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1.
Yakugaku Zasshi ; 140(8): 969-977, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741870

RESUMO

We developed a method of video-rate bioluminescence imaging to visualize proteins secreted from living cells. A protein of interest was fused to Gaussia luciferase (GLase), and the luminescence signals of secreted GLase with coelenterazine (luciferin) were visualized at a video-rate of 30-500 ms/frame by using a water-cooled EM-CCD camera. We established a subclonal rat INS-1E cell line, named iGL cells, stably expressing the fusion protein of insulin and GLase (Insulin-GLase). By stimulation with high glucose, 3D-cultured iGL cells showed synchronized oscillatory secretion of insulin for over 1 h, as similarly observed in an isolated rat pancreatic islet. In 2D-cultured iGL cells, the luminescence images indicated that synchronized insulin secretion was localized in intercellular spaces between cells. Further, the relative amount of insulin secretion from iGL cells was easily determined with a luminometer, and we demonstrated that cell-cell interaction of beta cells is fundamental to increase glucose-stimulated insulin secretion by synchronization. Thus, iGL cells would be valuable for studying oscillatory insulin secretion and evaluating anti-diabetic drugs. Our bioluminescence imaging method with GLase could be generally used for investigating protein secretion in 2D and 3D cell culture systems.


Assuntos
Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Medições Luminescentes/métodos , Imagem Molecular/métodos , Animais , Comunicação Celular , Linhagem Celular , Células Cultivadas , Humanos , Imidazóis , Luciferases , Pirazinas , Ratos
2.
PLoS One ; 15(7): e0236603, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706828

RESUMO

BACKGROUND AND OBJECTIVE: Dipeptidyl peptidase-4 (DPP-4) inhibitors have been suggested to have pancreatic beta-cell preserving effect according to studies using homeostatic model of assessment for beta-cell function (HOMA-ß). However, whether HOMA-ß is a suitable biomarker for comparisons between hypoglycemic drugs with different mechanisms of action remains unclear. Therefore, we conducted a meta-analysis to compare the effects of DPP-4 inhibitors and other classes of hypoglycemic drugs on HOMA-ß and proinsulin-to-insulin ratio (PIR). METHODS: We searched MEDLINE, CENTRAL, and Ichushi-web for the period of 1966 to May 2020. We collected randomized, controlled clinical trials in patients with type 2 diabetes mellitus comparing DPP-4 inhibitors and other classes of hypoglycemic agents [α-glucosidase inhibitors (α-GIs), glucagon-like peptide-1 (GLP-1) analogues, metformin, sodium-glucose cotransporter 2 (SGLT2) inhibitors, sulfonylureas, or thiazolidinediones]. Weighted mean differences and 95% confidence intervals of changes in HOMA-ß or PIR during study periods were calculated for pairwise comparisons. RESULTS: Thirty-seven and 21 relevant trials were retrieved for comparisons of HOMA-ß and PIR, respectively. HOMA-ß and PIR consistently showed superiority of DPP-4 inhibitors compared with α-GIs. Both biomarkers consistently supported inferiority of DPP-4 inhibitors compared with GLP-1 analogues. However, PIR showed inferiority of DPP-4 inhibitors compared with metformin, and superiority compared with SGLT2 inhibitors, whereas HOMA-ß showed no significant differences between DPP-4 inhibitors and the two other agents. CONCLUSION: DPP-4 inhibitors appear to be superior to α-GIs but inferior to GLP-1 analogues in preservation of beta-cell function assessed by either HOMA-ß or PIR. DPP-4 inhibitors seem to be superior to SGLT2 inhibitors but inferior to metformin on islet function assessed only by PIR. Because HOMA-ß and PIR may indicate different aspects of beta-cell function, results of beta-cell function preserving effects of hypoglycemic agents should be interpreted with caution.


Assuntos
Biomarcadores/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Proinsulina/metabolismo , Compostos de Sulfonilureia/farmacologia , Compostos de Sulfonilureia/uso terapêutico , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico
3.
Chem Biol Interact ; 328: 109197, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32710900

RESUMO

The present study was undertaken to assess the effect of imatinib mesylate; a tyrosine kinase inhibitor and a well-known anticancer with numerous medical benefits on blood sugar levels, insulin, and glucagon secretion in an experimental model of STZ-induced diabetes mellitus. Type 1 diabetes mellitus (T1DM) was induced by a single I.P. injection of Streptozotocin (STZ) (50 mg/kg) in male Sprague-Dawley rats. Daily oral imatinib (10 mg/kg) and (20 mg/kg) for 4 weeks induced a significant attenuation in signs of DM in rats reflected in their assessed lab values. Biomarkers of cell injury, tissue necrosis, and apoptosis; caspase-3 were significantly reduced with imatinib treatment. Furthermore, pancreatic antioxidants defenses of which; superoxide dismutase (SOD) and catalase activities, reduced glutathione (GSH) concentration, and total antioxidant capacity have significantly improved with a simultaneous reduction in malondialdehyde (MDA) content. Histopathologically, imatinib treatment was associated with a minimal pancreatic injury and marked restoration of insulin content in ß-cells. Moreover, imatinib treatment revealed a significant reduction in the infiltration of macrophages in ß-cells. Imatinib's ameliorative impact on DM may be attributed to it's mediated protection and preservation of pancreatic ß-cells function and the improvement in serum insulin levels and hence the improvement of blood glucose and overall glycemic control.


Assuntos
Diabetes Mellitus Experimental/patologia , Mesilato de Imatinib/farmacologia , Células Secretoras de Insulina/metabolismo , Administração Oral , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antioxidantes/metabolismo , Biomarcadores/sangue , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Caspase 3/metabolismo , Catalase/metabolismo , Diabetes Mellitus Experimental/sangue , Modelos Animais de Doenças , Glucagon/sangue , Glutationa/metabolismo , Insulina/sangue , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Estreptozocina , Superóxido Dismutase/metabolismo
4.
DNA Cell Biol ; 39(9): 1700-1710, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32721233

RESUMO

The increased secretion of glucagon-like peptide-1 (GLP-1) after Roux-en-Y gastric bypass (RYGB) is regarded as the main reason for the improvement of blood glucose. However, the single-nucleotide polymorphisms (SNPs) of GLP-1 Receptor (GLP1R) impair receptor function, subsequently affecting ß cell insulin secretion function, ultimately affecting the efficacy of RYGB. In this study, we revealed that two SNPs in GLP1R gene, rs3765467 and rs10305492, could significantly reduce the insulin secreted by ß cells and the cyclic AMP concentration, whereas promote ß cell apoptosis. Under high glucose exposure, rs3765467 and rs10305492 impaired ß cell secretion of insulin and ß cell viability in the same way; in other words, GLP1R rs3765467 and rs10305492 exert an effect on pancreatic ß cell glucose-stimulated insulin secretion. Moreover, GLP-1 antagonist Exendin (9-39) further enhanced, whereas GLP-1 agonist Exendin-4 partially attenuated the effects of SNPs on the functions and apoptosis of ß cells. In conclusion, the rs3765467 and rs10305492 SNPs in GLP1R show to exert a critical effect on regulating insulin secretory capacity of ß cells and ß cell mass. Through leading to the dysfunction and apoptosis of ß cells, GLP1R rs3765467 and rs10305492 might also impair GLP-1 interaction with GLP1R, therefore attenuating the therapeutic effect of RYGB.


Assuntos
Apoptose , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Polimorfismo de Nucleotídeo Único , Animais , Linhagem Celular , Células Cultivadas , Exenatida/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Mutação , Ratos
5.
Diab Vasc Dis Res ; 17(4): 1479164120945675, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722929

RESUMO

Activation of the prostaglandin E2 receptor EP4 alters polarization of adipose tissue macrophages towards the anti-inflammatory M2 phenotype to suppress chronic inflammation. However, the role of EP4 signalling in pancreatic macrophages that affect insulin secretion is unclear. We examined the role of EP4 signalling in islet inflammation in vitro and in vivo. Obese diabetic db/db mice were treated with an EP4-selective agonist or vehicle for 4 weeks. Islet morphology did not significantly differ and glucose-stimulated insulin secretion was increased, whereas the pancreatic M1/M2 ratio was decreased in the EP4 agonist-treated group compared to the vehicle group. Because EP4 activation in MIN6 cells did not affect insulin secretion, we used a MIN6/macrophage co-culture system to evaluate the role of EP4 signalling in islet inflammation and subsequent inhibition of insulin release. Co-culture with M1-polarized macrophages markedly suppressed insulin expression in MIN6 cells; however, modulation of M1 polarization by the EP4 agonist significantly reversed the negative impact of co-cultivation on insulin production. The enhanced expression levels of pro-inflammatory cytokines in co-cultured MIN6 cells were markedly inhibited by EP4 agonist treatment of M1 macrophages. Thus, EP4 activation may suppress islet inflammation and protect ß-cell function by altering inflammatory macrophages in the diabetic pancreas.


Assuntos
Plasticidade Celular , Diabetes Mellitus Tipo 2/metabolismo , Inflamação/metabolismo , Células Secretoras de Insulina/metabolismo , Macrófagos Peritoneais/metabolismo , Obesidade/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ativação de Macrófagos , Macrófagos Peritoneais/patologia , Camundongos , Obesidade/patologia , Fenótipo , Via Secretória , Transdução de Sinais
6.
Ecotoxicol Environ Saf ; 201: 110802, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32531573

RESUMO

Extended exposure to inorganic arsenic through contaminated drinking water has been linked with increased incidence of diabetes mellitus. The most common exposure occurs through the consumption of contaminated drinking water mainly through geogenic sources of inorganic arsenic. Epigenetic modifications are important mechanisms through which environmental pollutants could exert their toxic effects. Bisulfite sequencing polymerase chain reaction method followed by Sanger sequencing was performed for DNA methylation analysis. Our results showed that sodium arsenite treatment significantly reduced insulin secretion in pancreatic islets. It was revealed that the methylation of glucose transporter 2 (Glut2) gene was changed at two cytosine-phosphate-guanine (CpG) sites (-1743, -1734) in the promoter region of the sodium arsenite-treated group comparing to the control. No changes were observed in the methylation status of peroxisome proliferator-activated receptor-gamma (PPARγ), pancreatic and duodenal homeobox 1 (Pdx1) and insulin 2 (Ins2) CpG sites in the targeted regions. Measuring the gene expression level showed increase in Glut2 expression, while the expression of insulin (INS) and Pdx1 were significantly affected by sodium arsenite treatment. This study revealed that exposure to sodium arsenite changed the DNA methylation pattern of Glut2, a key transporter of glucose entry into the pancreatic beta cells (ß-cells). Our data suggested possible epigenetic-mediated toxicity mechanism for arsenite-induced ß-cells dysfunction. Further studies are needed to dissect the precise epigenetic modulatory activity of sodium arsenite that affect the biogenesis of insulin.


Assuntos
Arsenitos/toxicidade , Metilação de DNA/efeitos dos fármacos , Transportador de Glucose Tipo 2/genética , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Compostos de Sódio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Epigênese Genética/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Insulina/genética , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Transativadores/genética
7.
Nat Commun ; 11(1): 2742, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488111

RESUMO

Next generation sequencing studies have highlighted discrepancies in ß-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human ß-cells postnatally, while its expression is restricted to embryonic and neo-natal ß-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to ß-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide-positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.


Assuntos
Células Secretoras de Insulina/metabolismo , Fator de Transcrição MafB/genética , Fator de Transcrição MafB/metabolismo , Células Estreladas do Pâncreas/metabolismo , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Feminino , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Células-Tronco , Transcriptoma
8.
Expert Opin Pharmacother ; 21(13): 1565-1578, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32521177

RESUMO

INTRODUCTION: Recent advances in anti-diabetic medications and glucose monitoring have led to a paradigm shift in diabetes care. Newer anti-diabetic medications such as DPP-4 inhibitors, GLP-1 receptor agonists (GLP-1RAs), and SGLT2 inhibitors have enabled optimal glycemic control to be achieved without increasing the risk of hypoglycemia and weight gain. Treatment with GLP-1RAs and SGLT2 inhibitors has been demonstrated to improve cardiorenal outcomes, positioning these agents as the mainstay of treatment for patients with type 2 diabetes (T2DM). The development of these newer agents has also prompted a paradigm shift in the concept of T2DM, highlighting the importance of beta cell dysfunction in the pathophysiology of T2DM. AREAS COVERED: Recent advances in pharmacotherapy for diabetes are summarized with a focus on the role of incretin-based drugs and SGLT2 inhibitors. The importance of a paradigm shift from a glucose-centric to a beta cell-centric concept of T2DM is also discussed, given from an Asian perspective. EXPERT OPINION: Management of T2DM including lifestyle modification as well as pharmacotherapy should be focused on reducing beta cell workload, to preserve functional beta cell mass. A paradigm shift from a glucose-centric to a beta cell-centric concept of T2DM enhances the implementation of person-centered diabetes care.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Glicemia/análise , Automonitorização da Glicemia , Diabetes Mellitus Tipo 2/metabolismo , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Glucose/metabolismo , Humanos , Hipoglicemia/induzido quimicamente , Incretinas/metabolismo , Insulina/uso terapêutico , Células Secretoras de Insulina/metabolismo , Medicina de Precisão , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Compostos de Sulfonilureia/uso terapêutico
9.
Nat Commun ; 11(1): 2538, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439909

RESUMO

Compromised ß-cell identity is emerging as an important contributor to ß-cell failure in diabetes; however, the precise mechanism independent of hyperglycemia is under investigation. We have previously reported that mTORC1/Raptor regulates functional maturation in ß-cells. In the present study, we find that diabetic ß-cell specific Raptor-deficient mice (ßRapKOGFP) show reduced ß-cell mass, loss of ß-cell identity and acquisition of α-cell features; which are not reversible upon glucose normalization. Deletion of Raptor directly impairs ß-cell identity, mitochondrial metabolic coupling and protein synthetic activity, leading to ß-cell failure. Moreover, loss of Raptor activates α-cell transcription factor MafB (via modulating C/EBPß isoform ratio) and several α-cell enriched genes i.e. Etv1 and Tspan12, thus initiates ß- to α-cell reprograming. The present findings highlight mTORC1 as a metabolic rheostat for stabilizing ß-cell identity and repressing α-cell program at normoglycemic level, which might present therapeutic opportunities for treatment of diabetes.


Assuntos
Diferenciação Celular , Plasticidade Celular , Diabetes Mellitus/patologia , Células Secretoras de Insulina/patologia , Proteína Regulatória Associada a mTOR/metabolismo , Animais , Glicemia/metabolismo , Diferenciação Celular/genética , Plasticidade Celular/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Regulação da Expressão Gênica , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Fator de Transcrição MafB/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Proteína Regulatória Associada a mTOR/genética , Transdução de Sinais
10.
BMC Med Genet ; 21(1): 91, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32375679

RESUMO

BACKGROUND: Renal hypouricemia (RHUC) is a hereditary disorder where mutations in SLC22A12 gene and SLC2A9 gene cause RHUC type 1 (RHUC1) and RHUC type 2 (RHUC2), respectively. These genes regulate renal tubular reabsorption of urates while there exist other genes counterbalancing the net excretion of urates including ABCG2 and SLC17A1. Urate metabolism is tightly interconnected with glucose metabolism, and SLC2A9 gene may be involved in insulin secretion from pancreatic ß-cells. On the other hand, a myriad of genes are responsible for the impaired insulin secretion independently of urate metabolism. CASE PRESENTATION: We describe a 67 year-old Japanese man who manifested severe hypouricemia (0.7 mg/dl (3.8-7.0 mg/dl), 41.6 µmol/l (226-416 µmol/l)) and diabetes with impaired insulin secretion. His high urinary fractional excretion of urate (65.5%) and low urinary C-peptide excretion (25.7 µg/day) were compatible with the diagnosis of RHUC and impaired insulin secretion, respectively. Considering the fact that metabolic pathways regulating urates and glucose are closely interconnected, we attempted to delineate the genetic basis of the hypouricemia and the insulin secretion defect observed in this patient using whole exome sequencing. Intriguingly, we found homozygous Trp258* mutations in SLC22A12 gene causing RHUC1 while concurrent mutations reported to be associated with hyperuricemia were also discovered including ABCG2 (Gln141Lys) and SLC17A1 (Thr269Ile). SLC2A9, that also facilitates glucose transport, has been implicated to enhance insulin secretion, however, the non-synonymous mutations found in SLC2A9 gene of this patient were not dysfunctional variants. Therefore, we embarked on a search for causal mutations for his impaired insulin secretion, resulting in identification of multiple mutations in HNF1A gene (MODY3) as well as other genes that play roles in pancreatic ß-cells. Among them, the Leu80fs in the homeobox gene NKX6.1 was an unreported mutation. CONCLUSION: We found a case of RHUC1 carrying mutations in SLC22A12 gene accompanied with compensatory mutations associated with hyperuricemia, representing the first report showing coexistence of the mutations with opposed potential to regulate urate concentrations. On the other hand, independent gene mutations may be responsible for his impaired insulin secretion, which contains novel mutations in key genes in the pancreatic ß-cell functions that deserve further scrutiny.


Assuntos
Complicações do Diabetes/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Transportadores de Ânions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Erros Inatos do Transporte Tubular Renal/genética , Cálculos Urinários/genética , Idoso , Complicações do Diabetes/complicações , Complicações do Diabetes/patologia , Glucose/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Heterozigoto , Proteínas de Homeodomínio/genética , Homozigoto , Humanos , Insulina/biossíntese , Insulina/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Mutação/genética , Erros Inatos do Transporte Tubular Renal/complicações , Erros Inatos do Transporte Tubular Renal/patologia , Ácido Úrico/metabolismo , Cálculos Urinários/complicações , Cálculos Urinários/patologia , Sequenciamento Completo do Exoma
11.
Gene ; 753: 144785, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32445922

RESUMO

OBJECTIVE: Type 1 diabetes onset is preceded by a pre-inflammatory stage leading to insulitis and followed by targeted destruction of the insulin-producing beta cells of the pancreas. Osteopontin (OPN) is a secreted phosphoprotein with cytokine properties, implicated in many physiological and pathological processes, including infection and autoimmunity. We have previously identified up-regulated osteopontin transcripts in the pancreatic lymph nodes of the NOD (Non-Obese Diabetic) mouse at the pre-diabetic stages. Investigating the underlined disease initiating mechanisms may well contribute to the development of novel preventive therapies. Our aim was to construct opn null mice in a NOD autoimmune-prone genetic background and address the pathogenic or protective role of the osteopontin molecule in the early stages of type 1 diabetes. METHODS: We generated opn null mutant mice in a NOD genetic background by serial backcrossing to the existing C57BL/6 opn knockout strain. The presence of opn wild type or null alleles in the congenic lines was evaluated by PCR amplification. We used NOD opn-null mice to assess the phenotypic evolution of type 1 diabetes. The presence of OPN in the serum was evaluated by ELISA and by immunostaining on the mouse tissues. The primary gene structure of the NOD opn encoding gene and protein sequences were compared to the known alleles of other mouse strains. Evaluation of Single Nucleotide Polymorphisms (SNPs) variation between opn alleles of the opn gene is reported. RESULTS: In the absence of OPN, type 1 diabetes is accelerated, suggesting a protective role of this cytokine on the insulin-producing cells of the pancreatic islets. Conversely, in the presence of the opn gene, an increase of the OPN protein in the serum of young NOD mice indicates that this molecule might be involved in the immune regulatory events taking place at early stages, prior to disease onset. Our data support that OPN acts as a positive regulator of the early islet autoimmune damage, possibly by a shift of the steady-state of T1D pathogenesis. We report that the OPN protein structure of the NOD/ShiLtJ strain corresponds to the a-type allele of the osteopontin gene. Comparative analysis of the single nucleotide polymorphisms between the a-type and b-type alleles indicates that the majority of variations are within the non-coding regions of the gene. CONCLUSIONS: The construction of opn null mice in an autoimmune genetic background (NOD.B6.Cg-spp1-/-) provides important tools for the study of the implication of the OPN in type 1 diabetes, offering the possibility to address the significance of this molecule as an early marker of the disease and as a therapeutic agent in preclinical studies.


Assuntos
Diabetes Mellitus Tipo 1/genética , Osteopontina/genética , Alelos , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Pâncreas/metabolismo , Polimorfismo de Nucleotídeo Único/genética
12.
Toxicology ; 441: 152502, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32473187

RESUMO

Cigarette smoking is a well-recognized risk factor for type 2 diabetes (T2DM), and may result in islet ß cell damage and impaired insulin secretion. However, the underlying mechanisms remain largely elusive. In the present study, we demonstrated that nicotine induced premature senescence of pancreatic ß cells in vitro and in vivo. The senescence-associated ß-galactosidase (SA-ß-Gal) assay showed that nicotine exposure induced apparent senescence phenotype of ß-TC-6 cells at an initiating dose of 100 µM and starting from 12 h. In addition, 100 and 500 µM of nicotine exposure altered the expression of senescence marker proteins, such as p16, p19 and p21. Furthermore, we uncovered that the levels of intracellular Ca2+ and reactive oxygen species (ROS) were significantly elevated in ß-TC-6 cells following exposure to 100 and 500 µM nicotine, while calcium channel blocker can reverse this effect. Furthermore, the senescence-inducing phenotype was confirmed in rat insulinoma INS-1 cells at a similar dose range, whereas blockade of nAChRs, calcium and ROS led to apparent impairment of senescence. Finally, we found that administration with 100 and 200 µg/mL nicotine in drinking water for 28 days significantly exacerbated aberrant glucose homeostasis in a mouse model of fat-induced T2DM. Of great intrigue, pancreatic ß cells exhibited significantly enhanced senescence following nicotine administration. Taken together, this study suggests that premature senescence plays a pivotal role in nicotine-triggered ß cell destruction and glucose intolerance, providing a theoretical basis for targeted prevention and treatment of smoking-induced T2DM.


Assuntos
Senescência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/induzido quimicamente , Células Secretoras de Insulina/efeitos dos fármacos , Nicotina/toxicidade , Animais , Western Blotting , Cálcio/metabolismo , Progressão da Doença , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , beta-Galactosidase/metabolismo
13.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G36-G42, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32463335

RESUMO

After 50% proximal small bowel resection (SBR) in mice, we have demonstrated hepatic steatosis, impaired glucose metabolism without insulin resistance, and increased pancreatic islet area. We sought to determine the consequences of SBR on pancreatic ß-cell morphology, proliferation, and expression of a key regulatory hormone, glucagon-like peptide-1 (GLP-1). C57BL/6 mice underwent 50% SBR or sham operation. At 10 wk, pancreatic insulin content and secretion was measured by ELISA. Immunohistochemistry was performed to determine structural alterations in pancreatic α-and ß-cells. Western blot analysis was used to measure GLP-1R expression, and immunoassay was used to measure plasma insulin and GLP-1. Experiments were repeated by administering a GLP-1 agonist (exendin-4) to a cohort of mice following SBR. After SBR, there was pancreatic islet hypertrophy and impaired glucose tolerance. The proportion of α and ß cells was not grossly altered. Whole pancreas and pancreatic islet insulin content was not significantly different; however, SBR mice demonstrated decreased insulin secretion in both static incubation and islet perfusion experiments. The expression of pancreatic GLP-1R was decreased approximately twofold after SBR, compared with sham and serum GLP-1, was decreased. These metabolic derangements were mitigated after administration of the GLP-1 agonist. Following massive SBR, there is significant hypertrophy of pancreatic islet cells with morphologically intact α- and ß-cells. Significantly reduced pancreatic insulin release in both static and dynamic conditions demonstrate a perturbed second phase of insulin secretion. GLP-1 is a key mediator of this amplification pathway. Decreased expression of serum GLP-1 and pancreatic GLP-1R in face of no change in insulin content presents a novel pathway for enteropancreatic glucose regulation following SBR.NEW & NOTEWORTHY Metabolic changes occur following intestinal resection; however, the effects on pancreatic function are unknown. Prior studies have demonstrated that glucagon-like protein-1 (GLP-1) signaling is a crucial player in the improved insulin sensitivity after bariatric surgery. In this study, we explore the effect of massive small bowel resection on gut hormone physiology and provide novel insights into the enteropancreatic axis.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Intestinos/lesões , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Animais , Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Camundongos Endogâmicos C57BL , Pâncreas Exócrino/metabolismo
14.
Science ; 368(6494): 993-1001, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32467389

RESUMO

Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel CaV1.2 and the inwardly rectifying potassium channel Kir2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human ß cells (Electroß cells). Wireless electrical stimulation of Electroß cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.


Assuntos
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Estimulação Elétrica/instrumentação , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Tecnologia sem Fio/instrumentação , Animais , Biônica , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio , Engenharia Celular , Células HEK293 , Humanos , Masculino , Camundongos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Próteses e Implantes , Transcrição Genética , Transgenes
15.
Gene ; 746: 144649, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32251702

RESUMO

BACKGROUND: Studies have shown that vitamin D can enhance glucose-stimulated insulin secretion (GSIS) and change the expression of genes in pancreatic ß-cells. Still the mechanisms linking vitamin D and GSIS are unknown. MATERIAL AND METHODS: We used an established ß-cell line, INS1E. INS1E cells were pre-treated with 10 nM 1,25(OH)2vitamin D or 10 nM 25(OH)vitamin D for 72 h and stimulated with 22 mM glucose for 60 min. RNA was extracted for gene expression analysis. RESULTS: Expression of genes affecting viability, apoptosis and GSIS changed after pre-treatment with both 1,25(OH)2vitamin D and 25(OH)vitamin D in INS1E cells. Stimulation with glucose after pre-treatment of INS1E cells with 1,25(OH)2vitamin D resulted in 181 differentially expressed genes, whereas 526 genes were differentially expressed after pre-treatment with 25(OH)vitamin D. CONCLUSION: Vitamin D metabolites may affect pancreatic ß-cells and GSIS through changed gene expression for genes involved in ß-cell function and viability.


Assuntos
Apoptose , Regulação da Expressão Gênica/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Vitamina D/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Ratos , Vitamina D/farmacocinética , Vitamina D/farmacologia
16.
Life Sci ; 252: 117648, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275937

RESUMO

AIMS: This study was conducted to determine the relationship between mesencephalic astrocyte-derived neurotrophic factor (MANF), autophagy and endoplasmic reticulum (ER) stress, and whether liraglutide (LRG) can protect ß cells, promote autophagy and alleviate ER stress by regulating MANF expression. MAIN METHODS: Human serum samples were collected from healthy controls (NC), simple hyperlipidemia (HLD), and newly diagnosed type 2 diabetes (T2D). The MANF levels were detected using ELISA. In vitro, after the mouse islet MIN6 cells were treated with glucose (GLU), palmitate (PA), thapsigargin (TG), LRG, and chloroquine (CQ), cell proliferation was detected using cell counting kit-8 (CCK-8), apoptosis-related protein cleaved caspase 3 (C-cas-3), ER stress, and autophagy-related proteins were detected by Western blotting, MANF, insulin, and C-cas-3 proteins were detected via immunofluorescence. Subcellular structures and autophagosomes were examined using electron microscopy. KEY FINDINGS: Compared with the NC group, the MANF levels in the HLD and T2D groups increased significantly. After ER stress induced by GLU, PA, and TG, cell viability decreased, while MANF, c-cas3, ERS, and autophagy-related proteins increased, which was related to the concentration of GLU, PA, and TG. Compared with the BSA group, the number of mitochondria and autophagosomes in the PA group increased and the mitochondria were damaged. In the PA and TG plus CQ groups, the effect was further exaggerated. But after co-treatment with LRG, the effects of GLU, PA, and TG were attenuated. SIGNIFICANCE: LRG protects islet ß cells from ER stress by upregulating MANF to promote autophagy turnover.


Assuntos
Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Liraglutida/farmacologia , Fatores de Crescimento Neural/genética , Animais , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Hiperlipidemias/tratamento farmacológico , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/metabolismo , Camundongos , Regulação para Cima/efeitos dos fármacos
17.
Proc Natl Acad Sci U S A ; 117(16): 8912-8923, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32253320

RESUMO

Pancreatic islets regulate glucose homeostasis through coordinated actions of hormone-secreting cells. What underlies the function of the islet as a unit is the close approximation and communication among heterogeneous cell populations, but the structural mediators of islet cellular cross talk remain incompletely characterized. We generated mice specifically lacking ß-cell primary cilia, a cellular organelle that has been implicated in regulating insulin secretion, and found that the ß-cell cilia are required for glucose sensing, calcium influx, insulin secretion, and cross regulation of α- and δ-cells. Protein expression profiling in islets confirms perturbation in these cellular processes and reveals additional targets of cilia-dependent signaling. At the organism level, the deletion of ß-cell cilia disrupts circulating hormone levels, impairs glucose homeostasis and fuel usage, and leads to the development of diabetes. Together, these findings demonstrate that primary cilia not only orchestrate ß-cell-intrinsic activity but also mediate cross talk both within the islet and from islets to other metabolic tissues, thus providing a unique role of cilia in nutrient metabolism and insight into the pathophysiology of diabetes.


Assuntos
Cílios/metabolismo , Diabetes Mellitus/patologia , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Cálcio/metabolismo , Comunicação Celular/fisiologia , Cílios/genética , Cílios/patologia , Diabetes Mellitus/genética , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Feminino , Células Secretoras de Glucagon/metabolismo , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologia
18.
Nat Commun ; 11(1): 1822, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286278

RESUMO

B cell dysfunction due to obesity can be associated with alterations in the levels of micro-RNAs (miRNAs). However, the role of miRNAs in these processes remains elusive. Here, we show that miR-802 is increased in the pancreatic islets of obese mouse models and demonstrate that inducible transgenic overexpression of miR-802 in mice causes impaired insulin transcription and secretion. We identify Foxo1 as a transcription factor of miR-802 promoting its transcription, and NeuroD1 and Fzd5 as targets of miR-802-dependent silencing. Repression of NeuroD1 in ß cell and primary islets impairs insulin transcription and reduction of Fzd5 in ß cell, which, in turn, impairs Ca2+ signaling, thereby repressing calcium influx and decreasing insulin secretion. We functionally create a novel network between obesity and ß cell dysfunction via miR-802 regulation. Elucidation of the impact of obesity on microRNA expression can broaden our understanding of pathophysiological development of diabetes.


Assuntos
Secreção de Insulina/genética , Insulina/genética , MicroRNAs/metabolismo , Obesidade/genética , Transcrição Genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Dieta Hiperlipídica , Modelos Animais de Doenças , Proteína Forkhead Box O1/metabolismo , Receptores Frizzled/metabolismo , Deleção de Genes , Inativação Gênica , Insulina/metabolismo , Resistência à Insulina/genética , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , MicroRNAs/genética , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Regulação para Cima/genética
19.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140426, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32272193

RESUMO

Lipotoxicity, an important factor in the pathogenesis of diabetes, leads to defective ß-cell proliferation and increased apoptosis. Glucagon-like peptide-1 (GLP-1) analogs, which are used to treat type 2 diabetes, reduce endoplasmic reticulum stress and inflammation in pancreatic ß-cells and improve their survival. However, their effects on the heat shock response (HSR) have not been elucidated yet. We investigated whether the GLP-1 analog exendin-4 exerts its protective effect by modulating the HSR and mitogen-activated protein kinases (MAPKs) in BTC-6 mouse pancreatic cells under palmitic acid (PA) stress. Expression patterns were analyzed using mass spectrometry, Western blotting, and qRT-PCR in the presence of 250 or 400 µM PA and 100 nM exendin-4. Additionally, we measured MAPK expression and phosphorylation using qRT-PCR and Western blotting, respectively. Upregulation of heat shock protein (HSP), notably HSP72, in the presence of PA, was attenuated by exendin-4. Despite the absence of global effects on the HSR system, exendin-4 attenuated the expression of other non-classical HSPs (GRP94, DNAJA1, and DNAJB6) in the presence of PA. Regarding MAPKs, only extracellular signal-regulated kinase (ERK) phosphorylation was highly increased by exendin-4 in both the presence and absence of PA. Furthermore, exendin-4 significantly alleviated PA-induced cell death, which was further confirmed with proteomics analysis where key cellular functions, including cellular growth, assembly, and organization, were improved by exendin-4 treatment. Thus, our results expand the protective role of GLP-1 analogs to include other cellular mechanisms involved in restoring normal ß-cell homeostasis.


Assuntos
Exenatida/metabolismo , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Proteínas de Choque Térmico HSP72/metabolismo , Células Secretoras de Insulina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Exenatida/farmacologia , Proteínas de Choque Térmico HSP40 , Glicoproteínas de Membrana , Camundongos , Chaperonas Moleculares , Fosforilação , Substâncias Protetoras/farmacologia , Mapas de Interação de Proteínas , Regulação para Cima
20.
Nat Commun ; 11(1): 1950, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327658

RESUMO

BDNF signaling in hypothalamic circuitries regulates mammalian food intake. However, whether BDNF exerts metabolic effects on peripheral organs is currently unknown. Here, we show that the BDNF receptor TrkB.T1 is expressed by pancreatic ß-cells where it regulates insulin release. Mice lacking TrkB.T1 show impaired glucose tolerance and insulin secretion. ß-cell BDNF-TrkB.T1 signaling triggers calcium release from intracellular stores, increasing glucose-induced insulin secretion. Additionally, BDNF is secreted by skeletal muscle and muscle-specific BDNF knockout phenocopies the ß-cell TrkB.T1 deletion metabolic impairments. The finding that BDNF is also secreted by differentiated human muscle cells and induces insulin secretion in human islets via TrkB.T1 identifies a new regulatory function of BDNF on metabolism that is independent of CNS activity. Our data suggest that muscle-derived BDNF may be a key factor mediating increased glucose metabolism in response to exercise, with implications for the treatment of diabetes and related metabolic diseases.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Cálcio/metabolismo , Células Cultivadas , Glucose/metabolismo , Intolerância à Glucose , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor trkB/química , Receptor trkB/genética , Receptor trkB/metabolismo , Transdução de Sinais
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