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1.
Int J Mol Sci ; 21(21)2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-33143088

RESUMO

Diabetes mellitus is a very common chronic disease with progressively increasing prevalence. Besides the well-known autoimmune and inflammatory pathogenesis of type 1 diabetes, in many people, metabolic changes and inappropriate lifestyle favor a subtle chronic inflammatory state that contributes to development of insulin resistance and progressive loss of ß-cell function and mass, eventually resulting in metabolic syndrome or overt type 2 diabetes. In this paper, we review the anti-inflammatory effects of the extract of Hypericum perforatum L. (St. John's wort, SJW) and its main active ingredients firstly in representative pathological situations on inflammatory basis and then in pancreatic ß cells and in obese or diabetic animal models. The simultaneous and long-lasting inhibition of signal transducer and activator of transcription (STAT)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs)/c-jun N-terminal kinase (JNK) signaling pathways involved in pro-inflammatory cytokine-induced ß-cell dysfunction/death and insulin resistance make SJW particularly suitable for both preventive and therapeutic use in metabolic diseases. Hindrance of inflammatory cytokine signaling is likely dependent on the hyperforin content of SJW extract, but recent data reveal that hypericin can also exert relevant protective effects, mediated by activation of the cyclic adenosine monophosphate (cAMP)/protein kinase cAMP-dependent (PKA)/adenosine monophosphate activated protein kinase (AMPK) pathway, against high-fat-diet-induced metabolic abnormalities. Actually, the mechanisms of action of the two main components of SJW appear complementary, strengthening the efficacy of the plant extract. Careful quantitative analysis of SJW components and suitable dosage, with monitoring of possible drug-drug interaction in a context of remarkable tolerability, are easily achievable pre-requisites for forthcoming clinical applications.


Assuntos
Diabetes Mellitus Tipo 2/prevenção & controle , Hypericum/química , Inflamação/tratamento farmacológico , Floroglucinol/análogos & derivados , Extratos Vegetais/farmacologia , Terpenos/farmacologia , Animais , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/patologia , Humanos , Floroglucinol/farmacologia , Fitoterapia
2.
Nanomedicine ; 14(7): 2191-2203, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30016718

RESUMO

To improve the efficiency of pancreatic islet transplantation, we performed in-vitro and in-vivo experiments with isolated human pancreatic islets coated by multi-layer nano-encapsulation using differently charged polymers [chitosan and poly(sodium styrene sulfonate)] to obtain up to 9 layers. The islet coating (thickness: 104.2 ±â€¯4.2 nm) was uniform, with ≥ 90% cell viability and well preserved beta- and alpha-cell ultrastructure. Nano-encapsulated islets maintained physiological glucose-stimulated insulin secretion by both static incubation and perifusion studies. Notably, palmitate- or cytokine-induced toxicity was significantly reduced in nano-coated islets. Xenotransplantation of nano-encapsulated islets under the kidney capsule of streptozotocin-induced C57Bl/6J diabetic mice allowed long term normal or near normal glycemia, associated with minimal infiltration of immune cell into the grafts, well preserved islet morphology and signs of re-vascularization. In summary, the multi-layer nano-encapsulation approach described in the present study provides a promising tool to effectively protect human islets both in-vitro andin-vivo conditions.


Assuntos
Materiais Revestidos Biocompatíveis/química , Diabetes Mellitus Experimental/prevenção & controle , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/citologia , Nanoestruturas/administração & dosagem , Animais , Glicemia/análise , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/química , Transplante Heterólogo
4.
Diabetologia ; 57(2): 362-5, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24233056

RESUMO

AIMS/HYPOTHESIS: Previous work has demonstrated that beta cell amount (whether measured as beta cell mass, beta cell volume or insulin-positive area) is decreased in type 2 diabetes; however, recent findings suggest that mechanisms other than death may contribute to beta cell failure in this disease. To better characterise beta cell mass and function in type 2 diabetes, we performed morphological, ultra-structural and functional studies using histological samples and isolated islets. METHODS: Pancreases from ten non-diabetic (ND) and ten matched type 2 diabetic organ donors were studied by insulin, glucagon and chromogranin A immunocytochemistry and electron microscopy (EM). Glucose-stimulated insulin secretion was assessed using isolated islets and studies were performed using independent ND islet preparations after 24 h exposure to 22.2 mmol/l glucose. RESULTS: Immunocytochemistry showed that the fractional islet insulin-positive area was lower in type 2 diabetic islets (54.9 ± 6.3% vs 72.1 ± 8.7%, p < 0.01), whereas glucagon (23.3 ± 5.4% vs 20.2 ± 5.3%) and chromogranin A (86.4 ± 6.1% vs 89.0 ± 5.5%) staining was similar between the two groups. EM showed that the proportion of beta cells in type 2 diabetic islets was only marginally decreased; marked beta cell degranulation was found in diabetic beta cells; these findings were all reproduced after exposing isolated ND islets to high glucose. Glucose-stimulated insulin secretion was 40­50% lower from type 2 diabetic islets (p < 0.01), which again was mimicked by culturing non-diabetic islets in high glucose. CONCLUSIONS/INTERPRETATION: These results suggest that, at least in subgroups of type 2 diabetic patients, the loss of beta cells as assessed so far might be overestimated, possibly due to changes in beta cell phenotype other than death, also contributing to beta cell failure in type 2 diabetes.


Assuntos
Cromogranina A/metabolismo , Diabetes Mellitus Tipo 2/patologia , Glucagon/metabolismo , Células Secretoras de Insulina/patologia , Insulina/metabolismo , Pâncreas/patologia , Idoso , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica
5.
Biochim Biophys Acta ; 1833(8): 1904-13, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23545415

RESUMO

PRIMA-1 is a chemical compound identified as a growth suppressor of tumor cells expressing mutant p53. We previously found that in the MDA-MB-231 cell line expressing high level of the mutant p53-R280K protein, PRIMA-1 induced p53 ubiquitination and degradation associated to cell death. In this study, we investigated the ability of PRIMA-1 to induce autophagy in cancer cells. In MDA-MB-231 and HCT116 cells, expressing mutant or wild type p53, respectively, autophagy occurred following exposure to PRIMA-1, as shown by acridine orange staining, anti-LC3 immunofluorescence and immunoblots, as well as by electron microscopy. Autophagy was triggered also in the derivative cell lines knocked-down for p53, although to a different extent than in the parental cells expressing mutant or wild type p53. In particular, while wild type p53 limited PRIMA-1 induced autophagy, mutant p53 conversely promoted autophagy, thus sustaining cell viability following PRIMA-1 treatment. Therefore, the autophagic potential of PRIMA-1, besides being cell context dependent, could be modulated in a different way by the presence of wild type or mutant p53. Furthermore, since both cell lines lacking p53 were more sensitive to the cytotoxic effect of PRIMA-1 than the parental ones, our findings suggest that a deregulated autophagy may favor cell death induced by this drug.


Assuntos
Autofagia/efeitos dos fármacos , Proteínas de Membrana/farmacologia , Mutação , Neoplasias/genética , Proteínas do Tecido Nervoso/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Autofagia/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células HCT116 , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia
6.
J Nat Prod ; 77(3): 543-9, 2014 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-24417609

RESUMO

Garcinol (1), a polyisoprenylated benzophenone occurring in Garcinia species, has been reported to exert anti-inflammatory activity in LPS-stimulated macrophages, through inhibition of NF-κB and/or JAK/STAT-1 activation. In order to provide deeper insight into its effects on the cytokine signaling pathway and to clarify the underlying molecular mechanisms, 1 was isolated from the fruits of Garcinia cambogia along with two other polyisoprenylated benzophenones, guttiferones K (2) and guttiferone M (3), differing from each other in their isoprenyl moieties and their positions on the benzophenone core. The affinities of 1-3 for the STAT-1 protein have been evaluated by surface plasmon resonance and molecular docking studies and resulted in KD values in the micromolar range. Consistent with the observed high affinity toward the STAT-1 protein, garcinol and guttiferones K and M were able to modulate cytokine signaling in different cultured cell lines, mainly by inhibiting STAT-1 nuclear transfer and DNA binding, as assessed by an electrophorectic mobility shift assay.


Assuntos
Benzofenonas/isolamento & purificação , Benzofenonas/farmacologia , Garcinia cambogia/química , Macrófagos/efeitos dos fármacos , Fator de Transcrição STAT1/efeitos dos fármacos , Terpenos/química , Terpenos/farmacologia , Benzofenonas/química , Northern Blotting , Feminino , Frutas/química , Humanos , Lipopolissacarídeos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Conformação Molecular , Estrutura Molecular , NF-kappa B/antagonistas & inibidores , NF-kappa B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sri Lanka , Terpenos/isolamento & purificação
7.
Bioorg Med Chem Lett ; 22(11): 3810-5, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22565215

RESUMO

We describe a new class of NO-donor hypoglycemic products obtained by joining tolbutamide, a typical hypoglycemic sulfonylurea, with a NO-donor moiety through a hard link. As NO-donors we chose either furoxan (1,2,5-oxadiazole 2-oxide) derivatives or the classical nitrooxy function. A preliminary biological characterization of these compounds, including stimulation of insulin release from cultured rat pancreatic ß-cells and in vitro vasodilator and anti-aggregatory activities, is reported.


Assuntos
Hipoglicemiantes/síntese química , Doadores de Óxido Nítrico/química , Tolbutamida/análogos & derivados , Vasodilatadores/síntese química , Animais , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ratos , Compostos de Sulfonilureia/química , Tolbutamida/síntese química , Tolbutamida/farmacologia , Vasodilatadores/química , Vasodilatadores/farmacologia
8.
Physiol Rep ; 10(16): e15425, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35986504

RESUMO

eNOS-deficient mice were previously shown to develop hypertension and metabolic alterations associated with insulin resistance either in standard dietary conditions (eNOS-/- homozygotes) or upon high-fat diet (HFD) (eNOS+/- heterozygotes). In the latter heterozygote model, the present study investigated the pancreatic morphological changes underlying the abnormal glycometabolic phenotype. C57BL6 wild type (WT) and eNOS+/- mice were fed with either chow or HFD for 16 weeks. After being longitudinally monitored for their metabolic state after 8 and 16 weeks of diet, mice were euthanized and fragments of pancreas were processed for histological, immuno-histochemical and ultrastructural analyses. HFD-fed WT and eNOS+/- mice developed progressive glucose intolerance and insulin resistance. Differently from WT animals, eNOS+/- mice showed a blunted insulin response to a glucose load, regardless of the diet regimen. Such dysregulation of insulin secretion was associated with pancreatic ß-cell hyperplasia, as shown by larger islet fractional area and ß-cell mass, and higher number of extra-islet ß-cell clusters than in chow-fed WT animals. In addition, only in the pancreas of HFD-fed eNOS+/- mice, there was ultrastructural evidence of a number of hybrid acinar-ß-cells, simultaneously containing zymogen and insulin granules, suggesting the occurrence of a direct exocrine-endocrine transdifferentiation process, plausibly triggered by metabolic stress associated to deficient endothelial NO production. As suggested by confocal immunofluorescence analysis of pancreatic histological sections, inhibition of Notch-1 signaling, likely due to a reduced NO availability, is proposed as a novel mechanism that could favor both ß-cell hyperplasia and acinar-ß-cell transdifferentiation in eNOS-deficient mice with impaired insulin response to a glucose load.


Assuntos
Resistência à Insulina , Células Secretoras de Insulina , Animais , Glicemia/metabolismo , Transdiferenciação Celular , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Hiperplasia/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
Antioxidants (Basel) ; 10(1)2020 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33379141

RESUMO

In this paper we review the mechanisms of the antitumor effects of Hypericum perforatum L. (St. John's wort, SJW) and its main active component hyperforin (HPF). SJW extract is commonly employed as antidepressant due to its ability to inhibit monoamine neurotransmitters re-uptake. Moreover, further biological properties make this vegetal extract very suitable for both prevention and treatment of several diseases, including cancer. Regular use of SJW reduces colorectal cancer risk in humans and prevents genotoxic effects of carcinogens in animal models. In established cancer, SJW and HPF can still exert therapeutic effects by their ability to downregulate inflammatory mediators and inhibit pro-survival kinases, angiogenic factors and extracellular matrix proteases, thereby counteracting tumor growth and spread. Remarkably, the mechanisms of action of SJW and HPF include their ability to decrease ROS production and restore pH imbalance in tumor cells. The SJW component HPF, due to its high lipophilicity and mild acidity, accumulates in membranes and acts as a protonophore that hinders inner mitochondrial membrane hyperpolarization, inhibiting mitochondrial ROS generation and consequently tumor cell proliferation. At the plasma membrane level, HPF prevents cytosol alkalization and extracellular acidification by allowing protons to re-enter the cells. These effects can revert or at least attenuate cancer cell phenotype, contributing to hamper proliferation, neo-angiogenesis and metastatic dissemination. Furthermore, several studies report that in tumor cells SJW and HPF, mainly at high concentrations, induce the mitochondrial apoptosis pathway, likely by collapsing the mitochondrial membrane potential. Based on these mechanisms, we highlight the SJW/HPF remarkable potentiality in cancer prevention and treatment.

10.
Diabetes ; 69(3): 279-290, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32079704

RESUMO

Paraphrasing the Swiss physician and father of toxicology Paracelsus (1493-1541) on chemical agents used as therapeutics, "the dose makes the poison," it is now realized that this aptly applies to the calorigenic nutrients. The case here is the pancreatic islet ß-cell presented with excessive levels of nutrients such as glucose, lipids, and amino acids. The short-term effects these nutrients exert on the ß-cell are enhanced insulin biosynthesis and secretion and changes in glucose sensitivity. However, chronic fuel surfeit triggers additional compensatory and adaptive mechanisms by ß-cells to cope with the increased insulin demand or to protect itself. When these mechanisms fail, toxicity due to the nutrient surplus ensues, leading to ß-cell dysfunction, dedifferentiation, and apoptosis. The terms glucotoxicity, lipotoxicity, and glucolipotoxicity have been widely used, but there is some confusion as to what they mean precisely and which is most appropriate for a given situation. Here we address the gluco-, lipo-, and glucolipo-toxicities in ß-cells by assessing the evidence both for and against each of them. We also discuss potential mechanisms and defend the view that many of the identified "toxic" effects of nutrient excess, which may also include amino acids, are in fact beneficial adaptive processes. In addition, candidate fuel-excess detoxification pathways are evaluated. Finally, we propose that a more general term should be used for the in vivo situation of overweight-associated type 2 diabetes reflecting both the adaptive and toxic processes to mixed calorigenic nutrients excess: "nutrient-induced metabolic stress" or, in brief, "nutri-stress."


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Glucose , Humanos , Insulina , Nutrientes , Estresse Fisiológico
11.
J Pharm Pharmacol ; 71(1): 93-103, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28990659

RESUMO

OBJECTIVES: St John's wort extract (SJW) and its component hyperforin (HPF) were shown to potently inhibit cytokine-induced STAT-1 and NF-κB activation in pancreatic ß cells and protect them against injury. This study aimed at exploring the time course of STAT-1 inhibition afforded by these natural compounds in the ß-cell line INS-1E. METHODS: INS-1E cells were pre-incubated with SJW extract (2-5 µg/ml) or HPF (0.5-2 µm) and then exposed to a cytokine mixture. In some experiments, these compounds were added after or removed before cytokine exposure. STAT-1 activation was assessed by electrophoretic mobility shift assay, apoptosis by caspase-3 activity assay, mRNA gene expression by RT-qPCR. KEY FINDINGS: Pre-incubation with SJW/HPF for 1-2 h exerted a remarkable STAT-1 downregulation, which was maintained upon removal of the compounds before early or delayed cytokine addition. When the protective compounds were added after cell exposure to cytokines, between 15 and 90 min, STAT-1 inhibition also occurred at a progressively decreasing extent. Upon 24-h incubation, SJW and HPF counteracted cytokine-induced ß-cell dysfunction, apoptosis and target gene expression. CONCLUSIONS: SJW and HPF confer to ß cells a state of 'cytokine resistance', which can be elicited both before and after cytokine exposure and safeguards these cells from deleterious cytokine effects.


Assuntos
Hypericum/química , Células Secretoras de Insulina/efeitos dos fármacos , Floroglucinol/análogos & derivados , Extratos Vegetais/farmacologia , Terpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Floroglucinol/administração & dosagem , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Extratos Vegetais/administração & dosagem , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT1/metabolismo , Terpenos/administração & dosagem , Terpenos/isolamento & purificação , Fatores de Tempo
12.
Int J Biochem Cell Biol ; 40(8): 1509-21, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18226577

RESUMO

In both type 1 and type 2 diabetes, increased production of cytokines on autoimmune or metabolic basis is supposed to trigger an inflammatory process leading to dysfunction and death of pancreatic beta-cells. Therefore, anti-inflammatory pharmacological approaches aimed at blocking cytokine signalling pathways and consequent cytotoxicity in beta-cells are highly advisable. Based on previous evidence of cytokine antagonistic effects in other cell types, we explored the protective action of Hypericum perforatum (St-John's-wort) extract and its component hyperforin against cytokine-induced functional impairment and apoptosis in the INS-1E beta-cell line, searching for the underlying mechanisms. The results showed that either St-John's-wort extract or hyperforin (at 1-3 microM) prevented cytokine-induced impairment in glucose-stimulated insulin secretion and protected cells against apoptosis in a dose-dependent fashion. Inducible-NO-synthase expression was also potently hindered by the vegetal compounds. Interestingly, cytokine-induced activations of the signal-transducer-and-activator-of-transcription-1 (STAT-1) and the nuclear-factor-kappaB (NF-kappaB) were both down-regulated by SJW extract or HPF (range 0.5-5 microM) when evaluated by electrophoretic-mobility-shift-assay. Other transcription factors (CBF-1, SP-1) were unaffected. Components of SJW extract other than HPF were much less effective in down-regulating cytokine signalling. Significantly, inhibition of cytokine-elicited STAT-1 and NF-kappaB activation was confirmed in isolated rat and human islets incubated in the presence of these vegetal compounds. In conclusion, St-John's-wort extract and hyperforin are non-peptidyl compounds which, at low concentrations, target key mechanisms of cytokine-induced beta-cell injury, thereby improving beta-cell function and survival. Thus, they are potentially valuable for the prevention or limitation of beta-cell loss in diabetes.


Assuntos
Hypericum/química , Células Secretoras de Insulina/efeitos dos fármacos , Floroglucinol/análogos & derivados , Extratos Vegetais/farmacologia , Terpenos/farmacologia , Animais , Antracenos , Antineoplásicos , Apoptose/efeitos dos fármacos , Compostos Bicíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Humanos , Insulina/metabolismo , Secreção de Insulina , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Masculino , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Perileno/análogos & derivados , Perileno/farmacologia , Floroglucinol/farmacologia , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia
13.
Int J Biochem Cell Biol ; 38(5-6): 873-93, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16253543

RESUMO

Type 2 diabetes is increasingly viewed as a disease of insulin deficiency due not only to intrinsic pancreatic beta-cell dysfunction but also to reduction of beta-cell mass. It is likely that, in diabetes-prone subjects, the regulated beta-cell turnover that adapts cell mass to body's insulin requirements is impaired, presumably on a genetic basis. We still have a limited knowledge of how and when this derangement occurs and what might be the most effective therapeutic strategy to preserve beta-cell mass. The animal models of type 2 diabetes with reduced beta-cell mass described in this review can be extremely helpful (a) to have insight into the mechanisms underlying the defective growth or accelerated loss of beta-cells leading to the beta-cell mass reduction; (b) to investigate in prospective studies the mechanisms of compensatory adaptation and subsequent failure of a reduced beta-cell mass. Furthermore, these models are of invaluable importance to test the effectiveness of potential therapeutic agents that either stimulate beta-cell growth or inhibit beta-cell death.


Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Células Secretoras de Insulina/patologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ciclina D2 , Quinase 4 Dependente de Ciclina/deficiência , Ciclinas/deficiência , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/patologia , Exenatida , Retardo do Crescimento Fetal/fisiopatologia , Proteínas de Homeodomínio , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Peptídeos/uso terapêutico , Fosfoproteínas/deficiência , Proteínas Proto-Oncogênicas c-akt/deficiência , Ratos , Receptor de Insulina , Proteínas Quinases S6 Ribossômicas 70-kDa/deficiência , Transativadores/deficiência , Peçonhas/uso terapêutico , eIF-2 Quinase/deficiência
14.
Int J Biochem Cell Biol ; 81(Pt A): 92-104, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27780755

RESUMO

The extract of the herbaceous plant St. John's wort (SJW) and its phloroglucinol component hyperforin (HPF) were previously shown to inhibit cytokine-induced STAT-1 and NF-κB activation and prevent damage in pancreatic ß cells. To further clarify the mechanisms underlying their protective effects, we evaluated the phosphorylation state of various factors of cytokine signaling pathways and the expression of target genes involved in ß-cell function, inflammatory response and apoptosis induction. In the INS-1E ß-cell line, exposed to a cytokine mixture with/without SJW extract (2-5µg/ml) or HPF (1-5µM), protein phosphorylation was assessed by western blotting and expression of target genes by real-time quantitative PCR. SJW and HPF markedly inhibited, in a dose-dependent manner (from 60 to 100%), cytokine-induced activating phosphorylations of STAT-1, NF-κB p65 subunit and IKK (NF-κB inhibitory subunit IκBα kinase). MAPK and Akt pathways were also modulated by the vegetal compounds through hindrance of p38 MAPK, ERK1/2, JNK and Akt phosphorylations, each reduced by at least 65% up to 100% at the higher dose. Consistently, SJW and HPF a) abolished cytokine-induced mRNA expression of pro-inflammatory genes; b) avoided down-regulation of relevant ß-cell functional/differentiation genes; c) corrected cytokine-driven imbalance between pro- and anti-apoptotic factors, by fully preventing up-regulation of pro-apoptotic genes and preserving expression or function of anti-apoptotic Bcl-2 family members; d) protected INS-1E cells against cytokine-induced apoptosis. In conclusion, SJW extract and HPF exert their protective effects through simultaneous inhibition of multiple phosphorylation steps along various cytokine signaling pathways and consequent restriction of inflammatory and apoptotic gene expression. Thus, they have a promising therapeutic potential for the prevention or limitation of immune-mediated ß-cell dysfunction and damage leading to type 1 diabetes.


Assuntos
Apoptose/genética , Citocinas/metabolismo , Hypericum/química , Células Secretoras de Insulina/efeitos dos fármacos , Floroglucinol/análogos & derivados , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Terpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Floroglucinol/farmacologia , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Fator de Transcrição STAT1/metabolismo , Ativação Transcricional/efeitos dos fármacos
15.
J Biomed Nanotechnol ; 11(4): 730-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26310079

RESUMO

Limited tools are available for the non-invasive monitoring of transplanted islets. In this study, we have compared the widely used superparamagnetic iron oxide nanoparticle ferumoxide (Endorem) and multiwalled carbon nanotubes (MWCNTs) for islet cell labeling and tracking. INS-1 E cells and human pancreatic islets isolated from 12 non-diabetic cadaveric organ donors (age: 62 ±16 yr, BMI: 24.6 ± 3.3 kg/m2) were incubated with 50 µg/ml Endorem or 15 µg/ml MWCNTs and studied after 7 or 14 days to assess beta cell morphology, ultrastructure, function, cell survival and in-vitro and in-vivo magnetic resonance imaging (MRI). Light and electron (EM) microscopy showed the well-maintained morphology and ultrastructure of both INS-1 E and human islets during the incubation. EM also revealed the presence of Endorem and MWCNTs within the beta but not the alpha cells. The compounds did not affect beta cell function and viability, and in-vitro MRI showed that labeled INS-1 E cells and human islets could be imaged. Finally, MWCNT labeled human islets were successfully transplanted into the subcutis of rats localized in the desired site via magnetic field and tracked by MRI. These data suggest that MWCNTs can be an alternative labeling compound to be used with human islets for experimental and transplantation studies.


Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/efeitos dos fármacos , Nanotecnologia/métodos , Nanotubos de Carbono/química , Idoso , Animais , Sobrevivência Celular , Células Cultivadas , Meios de Contraste/química , Dextranos/química , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Microscopia Eletrônica , Microscopia de Fluorescência , Pessoa de Meia-Idade , Ratos
16.
Exp Gerontol ; 39(9): 1333-40, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15489056

RESUMO

Regular physical exercise has emerged, together with dietary restriction, as an effective intervention in delaying degenerative diseases and augmenting life span in rodents. The mechanisms involved remain largely unknown, although a beneficial influence on the age-related alteration of insulin sensitivity has been hypothesized. As muscle triglyceride (TG) accumulation is considered a reliable index of muscle insulin resistance, in this study we explored muscle TG content in 23-month-old male Sprague-Dawley rats subjected to life-long training. Plasma glucose, insulin, free fatty acid (FFA) and leptin levels were also measured. Both voluntary running in wheels (RW) and forced training in treadmill (TM) were studied. As RW rats weighed less than controls, a cohort of untrained animals, fed to pair weight (PW) with RW, was added to discriminate the effect of exercise from that of food restriction. Sedentary ad libitum fed rats served as controls. In 23-month-old RW rats, muscle TG content was reduced by 50% with respect to age-matched sedentary controls, while in TM group this reduction was smaller but still highly significant, and occurred independently on the changes in body fat mass. In both the trained rat groups, there was a significant decrease in circulating FFA levels and a trend to reduced insulin levels. In PW rats, muscle TG levels decreased similarly to RW rats, while plasma parameters were less modified. In particular, RW training was more effective than PW in preventing the age-related increase in circulating leptin levels. Our results suggest that voluntary exercise effectively counteracts the development of insulin resistance in the muscles of ageing rats as well as other related changes such as hyperlipacidaemia and compensatory hyperleptinaemia. Forced training or moderate food restriction appear slightly less effective than voluntary exercise in preventing age-dependent alterations in nutrient distribution and/or utilization.


Assuntos
Envelhecimento/metabolismo , Ácidos Graxos não Esterificados/sangue , Atividade Motora/fisiologia , Músculo Esquelético/metabolismo , Triglicerídeos/metabolismo , Tecido Adiposo/anatomia & histologia , Animais , Glicemia/metabolismo , Peso Corporal/fisiologia , Insulina/sangue , Leptina/sangue , Masculino , Ratos , Ratos Sprague-Dawley
17.
Acta Diabetol ; 51(1): 113-21, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24121871

RESUMO

The extract of Hypericum perforatum (St. John's wort, SJW) and its component hyperforin (HPF) were previously shown to inhibit cytokine-induced activation of signal transducer and activator of transcription-1 and nuclear factor κB and prevent apoptosis in a cultured ß-cell line. Objective of this study was to assess the protection exerted by SJW and HPF on isolated rat and human islets exposed to cytokines in vitro. Functional, ultrastructural, biomolecular and cell death evaluation studies were performed. In both rat and human islets, SJW and HPF counteracted cytokine-induced functional impairment and down-regulated mRNA expression of pro-inflammatory target genes, such as iNOS, CXCL9, CXCL10, COX2. Cytokine-induced NO production from cultured islets, evaluated by nitrites measurement in the medium, was significantly reduced in the presence of the vegetal compounds. Noteworthy, the increase in apoptosis and necrosis following 48-h exposure to cytokines was fully prevented by SJW and partially by HPF. Ultrastructural morphometric analysis in human islets exposed to cytokines for 20 h showed that SJW or HPF avoided early ß-cell damage (e.g., mitochondrial alterations and loss of insulin granules). In conclusion, SJW compounds protect rat and human islets against cytokine effects by counteracting key mechanisms of cytokine-mediated ß-cell injury and represent promising pharmacological tools for prevention or limitation of ß-cell dysfunction and loss in type 1 diabetes.


Assuntos
Citocinas/toxicidade , Citoproteção/efeitos dos fármacos , Hypericum , Ilhotas Pancreáticas/efeitos dos fármacos , Floroglucinol/análogos & derivados , Extratos Vegetais/farmacologia , Terpenos/farmacologia , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Glucose/farmacologia , Humanos , Hypericum/química , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Floroglucinol/farmacologia , Ratos , Ratos Sprague-Dawley
18.
PLoS One ; 9(8): e104156, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25093405

RESUMO

Abnormalities in eNOS gene, possibly interacting with high fat diet (HFD), affect peripheral vascular function and glucose metabolism. The relative role of eNOS gene, HFD and metabolic derangement on coronary function has not been fully elucidated. We test whether eNOS gene deficiency per se or in association with HFD modulates coronary function through mechanisms involving molecular pathways related to insulin signaling. Wild type (WT), eNOS-/- and eNOS+/- mice were studied. WT and eNOS+/- mice were fed with either standard or HF diet for 16 weeks and compared with standard diet fed eNOS-/-. Glucose and insulin tolerance tests were performed during the last week of diet. Coronary resistance (CR) was measured at baseline and during infusions of acetylcholine (Ach) or sodium-nitroprusside (SNP) to evaluate endothelium-dependent or independent vasodilation, in the Langendorff isolated hearts. Cardiac expression of Akt and ERK genes as evaluation of two major insulin-regulated signaling pathways involved in the control of vascular tone were assessed by western blot. HFD-fed mice developed an overt diabetic state. Conversely, chow-fed genetically modified mice (in particular eNOS-/-) showed a metabolic pattern characterized by normoglycemia and hyperinsulinemia with a limited degree of insulin resistance. CR was significantly higher in animals with eNOS gene deletions than in WT, independently of diet. Percent decrease in CR, during Ach infusion, was significantly lower in both eNOS-/- and eNOS+/- mice than in WT, independently of diet. SNP reduced CR in all groups except eNOS-/-. The cardiac ERK1-2/Akt ratio, increased in animals with eNOS gene deletions compared with WT, independently of diet. These results suggest that the eNOS genetic deficiency, associated or not with HFD, has a relevant effect on coronary vascular function, possibly mediated by increase in blood insulin levels and unbalance in insulin-dependent signaling in coronary vessels, consistent with a shift towards a vasoconstrictive pattern.


Assuntos
Vasos Coronários/fisiopatologia , Dieta Hiperlipídica , Deleção de Genes , Hiperinsulinismo/fisiopatologia , Insulina/metabolismo , Miocárdio/enzimologia , Óxido Nítrico Sintase Tipo III/genética , Transdução de Sinais , Animais , Glicemia/metabolismo , Peso Corporal , Vasos Coronários/metabolismo , Vasos Coronários/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Teste de Tolerância a Glucose , Hiperinsulinismo/sangue , Hiperinsulinismo/complicações , Técnicas In Vitro , Insulina/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Miocárdio/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resistência Vascular
19.
Eur J Pharmacol ; 729: 37-44, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24530416

RESUMO

We previously showed that the innovative radical scavenger bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) improves metabolic dysfunctions in a diabetic mouse model. Here, we compared the in vivo effects of IAC with those of the anti-diabetic drugs pioglitazone (PIO) and exendin-4 (EX-4). Diabetes was induced in C57Bl/6J mice by streptozotocin and nicotinamide administration. Paralleled by healthy controls, diabetic animals (D) were randomly assigned to four groups and treated daily for 7 consecutive weeks: D+saline, ip; D+IAC 30mg/kgb.w., ip; D+PIO 10mg/kgb.w. per os; and D+EX-4, 50µg/kgb.w., ip. Our results show that IAC reduced basal hyperglycemia and improved glucose tolerance better than PIO or EX-4. Interestingly, in the heart of diabetic mice, IAC treatment normalized the increased levels of GSSG/GSH ratio and thiobarbituric acid reactive substances, indexes of oxidative stress and damage, while PIO and EX-4 were less effective. As supported by immunohistochemical data, IAC markedly prevented diabetic islet ß-cell reduced density, differently from PIO and EX-4 that had only a moderate effect. Interestingly, in diabetic animals, IAC treatment enhanced the activity of pancreatic-duodenal homeobox 1 (PDX-1), an oxidative stress-sensitive transcription factor essential for maintenance of ß-cell function, as evaluated by quantification of its nuclear immunostaining, whereas PIO or EX-4 treatments did not. Altogether, these observations support the improvement of the general redox balance and ß-cell function induced by IAC treatment in streptozotocin-nicotinamide diabetic mice. Furthermore, in this model, the correction of diabetic alterations was better obtained by treatment with the radical scavenger IAC than with pioglitazone or exendin-4.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Sequestradores de Radicais Livres/uso terapêutico , Hipoglicemiantes/uso terapêutico , Peptídeos/uso terapêutico , Tiazolidinedionas/uso terapêutico , Peçonhas/uso terapêutico , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Exenatida , Sequestradores de Radicais Livres/farmacologia , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Niacinamida/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Peptídeos/farmacologia , Pioglitazona , Distribuição Aleatória , Estreptozocina/toxicidade , Tiazolidinedionas/farmacologia , Peçonhas/farmacologia
20.
J Med Chem ; 56(11): 4718-28, 2013 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-23662847

RESUMO

ATP-sensitive potassium (KATP) channels play a prominent role in controlling cardiovascular function. In this paper, a novel series of 4-(1-oxo-2-cyclopentenyl)-1,4-benzothiazine derivatives modified at the C-2, and C-6 positions were synthesized as openers of vascular KATP channels. Most of the tested compounds evoked vasorelaxing effects on rat aortic rings and membrane hyperpolarization in human vascular smooth muscle cells, with potency similar or superior to that of the reference levcromakalim (LCRK). The selective KATP blocker glibenclamide antagonized the above vascular effects, confirming that KATP channels are closely involved in the mechanism of action. The experimental results confirmed the 1,4-benzothiazine nucleus as an optimal scaffold for activators of vascular KATP channels; moreover, the high level of potency exhibited by the 6-acetyl substituted benzothiazine 8, along with the lack of any significant interference with insulin secretion from pancreatic ß-cells, paves the way to further develop a new series of potent activators of vascular KATP channels.


Assuntos
Ciclopentanos/síntese química , Canais KATP/metabolismo , Tiazinas/síntese química , Vasodilatadores/síntese química , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Ciclopentanos/química , Ciclopentanos/farmacologia , Humanos , Ligação de Hidrogênio , Técnicas In Vitro , Insulina/metabolismo , Secreção de Insulina , Ativação do Canal Iônico , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Tiazinas/química , Tiazinas/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/química , Vasodilatadores/farmacologia
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