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1.
Reprod Biol Endocrinol ; 17(1): 34, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953560

RESUMO

BACKGROUND: Women with Polycystic Ovary Syndrome (PCOS) present a heterogeneous reproductive and metabolic profile with an increased lifetime risk of Type 2 Diabetes (T2D). Early biomarkers of these metabolic disturbances in PCOS women have not been identified. The abundance of circulating insulin gene promotor cell-free DNA (INS cfDNA) was shown to be valuable as a predictive biomarker of ß-cell death in individuals with Type 1 diabetes (T1D) as well as with gestational diabetes. Since ß-cell death is common to the development of T1D as well as in T2D, we aimed to investigate if insulin-coding DNA is more abundant in circulation of PCOS women (vs Controls) and if their levels change after 6 yr. follow-up as a potential measure to predict future T2D. METHODS: A cohort of 40 women diagnosed with PCOS according to Rotterdam 2003 criteria and eight healthy controls were examined at baseline and 6 years follow-up. Clinical measurements for evaluation of glucose homeostasis as well as blood/serum samples were obtained at each visit. Methylated and unmethylated INS cfDNA were quantified using droplet digital PCR. Differences between groups were assessed using Kruskall-Wallis test and Wilcoxon Signed rank test. RESULTS: At baseline, there was no detectable difference in copy number (copies/µL) of methylated (p = 0.74) or unmethylated INS cfDNA (p = 0.34) between PCOS and Control groups. At follow up, neither methylated (p = 0.50) nor unmethylated INScfDNA levels (p = 0.48) differed significantly between these groups. Likewise, when pooling the groups, there was no difference between baseline and follow up, in terms of copies of methylated or unmethylated INS cfDNA (p = 0.38 and p = 0.52, respectively). There were no significant correlations between counts of unmethylated or methylated cfDNA and the clinical measurements of ß-cell function and pre-diabetes. CONCLUSION: The circulating level of unmethylated and methylated INScfDNA is similar between PCOS and Controls and cannot be used to predict islet ß-cell loss and progression to Type 2 diabetes in a 6-year follow-up. TRIAL REGISTRATION: The Danish Data Protection Agency (REG-31-2016. Approval: 01-12-2015) and by the Danish Scientific Ethical committee of Region Zealand (Journal no. SJ-525. Approval: 13-06-2016), Clinicaltrials.gov, ( NCT03142633 , registered 1. March, 2017, Retrospectively registered).


Assuntos
Ácidos Nucleicos Livres/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Insulina/genética , Síndrome do Ovário Policístico/metabolismo , Adulto , Biomarcadores/sangue , Metilação de DNA , Feminino , Humanos , Estudos Longitudinais
2.
J Agric Food Chem ; 67(17): 4774-4781, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30963762

RESUMO

Targeted analysis of Coffea arabica and Coffea canephora green coffees (total sample size n = 57) confirmed 2- O-ß-d-glucopyranosyl-carboxyatractyligenin (6) as the quantitatively dominating carboxyatractyligenin derivative. Its abundance in Arabicas (2425 ± 549 nmol/g, n = 48) exceeded that in Robustas (34 ± 12 nmol/g, n = 9) roughly by a factor of 70. Coffee processing involving heat (e.g., steam treatment and decaffeination) reduced concentrations of 6 and increased those of the decarboxylated derivative. The bioavailability of compound 6 in Caenorhabditis elegans was demonstrated by ultraperformance liquid chromatography-tandem mass spectrometry analysis of extracts prepared from nematode cultures incubated in a liquid medium containing 6. A toxicity assay performed to assess the impact of 6 in vivo showed a 20-fold higher median lethal dose (LD50 = 11.7 ± 1.2 mM) concentration compared to that of the known phytotoxic adenine-nucleotide transporters inhibitor carboxyatractyloside (2, LD50 = 0.61 ± 0.05 mM), whereas 1 mM 6 and 0.1 mM 2 were sufficient to decrease the survival of wild type C. elegans, already 10-20-fold lower doses reduced reproduction. Because the insulin/insulin-like growth factors signaling cascade (IIS) is a key regulator of life span and stress resistance, the impact of compound 6 on the survival of long-living daf-2 C. elegans was tested. As the susceptibility of these nematodes to 6 was as high as that in wild type, an impact on central metabolic processes independent of IIS was suggested. Analysis of the in vivo adenosine triphosphate (ATP) content of adult C. elegans revealed no changes after 1 and 24 h, but a 50% reduction after treatment with 1 mM 6 during the entire postembryonic development. These data speak for a developmental-stage-dependent modulation of the ATP pool by 6.


Assuntos
Atractilosídeo/análogos & derivados , Caenorhabditis elegans/efeitos dos fármacos , Coffea/química , Preparações de Plantas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Atractilosídeo/farmacocinética , Atractilosídeo/farmacologia , Disponibilidade Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Coffea/toxicidade , Café/química , Feminino , Insulina/genética , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Dose Letal Mediana , Masculino
3.
Environ Pollut ; 249: 822-830, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30953944

RESUMO

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that have been shown to be related to the occurrence of type 2 diabetes mellitus (T2DM). Nevertheless, it is necessary to further explore the development of T2DM caused by PCBs and its underlying mechanisms. In the present study, 21-day-old C57BL/6 male mice were orally treated with Aroclor 1254 (0.5, 5, 50 or 500 µg kg-1) once every three days. After exposure for 66 d, the mice showed impaired glucose tolerance, 13% and 14% increased fasting serum insulin levels (FSIL), and 63% and 69% increases of the pancreatic ß-cell mass in the 50 and 500 µg kg-1 groups, respectively. After stopping exposure for 90 d, treated mice returned to normoglycemia and normal FSIL. After re-exposure of these recovered mice to Aroclor 1254 for 30 d, fasting plasma glucose showed 15%, 28% and 16% increase in the 5, 50 and 500 µg kg-1 treatments, FSIL exhibited 35%, 27%, 30% and 32% decrease in the 0.5, 5, 50 or 500 µg kg-1 groups respectively, and there was no change in pancreatic ß-cell mass. Transcription of the pancreatic insulin gene (Ins2) was significantly down-regulated in the 50 and 500 µg kg-1 groups, while DNA-methylation levels were simultaneously increased in the Ins2 promoter during the course of exposure, recovery and re-exposure. Reduced insulin levels were initially rescued by a compensative increase in ß-cell mass. However, ß-cell mass eventually failed to make sufficient levels of insulin, resulting in significant increases in fasting blood glucose, and indicating the development of T2DM.


Assuntos
Glicemia/efeitos dos fármacos , /toxicidade , Poluentes Ambientais/toxicidade , Homeostase/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Animais , Glicemia/metabolismo , /administração & dosagem , Metilação de DNA/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Poluentes Ambientais/administração & dosagem , Insulina/sangue , Insulina/genética , Células Secretoras de Insulina/patologia , Masculino , Camundongos Endogâmicos C57BL
4.
Cell Physiol Biochem ; 52(4): 879-892, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958662

RESUMO

BACKGROUND/AIMS: CXCL14, a secreted chemokine peptide that promotes obesity-induced insulin resistance, is expressed by islets, but its effects on islet function are unknown. The aim of this study was to determine the role of CXCL14 in ß-cells and investigate how it transduces these effects. METHODS: Cxcl14 and Cxc-receptor mRNA expression was quantified by qPCR and CXCL14 expression in the pancreas was determined by immunohistochemistry. The putative function of CXCL14 at CXCR4 and CXCR7 receptors was determined by ß-arrestin recruitment assays. The effects of CXCL14 on glucose-stimulated insulin secretion, cAMP production, glucose-6-phosphate accumulation, ATP generation, apoptosis and proliferation were determined using standard techniques. RESULTS: CXCL14 was present in mouse islets, where it was mainly localised to islet δ-cells. Cxc-receptor mRNA profiling indicated that Cxcr4 and Cxcr7 are the most abundant family members in islets, but CXCL14 did not promote ß-arrestin recruitment at CXCR4 or CXCR7 or antagonise CXCL12 activation of these receptors. CXCL14 induced a concentration-dependent inhibition of glucose-stimulated insulin secretion, which was not coupled to Gαi signalling. However, CXCL14 inhibited glucose-6-phosphate generation and ATP production in mouse islets. CONCLUSION: CXCL14 is expressed by islet δ-cells where it may have paracrine effects to inhibit insulin secretion in a CXCR4/CXCR7-independent manner through reductions in ß-cell ATP levels. These observations, together with the previously reported association of CXCL14 with obesity and impaired glucose homeostasis, suggest that inhibition of CXCL14 signalling could be explored to treat type 2 diabetes.


Assuntos
Quimiocinas CXC/metabolismo , AMP Cíclico/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Animais , Quimiocinas CXC/genética , AMP Cíclico/genética , Insulina/genética , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Comunicação Parácrina , Receptores CXCR/genética , Receptores CXCR4/genética , Sistemas do Segundo Mensageiro
5.
Medicina (Kaunas) ; 55(4)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987250

RESUMO

Background and objectives: Mounting evidence shows that curcumin, a bioactive substance originating from turmeric root, has anticancer properties. Additionally, curcumin prevents the migration and metastasis of tumor cells. However, the molecular mechanism involved in the anti-metastatic action of curcumin is not clear. Most studies have suggested that migration inhibition is related to curcumin's anti-inflammatory properties. Curcumin possesses a regulatory effect on insulin and insulin-like growth factor-1 (IGF-1) receptors and signaling. Insulin signaling is one of the important pathways involved in tumor initiation and progression; therefore, we proposed that the anti-metastatic effect of curcumin may mediate the downregulation of insulin and insulin-like growth factor-1 receptors. Materials and Methods: Viable resistant cells resulting from treating SW480 cells with 5-fluorouracil (5-FU) were subjected to curcumin treatment to analyze the proliferation and migration capacity in comparison to the untreated counterparts. To test the proliferation and migration potential, MTT, colony formation, and wound healing assays were performed. Real-time polymerase chain reaction (RT-PCR) was performed to measure the mRNA expression of insulin-like growth factor-1R (IGF-1R), insulin receptor (IR), and avian myelocytomatosis virus oncogene cellular homolog (MYC). Results: Our findings showed that curcumin significantly decreased insulin and IGF-1 receptors in addition to MYC expression. Additionally, the downregulation of the insulin and insulin-like growth factor-1 receptors was correlated to a greater decrease in the proliferation and migration of chemoresistant colorectal cancer cells. Conclusions: These results suggest the possible therapeutic effectiveness of curcumin in adjuvant therapy in metastatic colorectal cancer.


Assuntos
Antineoplásicos/uso terapêutico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Curcumina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Genes myc/genética , Insulina/genética , Extratos Vegetais/uso terapêutico , Receptores de Somatomedina/genética , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Curcuma/química , Regulação para Baixo , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos
6.
Chemistry ; 25(36): 8513-8521, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31012517

RESUMO

Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, CysA6 and CysA11 (which form the internal intrachain A6-A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid-phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild-type B chain-di-S-sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long-standing challenge in chemical insulin synthesis. The affinity of the Se-insulin analogue for the lectin-purified insulin receptor was indistinguishable from that of WT-insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔGu ≈0.8 kcal mol-1 ). In accordance with such enhanced stability, reductive unfolding of the Se-insulin analogue and resistance to enzymatic cleavage by Glu-C protease occurred four times more slowly than that of WT-insulin. 2D-NMR and X-ray crystallographic studies demonstrated a native-like three-dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash.


Assuntos
Dissulfetos/química , Insulina/química , Selênio/química , Cristalografia por Raios X , Cisteína/química , Humanos , Insulina/genética , Insulina/metabolismo , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Selenocisteína/química , Termodinâmica
7.
Nutrients ; 11(3)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871106

RESUMO

Low birth weight is a risk factor for gestational and type 2 diabetes (T2D). Since mammalian target of rapamycin (mTOR) controls pancreatic ß-cell mass and hormone release, we hypothesized that nutritional insult in utero might permanently alter mTOR signaling. Mice were fed a low-protein (LP, 8%) or control (C, 20%) diet throughout pregnancy, and offspring examined until 130 days age. Mice receiving LP were born 12% smaller and ß-cell mass was significantly reduced throughout life. Islet mTOR levels were lower in LP-exposed mice and localized predominantly to α-rather than ß-cells. Incubation of isolated mouse islets with rapamycin significantly reduced cell proliferation while increasing apoptosis. mRNA levels for mTORC complex genes mTOR, Rictor and Raptor were elevated at 7 days in LP mice, as were the mTOR and Raptor proteins. Proglucagon gene expression was similarly increased, but not insulin or the immune/metabolic defense protein STING. In human and mouse pancreas STING was strongly associated with islet ß-cells. Results support long-term changes in islet mTOR signaling in response to nutritional insult in utero, with altered expression of glucagon and insulin and a reduced ß-cell mass. This may contribute to an increased risk of gestational or type 2 diabetes.


Assuntos
Dieta com Restrição de Proteínas , Proteínas na Dieta/administração & dosagem , Glucagon/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição Pré-Natal , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glucagon/genética , Insulina/genética , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Distribuição Aleatória , Serina-Treonina Quinases TOR/genética
8.
Gene ; 701: 125-130, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30910560

RESUMO

BACKGROUND AND AIM: Oxidative stress and impaired insulin secretion is an underlying major risk factor for the development of type 2 diabetes (T2D). Uncoupling protein-2 (UCP2) is involved in the regulation of reactive oxygen species production, insulin secretion, and lipid metabolism. Based on this we aimed to find an association of UCP2 (G-866A) polymorphism with the risk of T2D in South Indian population. METHODS: A total of 318 T2D patients and 312 controls were enrolled in this study. All the study subjects were genotyped for UCP2 (G-866A) polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Fasting blood glucose, HbA1c, serum lipid profile, systolic and diastolic blood pressure were measured by standard biochemical methods. Fasting serum insulin level was measured by ELISA. RESULTS: In UCP2 (G-866A) polymorphism, the distribution of GA (46%) and AA (14%) genotypes were significantly higher in T2D patients than the healthy controls. The frequency of GA and AA genotypes have high risk towards the development of T2D with an Odds Ratio (OR) of 1.55 (P = 0.01) and 2.04 (P = 0.01) respectively. Moreover, SNP-866 G>A allele was found to be significantly associated with T2D (OR = 1.48, P = 0.001, 95% CI = 1.16-1.88). Further, the UCP2 AA genotype showed significantly decreased level of insulin by the reduction in pancreatic ß-cell function in T2D patients. CONCLUSION: UCP2 (G-866A) polymorphism may play a crucial role in the pathogenesis of insulin secretion thus leads to the development of T2D.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Insulina/sangue , Polimorfismo Genético , Regiões Promotoras Genéticas , Proteína Desacopladora 2/genética , Adulto , Idoso , Feminino , Humanos , Insulina/genética , Masculino , Pessoa de Meia-Idade , Proteína Desacopladora 2/metabolismo
9.
Int J Mol Sci ; 20(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925781

RESUMO

We showed previously that the Schistosoma japonicum insulin-like peptide (SjILP) binds the worm insulin receptors, thereby, activating the parasite's insulin pathway and emphasizing its important role in regulating uptake of glucose, a nutrient essential for parasite survival. Here we show that SjILP is differentially expressed in the schistosome life cycle and is especially highly transcribed in eggs, miracidia, and adult female worms. RNA inference was employed to knockdown SjILP in adults in vitro, with suppression confirmed by significantly reduced protein production, declined adenosine diphosphate levels, and reduction in glucose consumption. Immunolocalization showed that SjILP is located to lateral gland cells of mature intra-ovular miracidia in the schistosome egg, and is distributed on the ciliated epithelium and internal cell masses of newly transformed miracidia. In schistosomula, SjILP is present on the tegument in two antero-lateral points, indicating highly polarized expression during cercarial transformation. Analysis of serum from S. japonicum-infected mice by ELISA using a recombinant form of SjILP as an antigen revealed IgG immunoreactivity to this molecule at 7 weeks post-infection indicating it is likely secreted from mature eggs into the host circulation. These findings provide further insights on ILP function in schistosomes and its essential roles in parasite survival and growth in different development stages.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Insulina/genética , Schistosoma japonicum/crescimento & desenvolvimento , Schistosoma japonicum/genética , Esquistossomose Japônica/parasitologia , Animais , Feminino , Proteínas de Helminto/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Insulina/imunologia , Estágios do Ciclo de Vida , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Schistosoma japonicum/imunologia , Esquistossomose Japônica/sangue , Esquistossomose Japônica/imunologia
10.
Biomed Res Int ; 2019: 7398063, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30805369

RESUMO

Background: Solute carrier family 2 member 4- (SLC2A4-) retinol binding protein-4- (RBP4-) phosphoenolpyruvate carboxykinase 1 (PCK1)/phosphoinositide 3-kinase (PI3K) is an adipocyte derived "signalling pathway" that may contribute to the pathogenesis of type 2 diabetes mellitus (T2DM). We explored whether single nucleotide polymorphisms (SNPs) of these "signalling pathway" genes are associated with gestational diabetes mellitus (GDM). Methods: Case-control studies were conducted to compare GDM and control groups. A total of 334 cases and 367 controls were recruited. Seventeen candidate SNPs of the pathway were selected. Chi-square tests, logistic regression, and linear regression were used to estimate the relationships of SNPs with GDM risk and oral glucose tolerance test (OGTT), fasting insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) levels. Model-based multifactor dimensionality reduction was used to estimate the adjusted interactions between genes. Regression and interaction analyses were adjusted by maternal age, prepregnancy BMI, and weekly BMI growth. The Bonferroni correction was applied for multiple comparisons. Results: RBP4 rs7091052 was significantly associated with GDM risk. SLC2A4 rs5435, RBP4 rs7091052, PCK1 rs1042531 and rs2236745, and PIK3R1 (coding gene of the PI3K P85 subunit) rs34309 were associated with OGTT, fasting insulin, and HOMA-IR levels in the linear regression analysis. The gene-gene interaction analysis showed that, compared with pregnant women with other genotype combinations, women with SLC2A4 rs5435 (CC/CT), RBP4 rs7091052 (CC), PCK1 rs1042531 (TT/TG) and rs2236745 (TT), and PIK3R1 rs34309 (AA) had lower GDM risk. Conclusion: SLC2A4, RBP4, PCK1, and PIK3R1 genes may be involved in the pathogenesis of GDM.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Diabetes Gestacional/genética , Transportador de Glucose Tipo 4/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfatidilinositol 3-Quinases/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Plasmáticas de Ligação ao Retinol/genética , Adulto , Glicemia/genética , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/genética , Feminino , Predisposição Genética para Doença/genética , Genótipo , Humanos , Insulina/genética , Resistência à Insulina/genética , Gravidez , Transdução de Sinais/genética
11.
In Vitro Cell Dev Biol Anim ; 55(4): 226-236, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30790128

RESUMO

The mechanism whereby 17ß-estradiol (E2) mediates insulin gene transcription has not been fully elucidated. In this study, exposure of hamster insulinoma (HIT-T15) cells to 5 × 10-9 to 1 × 10-7 M E2 led to a concentration-dependent decrease of insulin mRNA levels. Transient expression of the estrogen receptor (ER) in HIT-T15 cells revealed that estrogen receptor α (ERα) repressed transcription of the rat insulin II promoter in both ligand-dependent and ligand-independent manners. The N-terminal A/B domain of ERα was not required for either activity. However, the repression was absent with mutated ER lacking the DNA-binding domain. Moreover, introducing mutations in the D-box and P-box of the zinc finger of ER (C227S, C202L) also abolished the repression. Deletion of the insulin promoter region revealed that nucleotide positions - 238 to - 144 (relative to the transcriptional start site) were needed for ER repression of the rat insulin II gene. PDX1- and BETA2-binding sites were required for the repression, but an estrogen response element-like sequence or an AP1 site in the promoter was not involved. In conclusion, we found that estrogen repressed insulin mRNA expression in a beta cell line. In addition, the ER suppressed insulin gene transcription in a ligand-independent matter. These observations suggest ER may regulate insulin transcription by indirect genomic signaling.


Assuntos
Genoma , Células Secretoras de Insulina/metabolismo , Insulina/genética , Receptores Estrogênicos/metabolismo , Transcrição Genética , Animais , Bioensaio , Linhagem Celular , Cricetinae , Estradiol/farmacologia , Fulvestranto/farmacologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Ligantes , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Estrogênicos/química , Receptores Estrogênicos/genética , Deleção de Sequência , Tamoxifeno/farmacologia , Transcrição Genética/efeitos dos fármacos
12.
Biochimie ; 158: 191-198, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30677431

RESUMO

Arginine vasopressin (AVP), a peptide secreted from the posterior pituitary, is chiefly regarded as a hormone involved in the regulation of body fluid balance and osmolality. However, recent evidence has revealed that posterior pituitary hormones can exert important actions on endocrine pancreatic function. In the present study, the presence of AVP receptors, namely Avpr1a (V1a), Avpr1b (V1b) and Avpr2 (V2) was demonstrated in murine islets as well as rodent BRIN BD11 and human 1.1B4 beta-cells. Further to this, AVP was shown to induce significant concentration-dependent (10-12 - 10-6 M) increases of insulin release from both rodent and human beta-cells, as well as mouse islets. Insulinotropic actions of AVP were completely annulled by specific V1a or V1b receptor antagonists, and partially abolished by an oxytocin receptor antagonist. In addition, beta-cell insulin secretory actions of AVP were augmented by both IBMX (200 µM) and KCl (30 mM) and linked to significantly increased cAMP production and [Ca2+]i. AVP substantially increased proliferation of rodent and human beta-cells. Moreover, AVP fully protected against cytokine-induced beta-cell apoptosis. AVP had no effect on glucagon secretion. Immunohistochemical examination of beta- and alpha-cells revealed co-expression of AVP with glucagon, and particularly insulin. Finally, administration of AVP in combination with glucose to mice significantly reduced blood glucose, which was associated with increased plasma insulin. These data indicate that AVP possesses novel and potentially important effects on pancreatic endocrine function. Understanding disturbances in islet AVP receptor signalling could reveal insight into the beta-cell defects associated with diabetes.


Assuntos
Arginina Vasopressina/metabolismo , Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Arginina Vasopressina/genética , Arginina Vasopressina/farmacologia , Linhagem Celular , Glucagon/genética , Humanos , Insulina/genética , Células Secretoras de Insulina/citologia , Camundongos , Receptores de Vasopressinas/genética
13.
Biomed Pharmacother ; 111: 657-665, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30611990

RESUMO

A microRNA (miRNA) is a single-stranded, small and non-coding RNA molecule that contains 20-25 nucleotides. More than 2000 miRNAs have been identified in human genes since the first miRNA was discovered in Caenorhabditis elegans in the early 1990s. miRNAs play a crucial role in various biological processes by regulating gene expression through post-transcriptional mechanisms. The alterations of their levels are associated with various diseases, such as glucometabolic disorder and lipid metabolism disorder. In recent years, miRNAs have been proved to be involved in regulating the functions of pancreatic ß-cells, insulin resistance and other biological behaviors related to glucometabolic disorder and the pathogenesis of diabetes mellitus (DM). This review summarized specific miRNAs, including miRNA-375 (miR-375), miRNA-155 (miR-155), miRNA-21 (miR-21), miRNA-33 (miR-33), the let-7 family and some other miRNAs related to glucometabolic regulation, introduced the obstacles and challenges in miRNA therapy, and discussed the prospect of new treatment methods for glucometabolic disorder.


Assuntos
Glucose/metabolismo , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/metabolismo , MicroRNAs/metabolismo , Animais , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Glucose/genética , Humanos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/genética , Hiperglicemia/metabolismo , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Doenças Metabólicas/genética , MicroRNAs/administração & dosagem , MicroRNAs/genética
14.
Amino Acids ; 51(4): 619-626, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30604098

RESUMO

The insulin superfamily is a group of homologous proteins that are further divided into the insulin family and relaxin family according to their distinct receptors. All insulin superfamily members contain three absolutely conserved disulfide linkages and a nonchiral Gly residue immediately following the first B-chain cysteine. The functionality of this conserved Gly residue in the insulin family has been studied by replacing it with natural L-amino acids or the corresponding unnatural D-amino acids. However, such analysis has not been conducted on relaxin family members. In the present study, we conducted chiral mutagenesis on the conserved B11Gly of the chimeric relaxin family peptide R3/I5, which is an efficient agonist for receptor RXFP3 and RXFP4. Similar to the effects on insulin family foldability, L-Ala or L-Ser substitution completely abolished the in vitro refolding of a recombinant R3/I5 precursor; whereas, D-Ala or D-Ser substitution had no detrimental effect on refolding of a semi-synthetic R3/I5 precursor, suggesting that the conserved Gly residue controls the foldability of relaxin family members. In contrast to the effect on insulin family activity, D-Ala or D-Ser replacement had no detrimental effect on the binding and activation potencies of the mature R3/I5 towards both RXFP3 and RXFP4, suggesting that the conserved Gly residue is irrelevant to the relaxin family's activity. The present study revealed functionality of the conserved B-chain Gly residue for a relaxin family peptide for the first time, providing an overview of its contribution to foldability and activity of the insulin superfamily.


Assuntos
Glicina/metabolismo , Insulina/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Relaxina/metabolismo , Glicina/química , Glicina/genética , Humanos , Insulina/química , Insulina/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Relaxina/química , Relaxina/genética
15.
Mol Med Rep ; 19(3): 2220-2230, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664204

RESUMO

Polycystic ovary syndrome (PCOS) is a condition in which a woman's levels of the sex hormones (estrogen and progesterone) are out of balance, leading to the growth of ovarian cysts. PCOS can affect the menstrual cycle, fertility, cardiac function and even appearance of women. Therefore, we aimed to explore the genetic polymorphism of the melatonin receptors 1A and 1B in obese patients with PCOS to identify a new theoretical basis for its treatment. Patients presenting with PCOS (n=359) were enrolled and classified into an obese OB­PCOS group [body mass index (BMI) of PCOS patients ≥25 kg/m2] or a nonobese NOB­PCOS group, and 215 oviduct infertile patients who experienced normal ovulation were used as the control group. All baseline characteristics, endocrine hormone levels, lipid and glucose metabolism, and insulin indices were measured. The genotypes of rs2119882 within the MTNR1A gene and of rs10830963 within the MTNR1B gene were determined by PCR­RFLP; the genotype frequency and the difference in the distribution of allele frequency were compared. For rs2119882, C allele carriers who were not diagnosed with PCOS had an increased risk of developing PCOS, and C allele carriers with PCOS had an increased risk of developing OB­PCOS. For rs10830963, G allele carriers who were not diagnosed with PCOS had an increased risk of developing PCOS. The TT genotype in rs2119882 and the CC genotype in rs10830963 were protective factors for OB­PCOS, and increased levels of LH, testosterone, and estradiol and abnormal menstruation were key risk factors for PCOS. Furthermore, the TT genotype at the rs2119882 site was the key protective factor for OB­PCOS patients. Our study found that MTNR1A rs2119882 and MTNR1B rs10830963 could increase the risk for PCOS and cause glycolipid metabolism disorder in PCOS patients.


Assuntos
Obesidade/genética , Síndrome do Ovário Policístico/genética , Receptor MT1 de Melatonina/genética , Receptor MT2 de Melatonina/genética , Adulto , Glicemia , Índice de Massa Corporal , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Insulina/genética , Resistência à Insulina/genética , Metabolismo dos Lipídeos/genética , Obesidade/complicações , Obesidade/patologia , Síndrome do Ovário Policístico/complicações , Síndrome do Ovário Policístico/patologia , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco
16.
PLoS One ; 14(1): e0199483, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30689636

RESUMO

The nematode Caenorhabditis elegans has been used to examine the influence of epicatechin (EC), an abundant flavonoid in the human diet, in some stress biomarkers (ROS production, lipid peroxidation and protein carbonylation). Furthermore, the ability of EC to modulate the expression of some key genes in the insulin/IGF-1 signaling pathway (IIS), involved in longevity and oxidative or heat shock stress response, has also been explored. The final aim was to contribute to the elucidation of the mechanisms involved in the biological effects of flavonoids. The results showed that EC-treated wild-type C. elegans exhibited increased survival and reduced oxidative damage of biomolecules when submitted to thermal stress. EC treatment led to a moderate elevation in ROS levels, which might activate endogenous mechanisms of defense protecting against oxidative insult. The enhanced stress resistance induced by EC was found to be mediated through the IIS pathway, since assays in daf-2, age-1, akt-1, akt-2, sgk-1, daf-16, skn-1 and hsf-1 loss of function mutant strains failed to show any heat-resistant phenotype against thermal stress when treated with EC. Consistently, EC treatment upregulated the expression of some stress resistance associated genes, such as gst-4, hsp-16.2 and hsp-70, which are downstream regulated by the IIS pathway.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Catequina/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética
17.
Mol Med Rep ; 19(2): 974-983, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569116

RESUMO

Osteosarcoma (OS) is the most common pediatric primary bone tumor, with high malignancy rates and a poor prognosis following metastasis. At present, the role of microRNA (miR)­542­3p in OS remains to be elucidated. The purpose of the present study was to investigate the expression level of miR­542­3p in OS, and its potential molecular mechanisms, via a bioinformatics analysis. First, the expression of miR­542­3p in OS based on the continuous variables of the Gene Expression Omnibus database and PubMed was studied. Subsequently, the potential target genes of miR­542­3p were predicted using gene expression profiles and bioinformatics software. On the basis of the Database for Annotation, Visualization and Integrated Discovery, version 6.8, a study of gene ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway knowledge base was conducted to explore the biological value of miR­542­3p in OS. Finally, the protein­protein interaction (PPI) network was completed using the STRING database. The expression of miR­542­3p in OS was revealed to be significantly higher compared with that in normal tissue. In total, 1,036 target genes of miR­542­3p were obtained. The results of the GO enrichment analysis revealed that the significant terms were 'bone development', 'cell cycle arrest' and 'intracellular signal transduction'. The results of the KEGG analysis revealed the highlighted pathways that were targeted to miR­542­3p, including the sphingolipid signaling pathway (P=3.91x10­5), the phosphoinositide 3­kinase (PI3K)­AKT serine/threonine kinase (AKT) signaling pathway (P=3.17x10­5) and the insulin signaling pathway (P=1.04x10­5). The PPI network revealed eight hub genes: Ubiquitin­60S ribosomal protein L40, Ras­related C3 botulinum toxin substrate, mitogen­activated protein kinase 1, epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, PI3K regulatory subunit 1, AKT1, and actin­related protein 2/3 complex subunit 1A, which may be the key target genes of miR­542­3p in OS. Taken together, these results have demonstrated that miR­542­3p was overexpressed in OS. The potential target genes and biological functions of miR­542­3p may provide novel insights into the differentially expressed genes that are involved in OS.


Assuntos
Neoplasias Ósseas/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs/genética , Osteossarcoma/genética , Transdução de Sinais/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Insulina/genética , Insulina/metabolismo , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingolipídeos/metabolismo
18.
Methods Mol Biol ; 1916: 213-222, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30535698

RESUMO

This chapter describes the propagation and characterization of transplantable insulinoma cells as model of insulin-producing pancreatic islet cells in the rat. Here, the cells are propagated by transplantation into rats followed by harvesting after growth for approximately 1 month. The cells are then purified by Percoll density gradient centrifugation and characterized by pulse-chase radiolabelling and immunoprecipitation of the insulin-related peptides. The results show that the transplantable insulinoma cells produce insulin in a manner similar to that found in normal pancreatic islets.


Assuntos
Técnicas de Cultura de Células/métodos , Imunoprecipitação/métodos , Insulinoma/patologia , Neoplasias Pancreáticas/genética , Animais , Proliferação de Células/genética , Humanos , Insulina/genética , Secreção de Insulina/genética , Insulinoma/genética , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/patologia , Neoplasias Pancreáticas/patologia , Ratos
19.
Protein Expr Purif ; 153: 35-43, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30098414

RESUMO

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been used for the production of recombinant monomeric insulin precursor (MIP). Recombinant plasmids with one, two and four cassettes of the MIP gene have been successfully constructed in the pPICZαA expression vector to study the effects of gene copy number on MIP production. The MIP protein can be detected by dot-blot analysis from the culture broth of P. pastoris KM71H 24 h after placement in MMH induction medium. The secretion levels of MIP protein in culture broth at 72 h after induction indicated that P. pastoris KM71H with one cassette of the MIP gene had highest MIP protein levels (4.19 ±â€¯0.96 mg L-1). The transcription levels of the MIP gene increased proportionately with copy number. However, the amount of secreted MIP protein showed no correlation. The MIP molecular mass was 5756.951 Da, as confirmed by typical MALDI-TOF mass spectrometry. The MIP protein in culture broth was purified by two steps purification including SP Sepharose Fast Flow chromatography followed by ultrafiltration (10 kDa MW cutoff). The percentage of MIP recovery after the two-step purification was 70%, with a single band in a native-PAGE. The biological activity of tryptic hydrolyzed MIP was determined via the expression of the glucose transporter 4 gene (GLUT4) in H9c2 (2-1) cell line by RT-qPCR, and the results demonstrated that the MIP protein can induce glucose uptake and upregulation of GLUT4 mRNA transcription at 3 h and that this activity was related to Humalog® insulin.


Assuntos
Clonagem Molecular/métodos , Transportador de Glucose Tipo 4/agonistas , Glucose/metabolismo , Insulina/genética , Pichia/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Dosagem de Genes , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Humanos , Insulina/biossíntese , Insulina/farmacologia , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Pichia/metabolismo , Precursores de Proteínas/biossíntese , Precursores de Proteínas/farmacologia , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência
20.
Diabetes Obes Metab ; 21(1): 95-102, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30073765

RESUMO

AIM: To evaluate whether ß cells continue to undergo death in the later stages of type 1 diabetes (T1D). MATERIALS AND METHODS: Fasting banked sera from a cross-section of 90 participants in the T1D Exchange Registry with longstanding T1D (median duration of 9 years) were analysed. Subjects were determined to be C-peptide (-) or (+) based on mixed-meal tolerance testing. Results were compared with 54 adult non-diabetic controls. Stimulated samples were assayed in a subset of subjects. Levels of unmethylated and methylated preproinsulin (INS) DNA were analysed using digital droplet PCR. RESULTS: Fasting and stimulated circulating unmethylated INS DNA levels were increased among both C-peptide (-) and C-peptide (+) subjects with longstanding T1D compared with non-diabetic controls (P < 0.01). Consistent with prior reports, unmethylated INS DNA values correlated with methylated INS DNA values, which were also elevated among T1D subjects (P < 0.001). There was wide variation in the effects of mixed-meal stimulation on DNA levels, with fasting values in the highest quartiles decreasing with stimulation (P < 0.05). CONCLUSIONS: These results could reflect ongoing ß cell death in individuals with longstanding T1D, even in the absence of detectable C-peptide production, suggesting that therapies targeting ß cell survival could be beneficial among individuals with longstanding T1D.


Assuntos
DNA , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/epidemiologia , Insulina , Precursores de Proteínas , Adulto , Peptídeo C/sangue , Estudos de Casos e Controles , DNA/sangue , DNA/genética , Metilação de DNA , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Humanos , Insulina/sangue , Insulina/genética , Células Secretoras de Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/sangue , Precursores de Proteínas/genética , Adulto Jovem
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