type 2 diabetes.
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Morte Celular , Diabetes Mellitus Tipo 2/patologia , Resistência à Insulina/fisiologia , Ilhotas Pancreáticas/patologia , Células Secretoras de Somatostatina/patologia , Animais , Glicemia/metabolismo , Caspase 3/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Progressão da Doença , Feminino , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/metabolismo , Masculino , Papio hamadryas , Células Secretoras de Somatostatina/metabolismoRESUMO
Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-dependent transamidating and G protein activity. Previously we reported that the role of TG2 in insulin secretion may involve cytoplasmic actin remodeling and a regulative action on other proteins during granule movement. The aim of this study was to gain a better insight into the role of TG2 transamidating activity in mitochondria and in the nucleus of INS-1E rat insulinoma cell line (INS-1E) during insulin secretion. To this end we labeled INS-1E with an artificial donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8min. Biotinylated proteins of the nuclear/mitochondrial-enriched fraction were analyzed using two-dimensional electrophoresis and mass spectrometry. Many mitochondrial proteins involved in Ca(2+) homeostasis (e.g. voltage-dependent anion-selective channel protein, prohibitin and different ATP synthase subunits) and many nuclear proteins involved in gene regulation (e.g. histone H3, barrier to autointegration factor and various heterogeneous nuclear ribonucleoprotein) were identified among a number of transamidating substrates of TG2 in INS-1E. The combined results provide evidence that a temporal link exists between glucose-stimulation, first phase insulin secretion and the action of TG on histone H3 both in INS-1E and human pancreatic islets. BIOLOGICAL SIGNIFICANCE: Research into the role of transglutaminase 2 during insulin secretion in INS-1E rat insulinoma cellular model is depicting a complex role for this enzyme. Transglutaminase 2 acts in the different INS-1E compartments in the same way: catalyzing a post-translational modification event of its substrates. In this work we identify some mitochondrial and nuclear substrates of INS-1E during first phase insulin secretion. The finding that TG2 interacts with nuclear proteins that include BAF and histone H3 immediately after (2-5min) glucose stimulus of INS-1E suggests that TG2 may be involved not only in insulin secretion, as suggested by our previous studies in cytoplasmic INS-1E fraction, but also in the regulation of glucose-induced gene transcription.
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Núcleo Celular/enzimologia , Células Secretoras de Insulina/enzimologia , Transglutaminases/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação ao GTP , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Edulcorantes/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Transglutaminases/genéticaRESUMO
Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-dependent transamidating and G protein activity. Previously, we reported that tgm2 -/- mice have an impaired insulin secretion and that naturally occurring TG2 mutations associated with familial, early-onset type 2 diabetes, show a defective transamidating activity. Aim of this study was to get a better insight into the role of TG2 in insulin secretion by identifying substrates of TG2 transamidating activity in the pancreatic beta cell line INS-1E. To this end, we labeled INS-1E that are capable of secreting insulin upon glucose stimulation in the physiologic range, with an artificial acyl acceptor (biotinamido-pentylamine) or donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. In addition, subcellular localization of TG2 in human endocrine pancreas was studied by electron microscopy. Among several TG2's transamidating substrates in INS-1E, mass spectrometry identified cytoplasmic actin (a result confirmed in human pancreatic islet), tropomyosin, and molecules that participate in insulin granule structure (e.g., GAPDH), glucose metabolism, or [Ca(2+)] sensing (e.g., calreticulin). Physical interaction between TG2 and cytoplasmic actin during glucose-stimulated first-phase insulin secretion was confirmed by co-immunoprecipitation. Electron microscopy revealed that TG2 is localized close to insulin and glucagon granules in human pancreatic islet. We propose that TG2's role in insulin secretion may involve cytoplasmic actin remodeling and may have a regulative action on other proteins during granule movement. A similar role of TG2 in glucagon secretion is also suggested.
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Proteínas de Ligação ao GTP/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Transglutaminases/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação ao GTP/química , Glucose/metabolismo , Humanos , Secreção de Insulina , Células Secretoras de Insulina/química , Espectrometria de Massas , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Especificidade por Substrato , Transglutaminases/químicaRESUMO
The search for factors either promoting islets proliferation or survival during adult life is a major issue for both type 1 and 2 diabetes mellitus. Among factors with mitogenic activity on pancreatic ß-cells, human placental lactogen (hPL) showed stronger activity when compared to the other lactogen hormones: growth hormone (GH) and prolactin (PRL). The aim of the present work is to elucidate the biological and molecular events of hPL isoform A (hPL-A) activity on human cultured islets. We used pure human pancreatic islets and insulinoma cell lines (ßTC-1 and RIN, murine and rat respectively) stimulated with hPL-A recombinant protein and we compared hPL-A activity with that of hGH. We showed that hPL-A inhibits apoptosis, both in insulinoma and human islets, by the phosphorylation of AKT protein. Indeed, the antiapoptotic role of hPL-A was mediated by PI3K, p38 and it was independent by PKA, Erk1/2. Compared with hGH, hPL-A modulated at different intervals and/or intensity by the phosphorylation of JAKs/STATs and MAPKinases. Moreover, hPL-A induced PDX-1 intracellular expression, improving beta cell activity and ameliorating insulin secretion in response to high glucose stimulation. Our data support the idea that hPL-A is involved in the regulation of beta cells activity. Importantly, we found that hPL-A can preserve and improve the ability of purified human pancreatic islets cultured to secrete insulin in vitro.
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Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Lactogênio Placentário/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hormônio do Crescimento Humano/metabolismo , Hormônio do Crescimento Humano/farmacologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Neoplasias Pancreáticas , Lactogênio Placentário/farmacologia , Prolactina/metabolismo , Prolactina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Evaluation of the effects of tight glycemia control in critically ill patients should include temporal as well as punctual glycemia data. METHODS: Insulin drip was used to target intensive care unit (ICU) glucose levels between 80 and 126 mg dl⻹ in a consecutive series of adult cardiac surgery patients. ICU hourly glycemia was prospectively recorded. Glycemia standard deviation, hyperglycemia index (area under the curve for glycemia>126 mg dl⻹ divided by total hours in ICU), and hypoglycemic episodes were recorded and analyzed, together with outcomes. RESULTS: A total of 596 patients were included. Hypoglycemia occurred in 21% of the patients. In-hospital mortality was 2.6%. There was a univariate correlation between mortality and glycemia standard deviation, and hypoglycemia occurrence. At multivariate analysis, hypoglycemia was a determinant for mortality (p=0.002; odds ratio (OR)=20.0), respiratory failure (p=0.0001; OR=1.4), requirement of a tracheostomy (p=0.0001; OR=21.6), and hemodynamic instability requiring intra-aortic balloon pump (IABP) (p=0.01; OR=1.5). To clarify the determinants of hypoglycemia, a second multivariate model was built. Diabetes (p=0.0001; OR=23) and chronic renal failure (p=0.01; OR=25) were the sole determinants for hypoglycemia occurrence. CONCLUSION: Iatrogenic hypoglycemia secondary to ICU tight glycemia control correlates with hospital mortality, respiratory, and cardiac morbidity in patients undergoing cardiac surgery. ICU hyperglycemia index and glycemia temporal variability have no independent correlation with outcomes. Higher glycemia targets should be advised in the perioperative management of patients with diabetes and renal failure, as both conditions independently increase the risk of hypoglycemia occurrence.
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Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Hipoglicemia/induzido quimicamente , Hipoglicemiantes/efeitos adversos , Insulina/efeitos adversos , Idoso , Glicemia/metabolismo , Cuidados Críticos/métodos , Esquema de Medicação , Métodos Epidemiológicos , Feminino , Humanos , Hipoglicemia/sangue , Hipoglicemia/complicações , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Cuidados Pós-Operatórios/métodos , Insuficiência Respiratória/etiologia , TraqueostomiaRESUMO
In both Type 1 and 2 diabetes, insufficient numbers of insulin-producing beta-cells are a major cause of defective control of blood glucose and its complications. Restoration of damaged beta-cells by endocrine pancreas regeneration would be an ideal therapeutic option. The possibility of generating insulin-secreting cells with adult pancreatic stem or progenitor cells has been investigated extensively. The conversion of differentiated cells such as hepatocytes into beta-cells is being attempted using molecular insights into the transcriptional make-up of beta-cells. Additionally, the enhanced proliferation of beta-cells in vivo or in vitro is being pursued as a strategy for regenerative medicine for diabetes. Advances have also been made in directing the differentiation of embryonic stem cells into beta-cells. Although progress is encouraging, major gaps in our understanding of developmental biology of the pancreas and adult beta-cell dynamics remain to be bridged before a therapeutic application is made possible.
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Diabetes Mellitus/metabolismo , Diabetes Mellitus/terapia , Transplante de Células-Tronco/métodos , Animais , Glicemia/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Camundongos , Modelos Biológicos , Medicina Regenerativa/métodos , Células-Tronco/citologia , Resultado do TratamentoRESUMO
Early impairment of islet function and graft loss strongly limit the success of allogenic islet transplantation in insulin-dependent diabetes. Macrophages play a key role in this process thus the depletion of these cells may strongly affect islet survival. In this study, we have evaluated the effect of the depletion of macrophages in mouse allograft rejection using a new approach based on a single infusion of red blood cells loaded with the synthetic analogue of pyrophosphate clodronate. Graft survival was 19.4+/-0.89 and 20+/-2 days in the two control groups treated with physiological solution and unloaded erythrocytes, respectively; 25+/-1.9 days in the group treated with free-clodronate and 35+/-6 days in the erythrocytes-loaded group. Our results indicate clodronate selectively targeted to the macrophagic cells by a single administration of engineered erythrocytes can significantly prolong islet graft survival and open new therapeutic strategies in islet transplantation.
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Conservadores da Densidade Óssea/uso terapêutico , Ácido Clodrônico/uso terapêutico , Diabetes Mellitus Experimental/cirurgia , Sobrevivência de Enxerto/fisiologia , Transplante das Ilhotas Pancreáticas/fisiologia , Macrófagos/imunologia , Animais , Sobrevivência de Enxerto/efeitos dos fármacos , Terapia de Imunossupressão/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fatores de Tempo , Transplante HomólogoRESUMO
Among its pleiotropic actions, ghrelin modulates insulin secretion and glucose metabolism. Herein we investigated the role of ghrelin in pancreatic beta-cell proliferation and apoptosis induced by serum starvation or interferon (IFN)-gamma/TNF-alpha, whose synergism is a major cause for beta-cell destruction in type I diabetes. HIT-T15 beta-cells expressed ghrelin but not ghrelin receptor (GRLN-R), which binds acylated ghrelin (AG) only. However, both unacylated ghrelin (UAG) and AG recognized common high-affinity binding sites on these cells. Either AG or UAG stimulated cell proliferation through Galpha(s) protein and prevented serum starvation- and IFN-gamma/TNF-alpha-induced apoptosis. Antighrelin antibody enhanced apoptosis in either the presence or absence of serum but not cytokines. AG and UAG even up-regulated intracellular cAMP. Blockade of adenylyl cyclase/cAMP/protein kinase A signaling prevented the ghrelin cytoprotective effect. AG and UAG also activated phosphatidyl inositol 3-kinase (PI3K)/Akt and ERK1/2, whereas PI3K and MAPK inhibitors counteracted the ghrelin antiapoptotic effect. Furthermore, AG and UAG stimulated insulin secretion from HIT-T15 cells. In INS-1E beta-cells, which express GRLN-R, AG and UAG caused proliferation and protection against apoptosis through identical signaling pathways. Noteworthy, both peptides inhibited cytokine-induced NO increase in either HIT-T15 or INS-1E cells. Finally, they induced cell survival and protection against apoptosis in human islets of Langerhans. These expressed GRLN-R but showed also UAG and AG binding sites. Our data demonstrate that AG and UAG promote survival of both beta-cells and human islets. These effects are independent of GRLN-R, are likely mediated by AG/UAG binding sites, and involve cAMP/PKA, ERK1/2, and PI3K/Akt.
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Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Hormônios Peptídicos/farmacologia , Acilação , Animais , Sítios de Ligação , Linhagem Celular , Cricetinae , Meios de Cultura Livres de Soro/farmacologia , AMP Cíclico/fisiologia , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Grelina , Humanos , Técnicas In Vitro , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Interferon gama/farmacologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Hormônios Peptídicos/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Grelina , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Islet transplantation offers the potential to improve glycemic control in a subgroup of patients with type 1 diabetes mellitus who are disabled by refractory hypoglycemia. We conducted an international, multicenter trial to explore the feasibility and reproducibility of islet transplantation with the use of a single common protocol (the Edmonton protocol). METHODS: We enrolled 36 subjects with type 1 diabetes mellitus, who underwent islet transplantation at nine international sites. Islets were prepared from pancreases of deceased donors and were transplanted within 2 hours after purification, without culture. The primary end point was defined as insulin independence with adequate glycemic control 1 year after the final transplantation. RESULTS: Of the 36 subjects, 16 (44%) met the primary end point, 10 (28%) had partial function, and 10 (28%) had complete graft loss 1 year after the final transplantation. A total of 21 subjects (58%) attained insulin independence with good glycemic control at any point throughout the trial. Of these subjects, 16 (76%) required insulin again at 2 years; 5 of the 16 subjects who reached the primary end point (31%) remained insulin-independent at 2 years. CONCLUSIONS: Islet transplantation with the use of the Edmonton protocol can successfully restore long-term endogenous insulin production and glycemic stability in subjects with type 1 diabetes mellitus and unstable control, but insulin independence is usually not sustainable. Persistent islet function even without insulin independence provides both protection from severe hypoglycemia and improved levels of glycated hemoglobin. (ClinicalTrials.gov number, NCT00014911 [ClinicalTrials.gov].).
Assuntos
Diabetes Mellitus Tipo 1/terapia , Transplante das Ilhotas Pancreáticas/métodos , Adulto , Glicemia/metabolismo , Peptídeo C/sangue , Diabetes Mellitus Tipo 1/sangue , Estudos de Viabilidade , Seguimentos , Humanos , Hipoglicemiantes/uso terapêutico , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Infusões Intravenosas , Insulina/metabolismo , Insulina/uso terapêutico , Secreção de Insulina , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/normas , Isoanticorpos/sangue , Pessoa de Meia-Idade , Infecções Oportunistas/epidemiologia , Veia Porta , Reprodutibilidade dos Testes , Condicionamento Pré-Transplante/normasRESUMO
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)-positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Ilhotas Pancreáticas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/fisiologia , Animais , Sequência de Bases , Separação Celular , Células Cultivadas , Quimiocinas/genética , Quimiocinas/fisiologia , Quimiotaxia/fisiologia , DNA/genética , Expressão Gênica , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Several reports have been published on islet transplantation in humans, but few data are available on the effect of islet infusion on the hepatic structure. Our aim was to evaluate in a longitudinal study the impact on the liver of intrahepatic islet transplantation. Clinical outcome and liver imaging were evaluated in 31 cases of islet-kidney transplantation (follow-up 38 +/- 4 months, range 12-96 months). Patients were divided into three groups: full function (FF, 9 cases: established insulin independence); partial function (PF, 16 cases: transient insulin independence, prolonged C-peptide secretion): no function (NF, 6 cases: exhaustion of C-peptide secretion within the first year). Upper abdomen sonogram was regularly performed during the whole follow-up period. Percutaneous liver biopsy was performed in case of echographic abnormalities. Multiple small areas of focal hyperechogenicity were observed in nine cases after 6-12 months. These findings were observed only in FF (two) and in PF (seven) patients. Fasting C-peptide levels at the time of echography were higher in negative than in positive patients (2.42 +/- 0.16 vs. 1.51 +/- 0.10 ng/ml, p = 0,0001). Liver biopsies showed focal macrovesicular steatosis, surrounded by normal liver parenchyma. Normal liver function was maintained. In conclusion, our results indicate that islet transplantation can lead to structural changes of the liver parenchyma (focal steatosis). It is more often observed in patients with partial function. Sonogram can be considered a specific method to reveal liver changes after islet transplantation.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Fígado Gorduroso/etiologia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Fígado/patologia , Adulto , Biópsia , Peptídeo C/sangue , Movimento Celular , Diabetes Mellitus Tipo 1/tratamento farmacológico , Fígado Gorduroso/sangue , Fígado Gorduroso/patologia , Seguimentos , Humanos , Insulina/uso terapêutico , Transplante de Rim , Fígado/diagnóstico por imagem , Estudos Longitudinais , Pâncreas/fisiologia , Pâncreas/fisiopatologia , Resultado do Tratamento , Ultrassonografia Doppler em CoresRESUMO
The potential effects of islet transplantation on the renal function of 36 patients with type I diabetes mellitus and kidney transplants were studied with 4 yr of follow-up monitoring. Kidney-islet recipients were divided into two groups, i.e., patients with successful islet transplants (SI-K group) (n = 24, fasting C-peptide levels of >0.5 ng/ml for >1 yr) and patients with unsuccessful islet transplants (UI-K group) (n = 12, fasting C-peptide levels of <0.5 ng/ml). Kidney graft survival rates and function, urinary albumin excretion rates, and sodium handling were compared. Na(+)/K(+)-ATPase activity in protocol kidney biopsies and in red blood cells was cross-sectionally analyzed. The SI-K group demonstrated better kidney graft survival rates (100, 83, and 83% at 1, 4, and 7 yr, respectively) than did the UI-K group (83, 72, and 51% at 1, 4, and 7 yr, respectively; P = 0.02). The SI-K group demonstrated reductions in exogenous insulin requirements and higher C-peptide levels, compared with the UI-K group, whereas GFR values were similar. Microalbuminuria (urinary albumin index) increased significantly in the UI-K group only (UI-K, from 92.0 +/- 64.9 to 183.8 +/- 83.8, P = 0.05; SI-K, from 108.5 +/- 53.6 to 85.0 +/- 39.0, NS). In the SI-K group, but not in the UI-K group, natriuresis decreased at 2 and 4 yr (P < 0.01). The SI-K group demonstrated greater Na(+)/K(+)-ATPase immunoreactivity in renal tubular cells (P = 0.05) and higher activity in red blood cells (P = 0.03), compared with the UI-K group. The Na(+)/K(+)-ATPase activity in red blood cells was positively correlated with circulating C-peptide levels but not with glycated hemoglobin levels. Successful islet transplantation was associated with improvements in kidney graft survival rates and function among uremic patients with type I diabetes mellitus and kidney grafts.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Rim/fisiologia , Uremia/metabolismo , Albuminas/metabolismo , Pressão Sanguínea , Peptídeo C/química , Diabetes Mellitus Tipo 1/complicações , Nefropatias Diabéticas/patologia , Eritrócitos/metabolismo , Taxa de Filtração Glomerular , Rejeição de Enxerto , Sobrevivência de Enxerto , Humanos , Rim/metabolismo , Transplante de Rim , Sistema Renina-Angiotensina , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo , Uremia/complicaçõesRESUMO
Recent evidence suggests that insulin signaling through the insulin receptor A type (Ex11-), regulates insulin gene transcription. Because chronic hyperglycemia negatively affects insulin receptor function and regulates alternative splicing of the insulin receptor, we inquired whether chronic exposure of pancreatic beta-cells to high glucose results in alterations in insulin signaling due to changes in insulin receptor expression and relative abundance of its spliced isoforms. Our results demonstrate that the insulin receptor is localized in insulin secretory vescicles in human pancreatic beta-cells. Furthermore, we find that alterations in insulin expression and secretion caused by chronic exposure to high glucose are paralleled by decreased insulin receptor expression and increased relative abundance of the Ex11+ isoform in both human islets and RIN beta-cells. PDX-1 and HMGI(Y) transcription factors are down-regulated by high glucose. These changes are associated with defects in insulin signaling involving insulin receptor-associated PI 3-kinase/Akt/PHAS-I pathway in RIN beta-cells. Re-expression in RIN beta-cells chronically exposed to high glucose of the Ex11-, but not the Ex11+, isoform restored insulin mRNA expression. These data suggest that changes in early steps of insulin receptor signaling may play a role in determining beta-cell dysfunction caused by chronic hyperglycemia.