Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | https://hdl.handle.net/11600/64095 |
Resumo: | Purpose: Diabetic Keratopathy affects approximately 70% of diabetic patients. Mitochondrial dysfunction is considered a central feature underlying diabetic complications. The purpose of this study was characterized mitochondrial alterations in telomerase-immortalized human corneal epithelial (hTCEpi) cells exposed to hyperglycemic and hyperosmotic stress, also the changes in primary cultured diabetic human corneal epithelial cells (HCECs). Methods: To determine the effects of diabetes on mitochondria and metabolism, donor human corneas from diabetics were obtained from Tissue Transplant Services at UT Southwestern Medical Center. Primary cultures were generated using an established lab protocol. HCECs and hTCEpi cells were cultured in a defined serum-free keratinocyte growth media containing 6 mM of glucose. Cells were cultured for 24h, 3, 5, 7, 9 or 14 days in media with additional 19mM of glucose. Cells supplemented with 19 mM mannitol were used as an osmotic control. Mitochondrial morphology and polarization were assessed using MitoTracker Green, TMRE, and JC-1. Metabolic changes were quantified in real time using a Seahorse metabolic flux analyzer. Cell cycle were determined by staining with Propidium Iodide and analyzed using the Celigo imaging cytometer. Cell migration was evaluated by the wound healing assay and the cells proliferation was characterized by daily cell count for 7 or 14 days. Results: The HCECs cells from diabetic patients showed alterations in mitochondrial polarization and severe fragmentation. The hTCEpi cells exposed to acute hyperglycemia showed no abnormalities in cell cycle control and metabolism. Chronic exposure of hTCEpi cells to hyperglycemia (14 days) triggered cell cycle arrest in G0/G1 phase. At 14 days there was also a decrease in cell migration in the groups exposed to hyperglycemic and hyperosmotic stress. However, cell proliferation remained unchanged after both 7 and 14 days. Using JC-1, cells cultured in high glucose for 14 days showed a loss of polarization and mitochondrial fragmentation but this wasn’t significant in comparison to hyperosmolar stress. Metabolic activity was unchanged in cells cultured in high glucose for 24 hours. By day 3, cells showed a measurable drop in spare respiratory capacity that remained decreased through day 14. By day 5, glycolysis (ECAR) began to decrease and remained low through day 9. At days 7 and 9, OCR was increased, shifting cells towards a more respiratory phenotype. By day 14 OCR decreased and ECAR increased to normoglycemic levels and the phenotype became glycolytic again. There was also a sharp drop in others mitochondrial metabolic parameters. Discussion: Chronic, not acute, exposure to high glucose negatively impacts mitochondrial structure and function. The decline in mitochondrial activity was associated with a recovery of glycolysis, indicative of metabolic adaptation. |
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Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanasDiabetes mellitus: metabolic and mitochondrial changes in corneal epithelial cells.Diabetes MellitusCeratopatia diabéticaHiperglicemiaEpitélio corneanoDisfunção mitocondrialPurpose: Diabetic Keratopathy affects approximately 70% of diabetic patients. Mitochondrial dysfunction is considered a central feature underlying diabetic complications. The purpose of this study was characterized mitochondrial alterations in telomerase-immortalized human corneal epithelial (hTCEpi) cells exposed to hyperglycemic and hyperosmotic stress, also the changes in primary cultured diabetic human corneal epithelial cells (HCECs). Methods: To determine the effects of diabetes on mitochondria and metabolism, donor human corneas from diabetics were obtained from Tissue Transplant Services at UT Southwestern Medical Center. Primary cultures were generated using an established lab protocol. HCECs and hTCEpi cells were cultured in a defined serum-free keratinocyte growth media containing 6 mM of glucose. Cells were cultured for 24h, 3, 5, 7, 9 or 14 days in media with additional 19mM of glucose. Cells supplemented with 19 mM mannitol were used as an osmotic control. Mitochondrial morphology and polarization were assessed using MitoTracker Green, TMRE, and JC-1. Metabolic changes were quantified in real time using a Seahorse metabolic flux analyzer. Cell cycle were determined by staining with Propidium Iodide and analyzed using the Celigo imaging cytometer. Cell migration was evaluated by the wound healing assay and the cells proliferation was characterized by daily cell count for 7 or 14 days. Results: The HCECs cells from diabetic patients showed alterations in mitochondrial polarization and severe fragmentation. The hTCEpi cells exposed to acute hyperglycemia showed no abnormalities in cell cycle control and metabolism. Chronic exposure of hTCEpi cells to hyperglycemia (14 days) triggered cell cycle arrest in G0/G1 phase. At 14 days there was also a decrease in cell migration in the groups exposed to hyperglycemic and hyperosmotic stress. However, cell proliferation remained unchanged after both 7 and 14 days. Using JC-1, cells cultured in high glucose for 14 days showed a loss of polarization and mitochondrial fragmentation but this wasn’t significant in comparison to hyperosmolar stress. Metabolic activity was unchanged in cells cultured in high glucose for 24 hours. By day 3, cells showed a measurable drop in spare respiratory capacity that remained decreased through day 14. By day 5, glycolysis (ECAR) began to decrease and remained low through day 9. At days 7 and 9, OCR was increased, shifting cells towards a more respiratory phenotype. By day 14 OCR decreased and ECAR increased to normoglycemic levels and the phenotype became glycolytic again. There was also a sharp drop in others mitochondrial metabolic parameters. Discussion: Chronic, not acute, exposure to high glucose negatively impacts mitochondrial structure and function. The decline in mitochondrial activity was associated with a recovery of glycolysis, indicative of metabolic adaptation.Introdução: A ceratopatia diabética afeta aproximadamente 70% dos pacientes diabéticos e a disfunção mitocondrial é considerada uma das causas centrais dessas complicações. Objetivo: Caracterizar as alterações mitocondriais e metabólicas nas células epiteliais corneanas humanas (HCECs) de pacientes diabéticos e em cultura de células epiteliais da córnea humana imortalizadas (hTCEpi), submetidas a alta concentração de glicose. Métodos: As córneas humanas de pacientes diabéticos e de pacientes saudáveis, grupo controle, foram obtidas do Serviço de Transplante de Tecidos da UTSouthwestern Medical Center-Dallas. As culturas primárias foram geradas usando um protocolo já estabelecido pelo laboratório. As células hTCEpi e HCECs foram cultivadas em meio de cultura de crescimento sem soro contendo 6mM de glicose. As células hTCEpi foram cultivas por 24h, 3, 5, 7, 9 ou 14 dias em meio com acréscimo de 19mM de glicose e para controle osmótico o meio foi suplementado com 19mM de manitol. A polarização mitocondrial foi avaliada usando os corantes MitoTracker, TMRE e JC1. As alterações metabólicas foram avaliadas em tempo real usando o Seahorse Metabolic Flux Analyzed. O ciclo celular foi determinado usando Iodeto de propídio e analisado usando o citômetro Celigo. A migração celular foi avaliada pelo ensaio de wound healing e a proliferação caracterizada pela contagem celular diária por 7 ou 14 dias. Resultados: As células HCECs de pacientes diabéticos apresentaram alteração na polarização mitocondrial assim como fragmentação severa. As células hTCEpi submetidas à hiperglicemia aguda não apresentaram anormalidades no crescimento, controle do ciclo celular, migração e metabolismo. A exposição crônica das células hTCEpi à hiperglicemia (14 dias) desencadeou a parada do ciclo celular em G0/G1. Aos 14 dias também houve um lentificação da migração celular nos grupos expostos ao estresse hiperglicêmico e hiperosmótico. Porém a proliferação celular permaneceu inalterada tanto após 7 quanto 14 dias. As células cultivadas em alta glicose por 14 dias apresentaram perda de polarização e fragmentação mitocondrial com o corante JC-1, porém não houve diferença significativa quando comparada ao grupo do estresse hiperosmolar. A atividade metabólica permaneceu inalterada em células cultivadas em alta glicose por 24 horas. No dia 3, as células mostraram uma queda mensurável na capacidade respiratória excedente que permaneceu diminuída até o dia 14. No dia 5, a glicólise (ECAR) começou a diminuir e permaneceu baixa até o dia 9. Nos dias 7 e 9, o OCR (taxa de consumo de oxigênio) aumentou, deslocando as células para um fenótipo respiratório. No dia 14, o OCR diminuiu e o ECAR aumentou para níveis normoglicêmicos e o fenótipo celular tornou-se glicolítico novamente. Houve também uma queda acentuada dos outros parâmetros metabólicos mitocondriais. Discussão: A exposição crônica, não aguda, à glicose alta afeta negativamente a estrutura e a função mitocondrial. O declínio da atividade mitocondrial foi associado a uma recuperação da glicólise, indicativa de adaptação metabólica.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)88887.374521/2019-00Universidade Federal de São PauloCampos, Mauro Silveira de Queiroz [UNIFESP]http://lattes.cnpq.br/8668472375424523http://lattes.cnpq.br/7858613725199409Mussi, Natalia [UNIFESP]2022-07-19T17:57:03Z2022-07-19T17:57:03Z2022-03-23info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersion68 f.application/pdfMUSSI, N. Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas. São Paulo, 2022. 68 f. Tese (Doutorado em Oftalmologia e Ciências Visuais) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, 2022.https://hdl.handle.net/11600/64095porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-07-26T23:59:23Zoai:repositorio.unifesp.br/:11600/64095Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-07-26T23:59:23Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.none.fl_str_mv |
Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas Diabetes mellitus: metabolic and mitochondrial changes in corneal epithelial cells. |
title |
Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas |
spellingShingle |
Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas Mussi, Natalia [UNIFESP] Diabetes Mellitus Ceratopatia diabética Hiperglicemia Epitélio corneano Disfunção mitocondrial |
title_short |
Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas |
title_full |
Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas |
title_fullStr |
Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas |
title_full_unstemmed |
Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas |
title_sort |
Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas |
author |
Mussi, Natalia [UNIFESP] |
author_facet |
Mussi, Natalia [UNIFESP] |
author_role |
author |
dc.contributor.none.fl_str_mv |
Campos, Mauro Silveira de Queiroz [UNIFESP] http://lattes.cnpq.br/8668472375424523 http://lattes.cnpq.br/7858613725199409 |
dc.contributor.author.fl_str_mv |
Mussi, Natalia [UNIFESP] |
dc.subject.por.fl_str_mv |
Diabetes Mellitus Ceratopatia diabética Hiperglicemia Epitélio corneano Disfunção mitocondrial |
topic |
Diabetes Mellitus Ceratopatia diabética Hiperglicemia Epitélio corneano Disfunção mitocondrial |
description |
Purpose: Diabetic Keratopathy affects approximately 70% of diabetic patients. Mitochondrial dysfunction is considered a central feature underlying diabetic complications. The purpose of this study was characterized mitochondrial alterations in telomerase-immortalized human corneal epithelial (hTCEpi) cells exposed to hyperglycemic and hyperosmotic stress, also the changes in primary cultured diabetic human corneal epithelial cells (HCECs). Methods: To determine the effects of diabetes on mitochondria and metabolism, donor human corneas from diabetics were obtained from Tissue Transplant Services at UT Southwestern Medical Center. Primary cultures were generated using an established lab protocol. HCECs and hTCEpi cells were cultured in a defined serum-free keratinocyte growth media containing 6 mM of glucose. Cells were cultured for 24h, 3, 5, 7, 9 or 14 days in media with additional 19mM of glucose. Cells supplemented with 19 mM mannitol were used as an osmotic control. Mitochondrial morphology and polarization were assessed using MitoTracker Green, TMRE, and JC-1. Metabolic changes were quantified in real time using a Seahorse metabolic flux analyzer. Cell cycle were determined by staining with Propidium Iodide and analyzed using the Celigo imaging cytometer. Cell migration was evaluated by the wound healing assay and the cells proliferation was characterized by daily cell count for 7 or 14 days. Results: The HCECs cells from diabetic patients showed alterations in mitochondrial polarization and severe fragmentation. The hTCEpi cells exposed to acute hyperglycemia showed no abnormalities in cell cycle control and metabolism. Chronic exposure of hTCEpi cells to hyperglycemia (14 days) triggered cell cycle arrest in G0/G1 phase. At 14 days there was also a decrease in cell migration in the groups exposed to hyperglycemic and hyperosmotic stress. However, cell proliferation remained unchanged after both 7 and 14 days. Using JC-1, cells cultured in high glucose for 14 days showed a loss of polarization and mitochondrial fragmentation but this wasn’t significant in comparison to hyperosmolar stress. Metabolic activity was unchanged in cells cultured in high glucose for 24 hours. By day 3, cells showed a measurable drop in spare respiratory capacity that remained decreased through day 14. By day 5, glycolysis (ECAR) began to decrease and remained low through day 9. At days 7 and 9, OCR was increased, shifting cells towards a more respiratory phenotype. By day 14 OCR decreased and ECAR increased to normoglycemic levels and the phenotype became glycolytic again. There was also a sharp drop in others mitochondrial metabolic parameters. Discussion: Chronic, not acute, exposure to high glucose negatively impacts mitochondrial structure and function. The decline in mitochondrial activity was associated with a recovery of glycolysis, indicative of metabolic adaptation. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-07-19T17:57:03Z 2022-07-19T17:57:03Z 2022-03-23 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
MUSSI, N. Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas. São Paulo, 2022. 68 f. Tese (Doutorado em Oftalmologia e Ciências Visuais) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, 2022. https://hdl.handle.net/11600/64095 |
identifier_str_mv |
MUSSI, N. Diabetes mellitus: alterações metabólicas e mitocondriais em células epiteliais corneanas. São Paulo, 2022. 68 f. Tese (Doutorado em Oftalmologia e Ciências Visuais) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, 2022. |
url |
https://hdl.handle.net/11600/64095 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
68 f. application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de São Paulo |
publisher.none.fl_str_mv |
Universidade Federal de São Paulo |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
instname_str |
Universidade Federal de São Paulo (UNIFESP) |
instacron_str |
UNIFESP |
institution |
UNIFESP |
reponame_str |
Repositório Institucional da UNIFESP |
collection |
Repositório Institucional da UNIFESP |
repository.name.fl_str_mv |
Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP) |
repository.mail.fl_str_mv |
biblioteca.csp@unifesp.br |
_version_ |
1814268307363069952 |