Estudo do efeito e do mecanismo de ação de isoflavonas naturais com potencial efeito antidiabético

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2014.A diabetes é uma doença crônica que afeta milhões de pessoas no mundo. As previsões para 2035 são de que a doença atinja mais de 592 milhões de pessoas. Estes dados revelam um importante problema de saúde pública e reforçam a necessidade de buscar por alternativas de tratamento preventivo e terapêutico. As plantas medicinais são uma importante fonte de compostos bioativos. Dentre estas, as isoflavonas despontam como uma classe de biomoléculas com importante atividade antidiabética, associadas principalmente a efeitos fitoestrógenos. Dessa forma, este estudo objetivou caracterizar a atividade anti-hiperglicêmica das isoflavonas PMA2.31 e PMA19, extraídas da fração acetato de etila de Polygala molluginifolia, bem como, elucidar os mecanismos pelos quais estas ações ocorrem. Para tanto, ambas as isoflavonas foram submetidas ao teste de tolerância oral à glicose, no qual, ratos Wistar machos receberam o tratamento (PMA2.31 e PMA19) isoladamente ou combinado com sitagliptina por gavagem e, 30 min após, uma sobrecarga de glicose foi administrada e a glicemia determinada aos 15, 30, 60 e 180 min subsequentes a esta administração. A avaliação da atividade das dissacaridases intestinais in vivo e o conteúdo de glicogênio hepático e muscular foi realizado no tempo 180 min. A atividade de dissacaridases também foi avaliada mediante incubação in vitro. Amostras de soro foram utilizadas para a dosagem de insulina e GLP-1 sérico. A atividade de DPP-IV (Dipeptidilpeptidase-IV) foi mensurada in vitro. A fim de identificar os mecanismos pelos quais as isoflavonas agem secretando insulina e/ou GLP-1, o efeito de PMA2.31 e/ou PMA19 foi avaliado no influxo de cálcio em fatias do intestino (cólon) e em ilhotas pancreáticas. Ainda, PMA2.31 e PMA19 foram incubadas na presença de albumina e glicose ou frutose, para determinação da glicação. Os resultados obtidos demonstraram a redução glicêmica induzida por PMA2.31 e PMA19 após 15 e 30 min, respectivamente. Ainda, PMA2.31 apresentou o efeito anti-hiperglicêmico potenciado quando combinado com sitagliptina. Ambas as isoflavonas aumentaram os níveis séricos de insulina além de PMA2.31 aumentar GLP-1 (Glucagon Like Peptide-1) sérico. O mecanismo pelo qual PMA2.31 atua nestes efeitos envolve a inibição parcial de DPP-IV e aumento da secreção de GLP-1 via ativação de PKC, uma vez que, houve um aumento significativo do influxo de cálcio em fatias de intestino e inibição deste estímulo quando utilizado inibidor de PKC (Proteína cinase C). Diferente de PMA2.31, PMA19 age estimulando a secreção direta de insulina, devido ao incremento observado no influxo de cálcio em ilhotas pancreáticas, sendo estas ações semelhantes às do estradiol e envolvem efeitos no canal de potássio dependente de ATP, canal de cálcio dependente de voltagem e ativação de PKA (Proteína cinase A). Além das ações supramencionadas, PMA2.31 aumentou o conteúdo de glicogênio hepático e assim como a fração acetato de etila, inibiu a atividade de maltase. A fração acetato de etila e as isoflavonas também diminuíram a glicação com albumina demonstrando um potencial efeito antidiabético. Ambas as isoflavonas não produziram alterações na concentração sérica de LDH, sugerindo serem atóxicas na concentração e tempo de tratamento avaliado.Abstract : Diabetes is a chronic disease that affects approximately 382 million people worldwide, with predictions stating that this number will increase to 592 million in 2035. These data reveal an important public health problem and support the necessity to search for new targets for preventive and therapeutical treatment. Medicinal plants are an important source of bioactive compounds. Among those, the study of plants that contain isoflavones indicates these compounds as a class of biomolecules with important antidiabetic activity, mainly associated with phytoestrogens action. Therefore, this study aimed to characterize the anti-hyperglycemic activity of isoflavones PMA2.31 and PMA19, isolated from ethyl acetate fraction of P. molluginifolia, as well as to elucidate the mechanisms by which these actions occur. To this end, both isoflavones were submitted to oral glucose tolerance test, in which, male Wistar rats received the treatment (PMA2.31, PMA19) by gavage, with/without sitagliptin and, after 30 min, a glucose load was administered and blood glucose levels were determined at 15, 30, 60 and 180 min.The evaluation of in vivo intestinal disaccharidases activity, muscle and hepatic glycogen content was measured at 180 min. The disaccharidase activity was also performed by in vitro treatment. Serum samples were collected for insulin and GLP-1 dosage. In vitro DPP-IV activity was also measured. To investigate the mechanisms by which isoflavones act in insulin and/or GLP-1 secretion, PMA2.31 and/or PMA19 effect on calcium influx in pancreatic islets and intestine slices (colon) was evaluated. PMA2.31 and PMA19 were also tested for albumin glycation measurement. The results showed a significantly blood glucose reduction after treatment with PMA2.31 and PMA19 in 15 and 30 min, respectively. The effect of PMA2.31 was higher when co-administered with sitagliptin. Both isoflavones increased insulin serum levels and PMA2.31 was able to increase GLP-1 serum levels. The PMA2.31 effect on GLP-1 may involve an intermediate DPP-IV inhibition and augmented GLP-1 secretion via PKC activation, since there was a significant increase in calcium influx in intestine slices, and this effect was inhibited by co-incubation with a PKC inhibitor. On the other hand, PMA19 acts directly on insulin secretion due to its stimulus in pancreatic islets calcium influx, with a similar mechanism of action to estradiol, which involve effects on ATP-dependent potassium channel, voltage-gated calcium channel and PKA activation. In addition to the above actions, PMA2.31 increased hepatic glycogen content and both this isoflavone and ethyl acetate fraction, inhibited maltase activity. The ethyl acetate fraction and both isoflavones also decreased albumin glycation, demonstrating a potential antidiabetic effect. Both isoflavones did not produce change in serum LDH, indicating they are non-toxic in the concentration and treatment period studied

Neratinib protects pancreatic beta cells in diabetes

The loss of functional insulin-producing β-cells is a hallmark of diabetes. Mammalian sterile 20-like kinase 1 (MST1) is a key regulator of pancreatic β-cell death and dysfunction; its deficiency restores functional β-cells and normoglycemia. The identification of MST1 inhibitors represents a promising approach for a β-cell-protective diabetes therapy. Here, we identify neratinib, an FDA-approved drug targeting HER2/EGFR dual kinases, as a potent MST1 inhibitor, which improves β-cell survival under multiple diabetogenic conditions in human islets and INS-1E cells. In a pre-clinical study, neratinib attenuates hyperglycemia and improves β-cell function, survival and β-cell mass in type 1 (streptozotocin) and type 2 (obese Leprdb/db) diabetic mouse models. In summary, neratinib is a previously unrecognized inhibitor of MST1 and represents a potential β-cell-protective drug with proof-of-concept in vitro in human islets and in vivo in rodent models of both type 1 and type 2 diabetes

Fluconazole and amphotericin-B resistance are associated with increased catalase and superoxide dismutase activity in Candida albicans and Candida dubliniensis

Introduction Candida dubliniensis, a new species of Candida that has been recovered from several sites in healthy people, has been associated with recurrent episodes of oral candidiasis in AIDS and HIV-positive patients. This species is closely related to C. albicans. The enzymatic activity of C. dubliniensis in response to oxidative stress is of interest for the development of drugs to combat C. dubliniensis. Methods Fluconazole- and amphotericin B-resistant strains were generated as described by Fekete-Forgács et al. (2000). Superoxide dismutase (SOD) and catalase assays were performed as described by McCord and Fridovich (1969) and Aebi (1984), respectively. Results We demonstrated that superoxide dismutase (SOD) and catalase activities were significantly higher (p<0.05) in the fluconazole- and amphotericin B-resistant strains of C. dubliniensis and C. albicans than in the sensitive strains. The catalase and SOD activities were also significantly (p<0.01) higher in the sensitive and resistant C. albicans strains than in the respective C. dubliniensis strains. Conclusions These data suggest that C. albicans is better protected from oxidative stress than C. dubliniensis and that fluconazole, like amphotericin B, can induce oxidative stress in Candida; oxidative stress induces an adaptive response that results in a coordinated increase in catalase and SOD activities

E96V Mutation in the <i>Kdelr3</i> Gene Is Associated with Type 2 Diabetes Susceptibility in Obese NZO Mice

Type 2 diabetes (T2D) represents a multifactorial metabolic disease with a strong genetic predisposition. Despite elaborate efforts in identifying the genetic variants determining individual susceptibility towards T2D, the majority of genetic factors driving disease development remain poorly understood. With the aim to identify novel T2D risk genes we previously generated an N2 outcross population using the two inbred mouse strains New Zealand obese (NZO) and C3HeB/FeJ (C3H). A linkage study performed in this population led to the identification of the novel T2D-associated quantitative trait locus (QTL) Nbg15 (NZO blood glucose on chromosome 15, Logarithm of odds (LOD) 6.6). In this study we used a combined approach of positional cloning, gene expression analyses and in silico predictions of DNA polymorphism on gene/protein function to dissect the genetic variants linking Nbg15 to the development of T2D. Moreover, we have generated congenic strains that associated the distal sublocus of Nbg15 to mechanisms altering pancreatic beta cell function. In this sublocus, Cbx6, Fam135b and Kdelr3 were nominated as potential causative genes associated with the Nbg15 driven effects. Moreover, a putative mutation in the Kdelr3 gene from NZO was identified, negatively influencing adaptive responses associated with pancreatic beta cell death and induction of endoplasmic reticulum stress. Importantly, knockdown of Kdelr3 in cultured Min6 beta cells altered insulin granules maturation and pro-insulin levels, pointing towards a crucial role of this gene in islets function and T2D susceptibility