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1.
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
2.
J Steroid Biochem Mol Biol ; 201: 105694, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32437964

RESUMO

Progesterone receptor (PgR) inhibits cell proliferation in pancreatic neuroendocrine neoplasms (PanNEN). In non-neoplastic pancreas, loss of PgR induces ß-cell proliferation and insulin production. However, detailed association between PgR and insulin producing PanNENs is poorly understood. Insulin, proinsulin, and PgR were immunolocalized in 82 PanNENs (54 non-functioning PanNENs: NF-PanNENs and 28 insulinomas). The status of immunoreactivity was compared to the clinicopathological factors of the patients. Immunoreactivity was also confirmed by employing the double-immunohistochemistry. These results were also compared with those in non-neoplastic Langerhans islets. PgR immunoreactivity was significantly higher in insulinomas than that in NF-PanNENs (p < 0.001). Insulin and proinsulin immunoreactivity was also detected in 20 (37 %) of (single cell) insulin positive NFs (Inspos-NF-PanNEN), in which PgR expression was higher than in insulin negative NF-PanNENs (Insneg-NF-PanNEN, p = 0.03). The ratio of PgR-insulin double positive cells to overall insulin positive cells, as well as PgR-proinsulin double positive cells to proinsulin positive cells, was detected to the same degree in insulinoma (PgR-insulin 70 %, PgR-proinsulin 66 %), Inspos-NF-PanNENs (PgR-insulin 65 %, PgR-proinsulin 68 %) and normal islet (PgR-insulin 80 %, PgR-proinsulin 72 %). PgR and insulin expressing cells colocalize in tumor cells of the PanNENs regardless of the hormone-related symptoms of the patients. Inhibitory effect of PgR on tumor cells might be associated with the favourable clinical outcome of insulinoma patients.


Assuntos
Tumores Neuroendócrinos/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Progesterona/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proinsulina/metabolismo
3.
PLoS One ; 15(5): e0233502, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433667

RESUMO

The environment within the Endoplasmic Reticulum (ER) influences Insulin biogenesis. In particular, ER stress may contribute to the development of Type 2 Diabetes (T2D) and Cystic Fibrosis Related Diabetes (CFRD), where evidence of impaired Insulin processing, including elevated secreted Proinsulin/Insulin ratios, are observed. Our group has established the role of a novel ER chaperone ERp29 (ER protein of 29 kDa) in the biogenesis of the Epithelial Sodium Channel, ENaC. The biogenesis of Insulin and ENaC share may key features, including their potential association with COP II machinery, their cleavage into a more active form in the Golgi or later compartments, and their ability to bypass such cleavage and remain in a less active form. Given these similarities we hypothesized that ERp29 is a critical factor in promoting the efficient conversion of Proinsulin to Insulin. Here, we confirmed that Proinsulin associates with the COP II vesicle cargo recognition component, Sec24D. When Sec24D expression was decreased, we observed a corresponding decrease in whole cell Proinsulin levels. In addition, we found that Sec24D associates with ERp29 in co-precipitation experiments and that ERp29 associates with Proinsulin in co-precipitation experiments. When ERp29 was overexpressed, a corresponding increase in whole cell Proinsulin levels was observed, while depletion of ERp29 decreased whole cell Proinsulin levels. Together, these data suggest a potential role for ERp29 in regulating Insulin biosynthesis, perhaps in promoting the exit of Proinsulin from the ER via Sec24D/COPII vesicles.


Assuntos
Proteínas de Choque Térmico/fisiologia , Insulina/biossíntese , Proteínas de Transporte Vesicular/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Fibrose Cística/complicações , Diabetes Mellitus Tipo 2 , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Proinsulina/metabolismo , Transporte Proteico
4.
Am J Physiol Endocrinol Metab ; 318(6): E892-E900, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32255680

RESUMO

Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when ß-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard proteasome, while the roles of other proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active proteasome subunit, namely, the inducible ß5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of ß5i-containing intermediate proteasomes was significantly increased in these cells, as was ß5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon ß5i small interfering RNA-mediated knockdown. Finally, the fraction of ß-cells expressing the ß5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that ß5i is an inducible proteasome subunit dedicated to the degradation of mishandled proinsulin.


Assuntos
Diabetes Mellitus Tipo 2/genética , Estresse do Retículo Endoplasmático/genética , Degradação Associada com o Retículo Endoplasmático/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Proinsulina/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Animais , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Ilhotas Pancreáticas/metabolismo , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Ratos
5.
Diabetes ; 69(5): 954-964, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139596

RESUMO

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene-induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in ß-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.


Assuntos
Insulina/síntese química , Insulina/metabolismo , Proinsulina/química , Proinsulina/metabolismo , Animais , Linhagem Celular , Humanos , Insulina/química , Insulina/genética , Ilhotas Pancreáticas , Camundongos , Modelos Moleculares , Mutação , Proinsulina/genética , Conformação Proteica
6.
Am J Cardiol ; 125(5): 678-684, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31948661

RESUMO

Insulin resistance early after acute myocardial infarction is associated with increased heart failure and mortality. OMEGA-REMODEL was a prospective double-blind 1:1 randomized control trial of patients with AMI. We reported that 6-month treatment with omega-3 fatty acid (O-3FA) 4 g/day attenuated cardiac remodeling accompanied by reduction in inflammation. We hypothesized that insulin resistance modifies the therapeutic effect of O-3FA on post-MI cardiac remodeling. The OMEGA-REMODEL study group was dichotomized according to cohort- and gender-specific median cutoff value of leptin-to-adiponectin ratio (LAR) at baseline (LAR-Hi vs LAR-Lo). Mixed model regression analyses were used to evaluate effect modification of O-3FA on reduction of left ventricular end-systolic volume index (LVESVI) by LAR status. Baseline LAR was evaluated on 325 patients (59 ± 11 years, 81% male). A total of 168 patients were categorized in LAR-Lo, and 157 in LAR-Hi. O-3FA treatment resulted in significant LVESVI reduction in patients with LAR-Lo but not with LAR-Hi (p = 0.0002 vs 0.66, respectively). Mixed model regression analysis showed significant modification of LAR on O-3FA's treatment effect in attenuating LVESVI (p = 0.021). In conclusion, this post-hoc efficacy analysis suggests that LAR status significantly modified O-3FA's treatment effect in attenuating cardiac remodeling. During the convalescent phase of acute infarct healing, patients with lower insulin resistance estimated by LAR appear to derive more therapeutic response from O-3FA toward improvement of LVESVI.


Assuntos
Adiponectina/metabolismo , Ácidos Graxos Ômega-3/uso terapêutico , Resistência à Insulina , Leptina/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Remodelação Ventricular , Idoso , Peptídeo C/metabolismo , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/metabolismo , Prognóstico , Proinsulina/metabolismo , Volume Sistólico , Resultado do Tratamento
7.
Nat Commun ; 11(1): 296, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941883

RESUMO

Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in ß cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t6A37 in tRNALysUUU to ms2t6A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms2t6A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2-/- ß cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.


Assuntos
Insulina/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Ferro/metabolismo , RNA de Transferência de Lisina/metabolismo , tRNA Metiltransferases/metabolismo , Animais , Linhagem Celular Tumoral , Intolerância à Glucose/genética , Homeostase , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Insulinoma/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteínas Ferro-Enxofre/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proinsulina/genética , Proinsulina/metabolismo , RNA de Transferência de Lisina/genética , Ratos , Resposta a Proteínas não Dobradas/genética , tRNA Metiltransferases/genética
8.
J Cell Biol ; 219(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31895406

RESUMO

Escape of large macromolecular complexes from the endoplasmic reticulum (ER), such as a viral particle or cellular aggregate, likely induces mechanical stress initiated on the luminal side of the ER membrane, which may threaten its integrity. How the ER responds to this threat remains unknown. Here we demonstrate that the cytosolic leaflet ER morphogenic protein reticulon (RTN) protects ER membrane integrity when polyomavirus SV40 escapes the ER to reach the cytosol en route to infection. SV40 coopts an intrinsic RTN function, as we also found that RTN prevents membrane damage during ER escape of a misfolded proinsulin aggregate destined for lysosomal degradation via ER-phagy. Our studies reveal that although ER membrane integrity may be threatened during ER escape of large macromolecular protein complexes, the action of RTN counters this, presumably by deploying its curvature-inducing activity to provide membrane flexibility and stability to limit mechanical stress imposed on the ER membrane.


Assuntos
Retículo Endoplasmático/genética , Complexos Multiproteicos/genética , Proinsulina/genética , Estresse Mecânico , Citosol/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/genética , Proteínas de Membrana/genética , Complexos Multiproteicos/metabolismo , Proinsulina/metabolismo , Agregados Proteicos/genética
9.
Diabetes ; 69(1): 67-82, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31624142

RESUMO

Insulin secretion is tightly regulated by membrane trafficking. RILP (Rab7 interacting lysosomal protein) regulates the endocytic trafficking, but its role in insulin secretion has not been investigated. In this study, we found that overexpression of RILP inhibited insulin secretion in both the ß-cell lines and freshly isolated islets. Consequently, the expression of RILP in islets suppressed the ability to recover the glucose homeostasis in type 1 diabetes mice upon transplantation. Of physiological relevance is that RILP expression was upregulated in the diabetic mouse islets. Mechanistically, overexpression of RILP induced insulin granule clustering, decreased the number of proinsulin-containing granules in ß-cells, and significantly promoted proinsulin degradation. Conversely, RILP depletion sustained proinsulin and increased insulin secretion. The proinsulin degradation induced by RILP expression was inhibited by lysosomal inhibitors and was Rab7-dependent. Finally, we showed that RILP interacts with insulin granule-associated Rab26 to restrict insulin secretion. This study presents a new pathway regulating insulin secretion and mechanically demonstrates a novel function of RILP in modulating insulin secretion through mediating the lysosomal degradation of proinsulin.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Lisossomos/metabolismo , Proinsulina/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteólise , Ratos , Ratos Sprague-Dawley
10.
J Diabetes Investig ; 11(1): 75-79, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31222973

RESUMO

AIMS/INTRODUCTION: We investigated associations between glucose tolerance and ß-cell function using a series of estimation methods in a population-based study. MATERIALS AND METHODS: Data from the Dynamics of Lifestyle and Neighborhood Community on Health Study were analyzed. A total of 489 participants (263 women) were divided into three groups: normal glucose tolerance (NGT), prediabetes (PDM) and diabetes group. We estimated ß-cell function by the homeostasis model assessment of ß-cell function, proinsulin level (PI), C-peptide index, proinsulin-to-C-peptide ratio (PI/CPR) and proinsulin-to-insulin ratio. Because data on all five parameters of ß-cell function showed skewed distributions, the values of these parameters were normalized by natural logarithmic (ln) transformation. Next, the association between glucose tolerance and ß-cell function among participants without diabetes was examined. In this analysis, glucose tolerance was assessed based on glycated hemoglobin levels. RESULTS: In the crude analysis, ln(PI) and ln(PI/CPR) were significantly higher in the diabetes group than those in the PDM and NGT groups, and these parameters were significantly higher in the PDM group than in the NGT group. Only ln(PI) in the PDM group was significantly higher compared with that in the NGT group after adjustment for age, sex and body mass index (ln[PI]: PDM group 2.38 pmol/L, 95% confidence interval 2.29-2.47 pmol/L; NGT group 2.17 pmol/L, 95% confidence interval 2.12-2.22 pmol/L; P < 0.05). In addition, ln(PI) levels were significantly and positively correlated with glycated hemoglobin quartile in participants without diabetes. CONCLUSIONS: Our results showed that PI was the most sensitive to reflect glucose intolerance.


Assuntos
Biomarcadores/metabolismo , Glicemia/análise , Peptídeo C/metabolismo , Intolerância à Glucose/diagnóstico , Insulina/metabolismo , Proinsulina/metabolismo , Adulto , Idoso , Estudos Transversais , Feminino , Seguimentos , Intolerância à Glucose/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
11.
Sci Rep ; 9(1): 17451, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767891

RESUMO

Previous studies have demonstrated the important role of kisspeptin in impaired glucose-stimulated insulin secretion (GSIS). In addition, it was reported that the activation of autophagy in pancreatic ß-cells decreases insulin secretion by selectively degrading insulin granules. However, it is currently unknown whether kisspeptin suppresses GSIS in ß-cells by activating autophagy. To investigate the involvement of autophagy in kisspeptin-regulated insulin secretion, we overexpressed Kiss1 in NIT-1 cells to mimic the long-term exposure of pancreatic ß-cells to kisspeptin during type 2 diabetes (T2D). Interestingly, our data showed that although kisspeptin potently decreases the intracellular proinsulin and insulin ((pro)insulin) content and insulin secretion of NIT-1 cells, autophagy inhibition using bafilomycin A1 and Atg5 siRNAs only rescues basal insulin secretion, not kisspeptin-impaired GSIS. We also generated a novel in vivo model to investigate the long-term exposure of kisspeptin by osmotic pump. The in vivo data demonstrated that kisspeptin lowers GSIS and (pro)insulin levels and also activated pancreatic autophagy in mice. Collectively, our data demonstrated that kisspeptin suppresses both GSIS and non-glucose-stimulated insulin secretion of pancreatic ß-cells, but only non-glucose-stimulated insulin secretion depends on activated autophagic degradation of (pro)insulin. Our study provides novel insights for the development of impaired insulin secretion during T2D progression.


Assuntos
Autofagia/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Kisspeptinas/fisiologia , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/fisiopatologia , Genes Reporter , Glucose/farmacologia , Kisspeptinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Proinsulina/metabolismo , Proteínas Recombinantes/metabolismo , Transfecção
12.
Endocrinology ; 160(11): 2759-2772, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504428

RESUMO

Thyroid hormones (THs) are crucial regulators of glucose metabolism and insulin sensitivity. Moreover, inactivating mutations in type 2 deiodinase (DIO2), the major TH-activating enzyme, have been associated with type 2 diabetes mellitus in both humans and mice. We studied the link between Dio2 deficiency and glucose homeostasis in fasted males of two different Dio2 knockout (KO) zebrafish lines. Young adult Dio2KO zebrafish (6 to 9 months) were hyperglycemic. Both insulin and glucagon expression were increased, whereas ß and α cell numbers in the main pancreatic islet were similar to those in wild-types. Insulin receptor expression in skeletal muscle was decreased at 6 months, accompanied by a strong downregulation of hexokinase and pyruvate kinase expression. Blood glucose levels in Dio2KO zebrafish, however, normalized around 1 year of age. Older mutants (18 to 24 months) were normoglycemic, and increased insulin and glucagon expression was accompanied by a prominent increase in pancreatic islet size and ß and α cell numbers. Older Dio2KO zebrafish also showed strongly decreased expression of glucagon receptors in the gastrointestinal system as well as decreased expression of glucose transporters GLUT2 and GLUT12, glucose-6-phosphatase, and glycogen synthase 2. This study shows that Dio2KO zebrafish suffer from transient hyperglycemia, which is counteracted with increasing age by a prominent hyperplasia of the endocrine pancreas together with decreases in hepatic glucagon sensitivity and intestinal glucose uptake. Further research on the mechanisms allowing compensation in older Dio2KO zebrafish may help to identify new therapeutic targets for (TH deficiency-related) hyperglycemia.


Assuntos
Glucose/metabolismo , Iodeto Peroxidase/deficiência , Envelhecimento/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Homeostase , Hiperglicemia/genética , Iodeto Peroxidase/genética , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Masculino , Proglucagon/metabolismo , Proinsulina/metabolismo , Receptor de Insulina/metabolismo , Receptores de Glucagon/metabolismo , Peixe-Zebra
13.
Transl Res ; 213: 90-99, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31442418

RESUMO

We recently described the persistence of detectable serum proinsulin in a large majority of individuals with longstanding type 1 diabetes (T1D), including individuals with undetectable serum C-peptide. Here, we sought to further explore the mechanistic etiologies of persistent proinsulin secretion in T1D at the level of the islet, using tissues obtained from human donors. Immunostaining for proinsulin and insulin was performed on human pancreatic sections from the Network for Pancreatic Organ Donors with Diabetes (nPOD) collection (n = 24). Differential proinsulin processing enzyme expression was analyzed using mass spectrometry analysis of human islets isolated from pancreatic sections with laser capture microdissection (n = 6). Proinsulin processing enzyme mRNA levels were assessed using quantitative real-time PCR in isolated human islets (n = 10) treated with or without inflammatory cytokines. Compared to nondiabetic controls, immunostaining among a subset (4/9) of insulin positive T1D donor islets revealed increased numbers of cells with proinsulin-enriched, insulin-poor staining. T1D donor islets also exhibited increased proinsulin fluorescence intensity relative to insulin fluorescence intensity. Laser capture microdissection followed by mass spectrometry revealed reductions in the proinsulin processing enzymes prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE) in T1D donors. Twenty-four hour treatment of human islets with inflammatory cytokines reduced mRNA expression of the processing enzymes PC1/3, PC2, and CPE. Taken together, these data provide new mechanistic insight into altered proinsulin processing in long-duration T1D and suggest that reduced ß cell prohormone processing is associated with proinflammatory cytokine-induced reductions in proinsulin processing enzyme expression.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/metabolismo , Proinsulina/metabolismo , Adulto , Feminino , Humanos , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
14.
SLAS Technol ; 24(6): 569-582, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31288593

RESUMO

During process development, the experimental search space is defined by the number of experiments that can be performed in specific time frames but also by its sophistication (e.g., inputs, sensors, sampling frequency, analytics). High-throughput liquid-handling stations can perform a large number of automated experiments in parallel. Nevertheless, the experimental data sets that are obtained are not always relevant for development of industrial bioprocesses, leading to a high rate of failure during scale-up. We present an automated mini bioreactor platform that enables parallel cultivations in the milliliter scale with online monitoring and control, well-controlled conditions, and advanced feeding strategies similar to industrial processes. The combination of two liquid handlers allows both automated mini bioreactor operation and at-line analysis in parallel. A central database enables end-to-end data exchange and fully integrated device and process control. A model-based operation algorithm allows for the accurate performance of complex cultivations for scale-down studies and strain characterization via optimal experimental redesign, significantly increasing the reliability and transferability of data throughout process development. The platform meets the tradeoff between experimental throughput and process control and monitoring comparable to laboratory-scale bioreactors.


Assuntos
Automação Laboratorial/normas , Reatores Biológicos , Escherichia coli/crescimento & desenvolvimento , Robótica/instrumentação , Algoritmos , Biotecnologia , Escherichia coli/genética , Ensaios de Triagem em Larga Escala , Humanos , Isopropiltiogalactosídeo , Miniaturização , Proinsulina/genética , Proinsulina/metabolismo , Software
15.
FASEB J ; 33(10): 11338-11348, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31311313

RESUMO

Insulin resistance in classic insulin-responsive tissues is a hallmark of type 2 diabetes (T2D). However, the pathologic significance of ß-cell insulin resistance and the underlying mechanisms contributing to defective insulin signaling in ß cells remain largely unknown. Emerging evidence indicates that proinsulin misfolding is not only the molecular basis of mutant INS-gene-induced diabetes of youth (MIDY) but also an important contributor in the development and progression of T2D. However, the molecular basis of ß-cell failure caused by misfolded proinsulin is still incompletely understood. Herein, using Akita mice expressing diabetes-causing mutant proinsulin, we found that misfolded proinsulin abnormally interacted with the precursor of insulin receptor (ProIR) in the endoplasmic reticulum (ER), impaired ProIR maturation to insulin receptor (IR), and decreased insulin signaling in ß cells. Importantly, using db/db insulin-resistant mice, we found that oversynthesis of proinsulin led to an increased proinsulin misfolding, which resulted in impairments of ProIR processing and insulin signaling in ß cells. These results reveal for the first time that misfolded proinsulin can interact with ProIR in the ER, impairing intracellular processing of ProIR and leading to defective insulin signaling that may contribute to ß-cell failure in both MIDY and T2D.-Liu, S., Li, X., Yang, J., Zhu, R., Fan, Z., Xu, X., Feng, W., Cui, J., Sun, J., Liu, M. Misfolded proinsulin impairs processing of precursor of insulin receptor and insulin signaling in ß cells.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proinsulina/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dobramento de Proteína , Receptor de Insulina/metabolismo
16.
Elife ; 82019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31184302

RESUMO

Biosynthesis of insulin - critical to metabolic homeostasis - begins with folding of the proinsulin precursor, including formation of three evolutionarily conserved intramolecular disulfide bonds. Remarkably, normal pancreatic islets contain a subset of proinsulin molecules bearing at least one free cysteine thiol. In human (or rodent) islets with a perturbed endoplasmic reticulum folding environment, non-native proinsulin enters intermolecular disulfide-linked complexes. In genetically obese mice with otherwise wild-type islets, disulfide-linked complexes of proinsulin are more abundant, and leptin receptor-deficient mice, the further increase of such complexes tracks with the onset of islet insulin deficiency and diabetes. Proinsulin-Cys(B19) and Cys(A20) are necessary and sufficient for the formation of proinsulin disulfide-linked complexes; indeed, proinsulin Cys(B19)-Cys(B19) covalent homodimers resist reductive dissociation, highlighting a structural basis for aberrant proinsulin complex formation. We conclude that increased proinsulin misfolding via disulfide-linked complexes is an early event associated with prediabetes that worsens with ß-cell dysfunction in type two diabetes.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Proinsulina/química , Dobramento de Proteína , Animais , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Progressão da Doença , Dissulfetos/química , Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Proinsulina/genética , Proinsulina/metabolismo , Receptores para Leptina/deficiência , Receptores para Leptina/genética
17.
Elife ; 82019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31184304

RESUMO

Regulated proinsulin biosynthesis, disulfide bond formation and ER redox homeostasis are essential to prevent Type two diabetes. In ß cells, protein disulfide isomerase A1 (PDIA1/P4HB), the most abundant ER oxidoreductase of over 17 members, can interact with proinsulin to influence disulfide maturation. Here we find Pdia1 is required for optimal insulin production under metabolic stress in vivo. ß cell-specific Pdia1 deletion in young high-fat diet fed mice or aged mice exacerbated glucose intolerance with inadequate insulinemia and increased the proinsulin/insulin ratio in both serum and islets compared to wildtype mice. Ultrastructural abnormalities in Pdia1-null ß cells include diminished insulin granule content, ER vesiculation and distention, mitochondrial swelling and nuclear condensation. Furthermore, Pdia1 deletion increased accumulation of disulfide-linked high molecular weight proinsulin complexes and islet vulnerability to oxidative stress. These findings demonstrate that PDIA1 contributes to oxidative maturation of proinsulin in the ER to support insulin production and ß cell health.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Obesidade/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proinsulina/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Dilatação Mitocondrial , Obesidade/etiologia , Obesidade/genética , Estresse Oxidativo , Pró-Colágeno-Prolina Dioxigenase/genética , Isomerases de Dissulfetos de Proteínas/genética
18.
Mol Cell Endocrinol ; 493: 110470, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158417

RESUMO

Endoplasmic reticulum (ER) homeostasis is essential for cell function. Increasing evidence indicates that, efficient protein ER export is important for ER homeostasis. However, the consequence of impaired ER export remains largely unknown. Herein, we found that defective ER protein transport caused by either Sar1 mutants or brefeldin A impaired proinsulin oxidative folding in the ER of ß-cells. Misfolded proinsulin formed aberrant disulfide-linked dimers and high molecular weight proinsulin complexes, and induced ER stress. Limiting proinsulin load to the ER alleviated ER stress, indicating that misfolded proinsulin is a direct cause of ER stress. This study revealed significance of efficient ER export in maintaining ER protein homeostasis and native folding of proinsulin. Given the fact that proinsulin misfolding plays an important role in diabetes, this study suggests that enhancing ER export may be a potential therapeutic target to prevent/delay ß-cell failure caused by proinsulin misfolding and ER stress.


Assuntos
Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Proinsulina/química , Proinsulina/metabolismo , Adulto , Animais , Brefeldina A/farmacologia , Células Cultivadas , Retículo Endoplasmático/química , Estresse do Retículo Endoplasmático , Feminino , Humanos , Células Secretoras de Insulina/citologia , Camundongos , Pessoa de Meia-Idade , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico
19.
Gene ; 706: 52-61, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31039435

RESUMO

Insulin is a peptide hormone responsible for stable glycemia, is entirely secreting from pancreatic ß cells at the core of glucose homeostatic regulation. Upon synthesis as preproinsulin on rough endoplasmic reticulum (rER), proinsulin is directed to trans Golgi apparatus. Subsequently, proinsulin packaging into secretory granules occurs in a dynamic and highly efficient process. During maturation stage of secretory granules, proinsulin undergoes cleavage and produces insulin and C-peptide upon acidification of the granules due to the activation of ATP-deriven proton pump. Fusion of the insulin containing secretory granules with the plasma membranes takes place after an increase in intracellular Ca2+. Finally, insulin is co-secreting with other components that are present in the secretory granules, including C-peptide, ATP, γ-aminobutyric acid (GABA), ghrelin and amylin. The other accompanying components of the insulin vesicles play important roles in the insulin secretion, insulin receptor activation and other homeostatic effects.. Responding to the glucose stimulation or increases in cytoplasmic Ca2+ levels, insulin secretion is immediately starts. Whereas, the second phase of insulin secretion is slow and continued, which reaches a plateau within 1-3 hours and lasts for longer period. In contrast to the first phase, the second phase of insulin secretion is independent of the extracellular glucose level. Finally, sequential or compound exocytosis of insulin is repressed to prevent sugar crash arising from excessive and sudden insulin secretion. In this paper we have reviewed the recent progress of molecular scenarios which are behind insulin biogenesis, intracellular sorting and exocytosis events.


Assuntos
Exocitose/fisiologia , Insulina/metabolismo , Insulina/fisiologia , Animais , Transporte Biológico , Glicemia/metabolismo , Glucose/metabolismo , Complexo de Golgi/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proinsulina/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico/fisiologia
20.
Sci Rep ; 9(1): 6952, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061431

RESUMO

Persistent hyperglycemia is causally associated with pancreatic ß-cell dysfunction and loss of pancreatic insulin. Glucose normally enhances ß-cell excitability through inhibition of KATP channels, opening of voltage-dependent calcium channels, increased [Ca2+]i, which triggers insulin secretion. Glucose-dependent excitability is lost in islets from KATP-knockout (KATP-KO) mice, in which ß-cells are permanently hyperexcited, [Ca2+]i, is chronically elevated and insulin is constantly secreted. Mouse models of human neonatal diabetes in which KATP gain-of-function mutations are expressed in ß-cells (KATP-GOF) also lose the link between glucose metabolism and excitation-induced insulin secretion, but in this case KATP-GOF ß-cells are chronically underexcited, with permanently low [Ca2+]i and lack of glucose-dependent insulin secretion. We used KATP-GOF and KATP-KO islets to examine the role of altered-excitability in glucotoxicity. Wild-type islets showed rapid loss of insulin content when chronically incubated in high-glucose, an effect that was reversed by subsequently switching to low glucose media. In contrast, hyperexcitable KATP-KO islets lost insulin content in both low- and high-glucose, while underexcitable KATP-GOF islets maintained insulin content in both conditions. Loss of insulin content in chronic excitability was replicated by pharmacological inhibition of KATP by glibenclamide, The effects of hyperexcitable and underexcitable islets on glucotoxicity observed in in vivo animal models are directly opposite to the effects observed in vitro: we clearly demonstrate here that in vitro, hyperexcitability is detrimental to islets whereas underexcitability is protective.


Assuntos
Membrana Celular/patologia , Glucose/farmacologia , Células Secretoras de Insulina/patologia , Insulina/metabolismo , Canais KATP/fisiologia , Proinsulina/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Edulcorantes/farmacologia
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