0 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE MEDICINA Carolina Abreu Miranda Avaliação de fatores de crescimento envolvidos no pâncreas endócrino de ratas com diabete moderado em diferentes momentos da vida Tese apresentada à Faculdade de Medicina, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de Botucatu, para obtenção do título de Doutora em Tocoginecologia. Área de Concentração: Ciências da Saúde Orientadora: Profa. Dra. Débora Cristina Damasceno Coorientadora: Profa. Dra. Yuri Karen Sinzato Botucatu 2020 1 Carolina Abreu Miranda Avaliação de fatores de crescimento envolvidos no pâncreas endócrino de ratas com diabete moderado em diferentes momentos da vida Tese apresentada à Faculdade de Medicina, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de Botucatu, para obtenção do título de Doutora em Tocoginecologia. Área de Concentração: Ciências da Saúde. Orientadora: Profa. Dra. Débora Cristina Damasceno Coorientadora: Profa. Dra. Yuri Karen Sinzato Botucatu 2020 2 3 Dedicatória 4 Ao povo brasileiro que com seu suor e trabalho diário financiaram esse trabalho. 5 Agradecimentos 6 A Deus que permitiu que tudo isso acontecesse, me dando saúde, coragem e força para me fazer superar as dificuldades que apareceram ao longo do caminho. Aos meus pais Carlos Roberto de Miranda e Marlei Abreu Ferreira Miranda e irmãos Felipe, Rafael e Júlio, por sempre me apoiarem, sonharem junto comigo, pela confiança e por compreender a minha ausência ao longo de todos esses anos longe de casa. Por saber que independente da distância, estamos sempre juntos e conectados pelos milhões de cabos e conexões que nos faz sentir mais próximos. Aos meus familiares que tanto lutaram e sonharam comigo cada passo que tenho dado desde a minha graduação. Em especial aos meus avós Osmar Dias de Miranda e Maria Clélia de Miranda, minha tia e madrinha querida Juscélia Dias de Miranda Dantas e meus primos queridos, quase irmãos Tiago e Vinícius. Que sempre me acolhem com tanto carinho, mesmo depois de tantos dias longe e me alegram com seus áudios e chamadas de vídeo quando a saudade aperta. À minha orientadora, Profa. Dra. Débora Cristina 7 Damasceno, que me recebeu e me acolheu em uma das etapas mais importantes da minha vida profissional. Pela confiança no meu trabalho e pelas puxadas de orelha tão necessárias. Pela dedicação em me ensinar um pouquinho do que é ser cientista e mulher. Muito obrigada! À minha coorientadora, Profa. Dra. Yuri Karen Sinzato, pela ajuda, ensinamentos e paciência ao longo desse percurso. Obrigada! À querida amiga e parceira de experimentos, Franciane Quintanilha Gallego Souza, por responder minhas mensagens desesperadas de ajuda e por todo o apoio em todos os momentos que pensei em desistir ou deixar de tentar. Devo muito dessa conquista a você! Muito obrigada! Aos meus amigos queridos Tatiele Schonholzer, Lucas Venturini, Kleber Eduardo, João Pedro, Carolina Saullo, Loraine Gollino, Thierres Santos, Juliana de Carvalho, Pedro Manzi que fazem parte dessa trajetória chamada vida e faz com que as coisas pareçam mais leves e fáceis de se encarar. Agradeço especialmente à Verônyca Gonçalves que 8 dividiu um pedacinho da vida dela comigo e que guardo um carinho especial pelo tempo compartilhado, pelas conversas e desabafos. Ao Eduardo Klöppel pela amizade e pela companhia. Por me apoiar nos meus momentos mais difíceis e sempre me confortar. Além de compartilhar comigo o amor da nossa cachorrinha “Pizza”. Às amigas que fiz durante meu período de intercambio Vanessa Iurif e Leda Muzzi, que fizeram com que os dias frios e escuros de Copenhagen fossem mais fáceis de suportar. Obrigada pela companhia e pelas risadas. Aos assistentes de suporte acadêmico (ASA) da Unidade de Pesquisa Experimental (UNIPEX) da Faculdade de Medicina de Botucatu, especialmente aos Srs. Danilo Chaguri, Carlos Roberto Gonçalves de Lima, José Márcio Cândido e Jurandir Antônio, pela manutenção dos biotérios, limpeza e cuidados com os animais, Marta Regina Russo Sarzi pelo auxílio nas técnicas morfológicas, Leandro Alves dos Santos e Ana Paula Dória P. da Cruz pelo auxílio com as análises de microscopia confocal. 9 À Profa. Dra. Luciane Alarcão Dias-Melício por ceder o aparelho de microscopia confocal e orientações quanto às análises e qualidade das imagens. Ao Prof. Dr. Sérgio Luís Felisbino, por ter cedido os reagentes de imunofluorescência e ao Caio César Damasceno Monção pela assistência durante a padronização da técnica de imunofluorescência. À Profa. Dra. Noeme Sousa Rocha por ceder seu laboratório e equipamentos para a realização deste projeto e à técnica do Laboratório de Rotina de Patologia da FMVZ_Botucatu, Maria Valéria Dalanezi, pela ajuda no processo de inclusão em parafina dos materiais. Ao Prof. Dr. Rogelio Hernandez Pando por ceder os anticorpos BMP-2 e BMP-7 e colaboração na elaboração dos escritos. Ao Prof. Dr. Sebastían SanMartín, que me recebeu de braços abertos para um estágio de curta-duração em seu laboratório na Universidade de Valparaíso – Chile, por ceder o espaço, reagentes e tempo para a discussão do meu trabalho. Agradeço também ao Prof. Juan Varas que me ajudou na padronização de alguns dos anticorpos, me 10 ensinando sobre técnicas de coloração histológicas. E ao Pós-Doc Ivo Carrasco que me orientou nos meus primeiros dias em Valparaíso. Ao Prof. Dr. Jens Høirris Nielsen e Nils Billestrup, que me receberam com muito afeto por 6 meses na Universidade de Copenhagen – Dinamarca. Por cederem o espaço e contribuírem significantemente nas discussões relacionadas ao meu projeto e pela oportunidade de aprender novas técnicas que muito agregam à minha experiencia acadêmica. Agradeço também à responsável técnica Sra. Helle Fjvordvang que com muita paciência me ensinou as mais diversas técnicas e me apresentou um pouquinho da cultura dinamarquesa. À Pós Doc Michala Prause pela paciência e boas discussões científicas. Às colegas de pesquisa e laboratório que fiz lá Min Qiao e Rikke Rejnholdt Jensen pelas conversas e jantares tão agradáveis. À Faculdade de Medicina de Botucatu – UNESP, em especial ao Departamento de Ginecologia e Obstetrícia e ao Laboratório de Pesquisa Experimental de Ginecologia e Obstetrícia (LAPGO) pela acolhia e concessão das dependências e aparelhos durante a realização deste trabalho. 11 À toda a equipe do Escritório de Apoio à Pesquisa (EAP) da Faculdade de Medicina, especialmente ao Prof. Dr. José Eduardo Corrente, pelo auxílio no cálculo do delineamento experimental e das análises estatísticas do estudo. Aos funcionários da Seção de Pós-Graduação da Faculdade de Medicina de Botucatu, em particular à secretária do Programa de Pós-Graduação em Tocoginecolgia, Sra. Solange Sako. Aos membros da banca de qualificação e de defesa da dissertação, Prof. Dr. Luis Antonio Justulin Junior, Prof. Dr. Sebastían SanMartín, Profa. Dra. Franciane Quintanilha, Prof. Dr. Rogelio Hernandez Pando pela disponibilidade, críticas e sugestões. À CAPES pela concessão da bolsa de pesquisa, Processo nº 88882.432893/2019-01, permitindo total dedicação a execução desse estudo. À CAPES pela concessão da bolsa do Programa de Doutorado Sanduíche no Exterior (PDSE), Processo nº 88881.187613/2018-01 que permitiu a realização do estágio 12 de pesquisa no exterior, ampliando meus conhecimentos e horizontes dentro da pesquisa. Aos animais, que cederam suas vidas para a realização deste trabalho, contribuindo cada dia mais com a ciência. 13 Sumário 14 CAPÍTULO 1 - "INFLUENCE OF THE DIABETS ON GROWTH AND TRANSCRIPTION FACTORS INVOLVED IN THE CELL REMODELING ON NEONATAL RATS” ............................................................... 16 ABSTRACT ................................................................................................................................ 17 INTRODUCTION ....................................................................................................................... 18 METHODS ................................................................................................................................. 19 Animals ............................................................................................................................... 19 Diabetes Induction .............................................................................................................. 20 Experimental Groups ........................................................................................................... 20 Blood and pancreas sample collection ................................................................................ 22 Serum insulin measurement ............................................................................................... 22 Islet pancreatic cell morphology ......................................................................................... 22 Statistical analysis ............................................................................................................... 26 RESULTS ................................................................................................................................... 26 DISCUSSION ............................................................................................................................. 34 REFERENCES ............................................................................................................................. 39 CAPÍTULO 2 - "GROWTH AND TRANSCRIPTION FACTORS INVOLVED IN PANCREATIC ENDOCRINE CELL ADAPTATION OF MILDLY DIABETIC ADULT FEMALE RATS” .................................................................. 49 ABSTRACT ................................................................................................................................ 50 INTRODUCTION ....................................................................................................................... 51 METHODS ................................................................................................................................ 52 Animals ............................................................................................................................... 53 Diabetes Induction .............................................................................................................. 53 Experimental Groups ........................................................................................................... 53 Oral Glucose Tolerance Test ................................................................................................ 54 Blood and pancreas sample collection ................................................................................ 54 Serum insulin, glucagon and glycated hemoglobin determinations ................................... 54 Islet pancreatic cell morphology ......................................................................................... 55 Statistical analysis ............................................................................................................... 57 RESULTS ................................................................................................................................... 60 DISCUSSION ............................................................................................................................. 67 CONCLUSION ........................................................................................................................... 69 REFERENCES ............................................................................................................................. 71 ATTACHMENT ...................................................................................................................................... 76 ETHICS COMMITTEE CERTIFICATE ............................................................................................ 77 15 Capítulo 1 16 INFLUENCE OF THE DIABETS ON GROWTH AND TRANSCRIPTION FACTORS INVOLVED IN THE CELL REMODELING ON NEONATAL RATS Carolina Abreu Miranda1, Franciane Quintanilha Gallego1, Yuri Karen Sinzato1, Juan Varas2, Rogélio Hernandez Pando3, Sebastian SanMartín2, Débora Cristina Damasceno1* 1Laboratory of Experimental research on Gynecology and Obstetrics, Postgraduate Course on Tocogynecology, Botucatu Medical School, São Paulo State University_UNESP, Botucatu, São Paulo State, Brazil. 2Biomedical Research Centre, Universidad de Valparaíso, Valparaíso, Chile. 3Department of Pathology, National Institute of Medical Sciences and Nutrition “Salvador Zubíran”, Mexico City, Mexico. Corresponding author: * Débora Cristina Damasceno, PhD, Departamento de Ginecologia e Obstetrícia, Faculdade de Medicina de Botucatu, UNESP, Distrito de Rubião Júnior, s/n, CEP 18.618-970, Botucatu, São Paulo, Brasil Phone: +55 14 38801630 E-mail: debora.damasceno@unesp.br ______________________________________________________________________________ Este manuscrito foi submetido à revista Pancreatology (Fator de Impacto = 3,241). 17 ABSTRACT Background/objective: The neonatal period of life is a critical window for the pancreatic development. The streptozotocin (STZ) is one of beta-cytotoxic agents to induce diabetes. In the STZ-induced diabetic model in neonatal period, the rats present a partial recovery of pancreatic islet cell, and the mild diabetes status at adulthood. To better understand how this occur we investigate the relationship between PDX-1, Ki-67, Ngn-3, BMP-2 and -7 in different moments in first month of their life. Methods: Female newborn Wistar rats received STZ at birth for diabetes induction, and the control (nondiabetic) received the vehicle. Then, the animals were euthanized at different life days: D5, D10, D15, and D30. The blood samples were collected for insulin measurement and pancreas was collected and processed for immunohistochemical analysis of PDX-1, Ki-67, Ngn-3, BMP- 2 and –7 and co-localization for labeling of insulin and glucagon. Results: The diabetic animals presented a lower immunostaining of PDX-1 on D5, D15 and D30, a higher labeling of cell proliferation on D5 and D10 and higher immunostaining of nuclear Ngn-3 in all moments in pancreatic islet cell. Besides, the ratio for BMP-2 and –7 immunostaining was increased in pancreatic islet cell in diabetic groups compared to respective control. Conclusion: During neonatal period, PDX-1, Ngn-3, BMP-2 and –7 participate in intricate approach for physiological pancreatic remodeling in control and diabetic rats. However, diabetic animals present a reduced capability for pancreatic islet recovery and have a functional impairment of the pancreatic endocrine cells, leading to diabetes at adulthood. Keywords: development, diabetes, islet cell, pancreas, rats 18 INTRODUCTION Pancreas is composed by endocrine and exocrine cells originated from the same progenitor cells [1]. During the early pancreatic development, mesenchyme cells are responsible for secretion of growth factors such as FGF (fibroblast growth factor), BMP (bone morphogenetic protein) and activins, which belongs to TGF-β superfamily (transforming growth factor-β). These factors stimulate proliferation of pancreatic precursor cells that express PDX-1 (pancreatic and duodenal homeobox 1) [2–7]. This proliferative process leads to pancreatic bud development that expand and branch [8–10], while TGF-β/BMP- 7 are secreted by mesenchyme and determine the endocrine-exocrine proportion in pancreatic tissue [11,12]. PDX-1 is the first transcription factor expressed in endocrine pancreatic tissue and induces Ngn-3 (neurogenin 3) activation [13–15]. Ngn-3 is necessary for endocrine cell specification during pancreas development and originates five types of endocrine cells that compose pancreatic islets [6,16,17]. These cells appear in the following chronological order in rodents: alpha (α), beta (β), delta (δ), epsilon (ε) and PP cells, which synthesize and secrete hormones as glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide (PP), respectively [10,16]. It is important to highlight that pancreatic development occurs in similar ways in human and rodent with few differences (revised in [18]). After birth (neonatal period) the pancreatic islets undergo physiological changes in structure and function. This period is considered a critical window of pancreatic development in mammals [19,20]. Moreover, for identifying immature β-cells some factors are observed as the low sensitivity to glucose and as the presence of markers as Ngn-3 [21–23]. Therefore, unfavorable events during pancreatic remodeling in this period can lead to onset of diseases in adulthood, including diabetes [24]. Studies on pancreatic development and islet function in humans are insufficient, and most of the acquired knowledge are originated from investigations using experimental models, mostly with rodents [3,18,25]. Although the rodent and human pancreatic islets have some differences, the cell architecture is similar between species [18,26,27]. This similarity indicates that studies using rodents are adequate to expand knowledge about islet 19 biology, substantially contributing to the development of strategies for the prevention, treatment and diagnosis of diseases such as diabetes [27,28]. Gallego et al. [29] demonstrated that diabetic rats, who received streptozotocin (STZ) on neonatal period, had a massive loss of β-cells at this period followed by a partial β-cell percentage recovery throughout life of these animals (15 days of life and pregnancy period) [29,30]. Despite that, the rats continued to present a lower percentage of β-cells, reduced pancreatic islet size and hyperglycemic status. PDX-1, Ngn3, BMP-2 and BMP-7 are important factors for pancreatic development. However, few studies address these markers in postnatal life associated with hyperglycemia. Therefore, we hypothesized that these factors are essential for pancreatic development and could participate in the recovery process of pancreatic β-cell in diabetic rats during neonatal period. In order to comprehend the changes in endocrine pancreas after diabetic status in rats, we evaluated the relationship between BMP-2, BMP-7, PDX-1, Ki-67 and Ngn3 in different moments during the first month of life. METHODs Animals Wistar rats were used and kept in the vivarium in the Laboratory of Experimental Research in Gynecology and Obstetrics in controlled conditions of temperature (22 ± 2ºC), light (12h light/dark cycle) and relative humidity (50 ± 10%), with tap water and fed laboratory chow ad libitum. Female rats were mated with males, weighing approximately 220-260g, (ratio of three females and one male rat) to obtain female newborns (NB) for the composition of the non-diabetic (control) and diabetic groups. The Ethics Committee on Animal Use (CEUA) of Botucatu Medical School, Unesp, approved all the experimental procedures applied in this study (Protocol Number: 1219/2017). 20 Diabetes Induction Female Wistar rats bred in our animal facility were fed ad libitum with commercial rat chow (Purina®, Brazil). After obtaining pups, streptozotocin (STZ - 100 mg/kg, subcutaneous route – sc., Sigma–Aldrich®, St. Louis, MO, USA) diluted in citrate buffer (0.10 M, pH 4.5, Sigma–Aldrich®, St. Louis, MO, USA) was injected in the female offspring, weighing approximately 5.0±1.0 g for diabetes induction [29] on day of birth (D1). Nondiabetic (Control) animals were injected only with citrate buffer (vehicle) under similar conditions to those performed for diabetic group. Blood glucose level was used as inclusion criteria on day 5 of life (D5) for each newborn. A small puncture at the distal end of the animal's tail was made in order to obtain a drop of blood, which was then used to determine the blood glucose levels using a conventional glucometer. Rats given STZ with glycemia level equal to or greater than 400 mg/dL on D5 were considered as diabetic, and female offspring that presented lower glycemia were excluded and discarded of the experiment. For the control group, female offspring with glycemia lesser than 120 mg/dL on D5 were included, and those presenting blood glucose levels above 120 mg/dL were also discarded [31]. Experimental Groups The control and diabetic female newborn rats (n = 115) were euthanized at days 5 (n= 40 newborns) for diabetes confirmation; on D10 (n= 40 newborns) and on D15 (n = 25 newborns) related to breastfeeding phase, and on D30 (n= 10 rats) related to post-weaning period. The D5 corresponded to hyperglycemic peak in diabetic animals, D10 and D15 resembled to the pancreatic regeneration period, while D30 represented the period immediately after cell regeneration and possible changes related to the weaning period [19,20,29,32] (Fig. 1). The newborns were maintained with their dam until the death day (D5, D10 and D15) or until weaning (D30) in individual polypropylene cages with wood shavings. After weaning (D21), the female rats were kept in polypropylene cages with wood shavings containing three rats/cage until the death day. For the serum measurements of control groups, we collected blood samples of pool of three female newborns/mother (weighing approximately 10±1 g) on D5; and on D10, weighing approximately 21±2 g; two newborns/mother (weighing approximately 28±4 g) on D15; and one 21 newborn/mother (weighing approximately 105±10g on D30). In the diabetic groups, it was necessary to increase of number of newborns for blood sample collection to equal to the blood amount withdrawn of control animals due to the lower body weight of the diabetic newborns compared to those of control group. On D5 and D10, five female newborns were used for each experimental day, weighing approximately 8±1 g and 15±3 g, respectively. On D15, three females weighing approximately 23±5 g; and on D30, one female newborn, weighing approximately 75±10 g, was used. For morphological analyses of control groups, a maximum of two female pups/mother for all experimental moments (D5, D10, D15 and D30) was used in order to ensure the genetic variability (Fig. 1). Figure 1. Experimental design. 22 Blood and pancreas sample collection After 6 hours of fasting, at the experimental periods (D5, D10, D15 and D30) the female rats were anesthetized with sodium thiopental (Thiopentax®, Cristália, Brazil) intraperitoneal route (120 mg/Kg according to Ethical Committee’s protocols) and euthanized by decapitation. The blood samples were collected and used to determine serum insulin levels. Following, the animals were submitted to laparotomy for the collection of the pancreas for morphological analysis of pancreatic tissues and immunolabeling to identify Ki- 67 and PDX-1 for pancreatic proliferation of islet cells; BMP-2 and BMP-7 for pancreatic differentiation; Ngn-3 for pancreatic endocrine cell differentiation after birth; and insulin and glucagon labeling to verify if there was overlapping of these hormones in pancreatic islet cells. Serum insulin measurement The whole blood samples were centrifuged at 1,575 x g for 10 minutes (min) at 4ºC, and the obtained serum was stored in a freezer at -80ºC until the measurement moment. The serum insulin level was determined according to manufacturer instructions of the specific commercial kit (Crystal Chemical® - Code: 90060, USA). Islet pancreatic cell morphology Histological analysis After dissection, the pancreas was fixed in 4% formaldehyde for 24 hours (h). The fragments were paraffin-embedded and cut into sections of 5μm using a rotary microtome and stained with hematoxylin and eosin for conventional morphological analysis of pancreatic tissue. The pancreatic islets were identified and the images were captured using the computerized image system (Software KS-300, version 3.0, Zeiss®), integrated with the digital camera image (CCD-IRIS/RGB, Sony®, China) microscope (DMR, Leica®, Brazil). The images were analyzed regarding to pancreatic islet size, atrophy, shape, and cell limit [33–35]. 23 Immunohistochemistry procedures The slides were rinsed and rehydrated through a grade ethanol series. In general, the immunohistochemical procedures included following steps: (1) tissue antigen retrieval; (2) blocking of endogenous peroxidase activity; (3) blocking non-specific proteins; (4) incubation with the primary antibody; (5) incubation with the secondary antibody; (6) peroxidase revelation using a specific chromogen; and (7) counterstaining with Harris’s Hematoxylin. Negative controls were performed for all antibodies by omitting the primary antibody step similarly to described by Gallego et al. [29]. Supplementary Chart 1 described the stages of antigen retrieval, primary and secondary antibodies, dilutions, incubations and chromogens used for all immunohistochemical procedures used in this study (Supplementary Chart 1). The images were captured through the computerized image system (Software KS-300, version 3.0, Zeiss®), which receives a digital camera image (CCD-IRIS/RGB, Sony®), coupled under a microscope (DMR, Leica®). The immunolabeled sections were semi-quantitatively evaluated for Ki-67, PDX-1, Ngn3, BMP-2 and BMP-7. The Ki-67 is a marker for cellular proliferation, is present during all active phases of the cell cycle, but is absent in quiescent cells [36,37]. The previous analysis revealed Ki-67 staining in pancreatic cell nuclei. Therefore, the number of Ki-67-positive immunostained cell nuclei were counted in 10 microscopic fields at a final magnification 400 x (10 x eyepiece and 40 x/0.65/∞/0.17 objective) from five different animals from each experimental group. The results are expressed as a Ki-67 ratio [(number of labeled nuclei/total number of cell in islet)/islet area]. The counting was performed using Image J® software (NIH – public access). PDX-1 and Ngn3 have been present in the cytoplasm and nuclei of all endocrine pancreatic cells. For the purpose of this study, only the staining in pancreatic nuclei was considered because the transcription factors are activated in the cell nuclei [38]. The PDX-1 and Ngn3 immunostaining analyses were performed by ratio between nuclei counting and number of total cells, and it was presented as a ratio of PDX-1 and Ngn3-labeled cells. The immunolabeling for BMP-2 and BMP-7 was not observed in cell nuclei, but only in the cytoplasm. Following, the true color image analysis by 24 Image Pro-Plus System software was applied to determine the integrated optical density (IOD) values for BMP-2 and BMP-7 [39]. In addition, the calculation of ratio between IOD and islet area (pixels²) for BMP-2 and BMP-7 was determined. Immunofluorescence procedure for insulin and glucagon double staining Double immunostaining was performed in this experiment for insulin and glucagon co-localization. The slides were dehydrated, and antigen retrieval was performed as described above for 20 min. The sections were incubated in 0.01% sodium borohydride (vol/vol) for 10 min and 5% BSA in PBS for 1h at room temperature to block nonspecific binding. Then, for the primary antibody incubation a monoclonal anti-insulin mouse antibody (1:10,000; Abcam®, Code: ab8304, Carlsbad, CA, USA), and a polyclonal anti-glucagon rabbit antibody (1:500; Abcam®, Code: ab8055, Carlsbad, CA, USA) was used overnight at 4ºC. Alexa Fluor 568 goat anti-mouse IgG (red color) and Alexa Fluor 488 goat anti-rabbit IgG (green color) (1:500, Abcam®, Code: ab175473 and ab150077, Carlsbad, CA, USA, respectively) were used as secondary antibody for 1h at room temperature. The sections were mounted for fluorescence with antifade mounting media with DAPI (Vecta Shield®, Burlingame, CA, USA). Immunostaining was visualized using a Leica TCS SP8 Confocal microscope from Experimental Research Unit (Unipex), Botucatu Medical School, UNESP, Brazil. For analysis of these parameters, the number of insulin and glucagon positive cells were counted in four microscopic fields at a final magnification (630x) from five different animals from each experimental group. The results were presented as image demonstrating by the overlapped cells (yellow color) in both groups. 25 Chart 1. Description of the primary and secondary antibodies, stages of antigen retrieval, dilution, incubation and chromogen 26 Statistical analysis For the calculation of the sample size (n), a completely randomized design with the factorial design of the Research Support Office (EAP) of Botucatu Medical School was used. For immunohistochemical analysis, the calculation was estimated in five animals or pool/group, with a minimum of 10 islets/animal/pancreas. Poison’s Distribution was used for comparison of percentage of the ratio of IOD/islet area of BMP-2 and BMP-7. Gamma Distribution was used for non-homogeneous data, as comparison of serum insulin concentration and the ratio of (positive cells/total cells)/islet area of Ki- 67, PDX-1 and Ngn-3. RESULTS Streptozotocin (STZ) action in pancreatic β-cells was confirmed by fasting glycemia at day 5 of life (D5) in all animals that received STZ at first day of life (D1). This finding has already been verified in our laboratory and published by Gallego et al. (2019). On D5, the STZ-induced diabetic animals presented higher fasting glycemia (diabetic= 520 ± 118 mg/dL versus control= 92 ± 20 mg/dL) in comparison to nondiabetic (control) animals. These rats also showed decreased pancreatic islet area in this life day (data not shown). From D5 to D30, the control rats presented a progressive increase of serum insulin concentrations, while diabetic rats presented higher insulin concentration at D5 and D30, but only at D5, the rats presented higher serum insulin concentration compared to control group (Fig. 2). Considering morphological analyses of experimental groups in different moments by HE staining (Fig. 3), the control group presented no differences in the pancreatic islet size among analyzed days, and the shape of islets was spherical with well-defined margins, except at D15 and D30 when the islets had an irregular shape and poorly defined margins. The diabetic group presented no difference concerning to pancreatic islet size, but atrophied islets compared to control group. The islet shape in diabetic animals showed irregularly and poorly defined margins at D5, D10 and D15 (Fig. 3). To evaluate the endocrine cell identity, proliferation and differentiation, 27 the PDX-1 ratio (Fig. 4A), Ki-67 ratio (Fig. 4B), BMP-2 IOD/islet area ratio (Fig. 4C) were evaluated. Other markers, such as BMP-7 IOD/islet area ratio (Fig. 4D) and Ngn-3 ratio (Fig. 4E) were analyzed. For these parameters, the comparison between control and diabetic groups at the same day of life was performed. In addition, the comparisons among days of life (D5 x D10 x D15 x D30) were performed into each group. Similar to the response pattern of the serum insulin levels, the PDX-1 ratio of control rats was significantly rising from D10 to D30. The diabetic rats had higher PDX-1 ratio at D30. At D5 and D30, the diabetic rats presented a higher PDX-1 ratio related to those of control females (Fig. 4A). Concerning to the Ki-67 ratio (Fig. 4B), the control and diabetic rats showed significant rising on days 15 and 30 of life. The diabetic animals showed a higher Ki-67 ratio in all moments of life compared to those of control group. The BMP-2 ratio (IOD/islet area) in the control animals was higher at D10 compared to other days. In diabetic animals, this ratio was lower at D15 when compared to D5, D10 and D30 (Fig. 4C). For the BMP-7 staining ratio (IOD/islet area) in the diabetic and control animals, there were higher values at D15 in relation to other life days (Fig. 4D). In diabetic group, BMP-7 ratio was higher in all evaluated days compared to those of control females. In control rats, a lower Ngn-3 ratio at D15 and a higher rate at D30 were verified as compared to other days. Ngn-3 ratio (Fig. 4E) was significantly different on D10, D15 and D30 in diabetic rats. There was a lower Ngn3 ratio on D15 compared with D5 and D10. The diabetic rats also showed higher values of Ngn-3-immunolabeled cells in relation to those of control rats. The double staining of insulin- plus glucagon-positive cells was more evident in both groups at D10 in pancreatic islet, and no statistical differences were observed between control and diabetic groups in this life day (Fig. 5). On day 5 of life, the diabetic animals showed a significant higher percentage of co- localization when compared to control animals at D5 (Fig. 5). 28 Figure 2. Serum insulin concentrations (ng/mL) of the control and diabetic animals at different moments of life (days 5, 10, 15 and 30 of life) (n=5 animals or pool/group). Data expressed as the mean ± standard deviation *p<0.05 – compared with the control group considering the same day and analyzed variable (Gamma Distribution Test). ap<0.05 – compared with D5; bp<0.05 – compared with D10; cp<0.05 – compared with D15 (Gamma Distribution Test) 29 Figure 3. Representative photomicrographs showing Hematoxylin and Eosin staining of control and diabetic animals at different moments of life (days 5, 10, 15 and 30 of life) (n=5 animals/group). Scale bar: 50μm. Total magnification: 40x. 30 31 32 Figure 4. Pancreatic islet of Control and Diabetic animals at different moments (days 5, 10, 15 and 30 of life). Representative photomicrographs of pancreatic islets showing (A) nuclear PDX-1 immunostaining (red arrows), negative control and comparison between the PDX-1 ratio (x10-6) for both experimental groups at different moments; (B) nuclear Ki-67 immunostaining (yellow arrows), negative control and comparison between the Ki-67 ratio (x10-6) for both experimental groups at different moments; (C) cytoplasmic BMP-2 immunostaining, negative control and comparison between the IOD/islet area of BMP-2 for both experimental groups at different moments; (D) cytoplasmic BMP-7 immunostaining, negative control and comparison between the IOD/islet area of BMP-7 for both experimental groups at different moments; (E) nuclear Ngn-3 immunostaining (green arrows), negative control and comparison between Ngn-3 ratio (x10-6) for both experimental groups at different moments. Total magnification: 40x. Scale bar: 50μm *p<0.05 – compared with the Control group considering the same day and analyzed variable (Gamma Distribution Test). p<0.05 – lower letters represent significant differences compared with the analyzed days in the same group: acompared with D5, bcompared with D10, ccompared with D15 (Gamma Distribution Test) 33 Figure 5. Representative images of for nuclei staining (DAPI-blue), glucagon (green) and insulin (red) and overlapped images for control and diabetic groups at different moments of life. The double immunostaining is demonstrating by yellow color at early postnatal life in rats. Total Magnification: 63x. Scale bar: 50µm 34 DISCUSSION During neonatal period, the pancreatic islet cells of control animals experienced changes in the activation of growth and transcription factors. This occurs to adapt for new metabolic and nutritional demand, which are necessary due to food introduction. In general, all markers evaluated in this study (Ki-67, PDX-1, Ngn-3, BMP-2 and BMP-7) seem to act in an integrated way on the proliferation and differentiation of pancreatic islet endocrine cells, which was observed throughout of the entire first month of life of control and diabetic animals. This occurs for an adequate adaptation, development of cellular identity and establishment of adequate pancreatic function, to allow glycemic homeostasis in control animals. However, the diabetic animals were not able to recover the pancreatic islet function at the end of first month of life (Fig. 6), contributing to diabetes onset. In this study, the pancreatic islets of the nondiabetic (control) rats presented an expected morphological characteristic, as spherical shape with well-defined margins, throughout life corroborating the studies of Cabrera et al. [40] and Di Cairano et al. [41,42]. During early postnatal period, the islet cells undergo a physiological remodeling process, which consists in a functional maturation of cells formed in fetal period and cell proliferation to increase the islet cell mass [43]. Consequently, the pancreas is capable to respond properly to the metabolic challenges inherent to body growth [44]. On D5 and D10, our control animals presented a lower cell proliferative activity in pancreatic islets as evidenced by the lower ratio of nuclear immunostaining with Ki-67/islet area. Kaung [45] observed a higher pancreatic endocrine cell (α, β, δ and PP) proliferation at the end of fetal period. However, there is evidence that proliferative capability of endocrine cells decreases after birth, but continues throughout first month of postnatal life [45], confirming our findings during the first 30 days old of control rats. Besides, the lower proliferation in endocrine cells in the control group on D10 could be related to higher intensity of BMP-2, since its action has been described for proliferative inhibiting in β-cell [46]. We verified a decreased proliferation on D5 and D10 followed by an increase on D15 and D30 in control 35 animals according to Puri et al. [47], who observed an intense proliferation of β- cells in rodents in the early postnatal stages (at postnatal day 16). At the early postnatal life, as on days 5 and 10, we observed the presence of overlapped cells for insulin and glucagon, which suggests cell immaturity. This indicates that these cells could be undergoing a remodeling process due to an extra uterine life adaptation. Ngn-3 is another marker that determines the cell immaturity [23]. Wang et al. [48] found that Ngn-3 inactivation in β-cells of newborn mice negatively regulated the expression of necessary several genes for endocrine differentiation and function, being the most affected the insulin and MafA genes, suggesting that Ngn-3 can be expressed in immature β-cells after birth [23,48,49]. Even with the Ngn-3 identification in pancreatic endocrine cells, the role of this factor after endocrine differentiation is still unclear, since the amount of this protein in newborn cells is much less than in embryonic endocrine progenitor cells [48]. However, the Ngn3 presence could be an indicative that some dedifferentiation process is happening to cells since the ratio of nuclear immunostaining with Ngn3/islet area significantly increased on day 30 of life in control animals. Dedifferentiation process is a regression from a mature endocrine cell to an endocrine progenitor-like cell [50]. The BMP-7 role in pancreatic islet cells after birth is unclear, and our results indicate some association with dedifferentiation process. It was demonstrated in vitro that non-endocrine human pancreatic tissue (hNEPTs), when stimulated by BMP-7, promoted the conversion of exocrine tissue into insulin-producing cells [51,52]. We observed immunostaining for BMP-7 in islet periphery, suggesting that it could act preferentially in non β-cells of control animals, with greater evidence on D15. Thus, in line with in vitro findings [51,52], we suggest that BMP-7 presence in periphery islet might stimulate non β-cells to become insulin producers. Related to pancreatic β-cell functional capability and cell identity, the ratio of nuclear immunostaining with PDX-1/islet area was determined. The PDX-1 stimulates the transcription of insulin gene for synthesis in β-cells [53,54]. Then, the PDX-1 presence in nuclei of endocrine cells in control animals indicated that they were stimulated for insulin synthesis and secretion. This can be confirmed by a response pattern between the findings of PDX-1 nuclear immunostaining 36 and serum insulin concentration, which showed a progressive increase over the first month of life. Regarding the findings in diabetic animals, there was β-cells loss on the first day of life, which led to hyperglycemia on D5, a smaller islet area and fewer number of endocrine cells in all periods studied [29]. The pancreatic tissue presented atrophic islets with irregular shape and poorly defined margins during the life of diabetic animals. A temporal response in the diabetic group followed the similar pattern of cell proliferation in the control group. However, the proliferative activity was greater than in control animals in all studied moments, as a compensatory mechanism to reverse the damage caused by hyperglycemia. It is suggested that these surviving cells belong to a population of less differentiated immature cells that retains a higher mitotic potential [55], which might explain the greater proliferative capacity observed in diabetic animals. The findings of BMP-2 intensity in pancreatic islet cells of diabetic animals seems to contribute to preserve the function of remaining β-cells because can act by inhibiting the proliferation of this cell type [46]. This fact indicates that this growth factor can affect β-cell function, contributing to the dedifferentiation of non β-cells. In addition, it was observed the presence of overlapped cells for insulin and glucagon like control group more evident at early postnatal life (D5 and D10). At this same moment of life, diabetic animals had a higher Ngn-3 ratio, which is related to the presence of immature cells [23]. However, the lower Ngn- 3 ratio on D15 followed by an increase on D30 could be an indicative of dedifferentiated cells [50]. The dedifferentiation process seems to be evident by increase of BMP-7 on D15 in diabetic group, which acts for an attempting to identity endocrine cell recovery [51,52]. Therefore, the functional capability of pancreatic β-cells is demonstrated by PDX-1 immunostaining, and diabetic animals presented an increased value of this factor at the end of the first month of life. Similar to increase of PDX, there was increase of the serum insulin concentrations, confirming a stimulus for insulin synthesis and secretion in the late neonatal phase of these animals. Our results demonstrate that diabetic animals show a hyperglycemic peak at 5 days of life, which is transient throughout the first month of life of 37 these animals. Although fasting glycemic changes are not observed, these animals showed higher glycemic levels after glucose overload during oral glucose tolerance test at D30 [29]. A factor that contributes to the impairment of β-cells is related to the formation and accumulation of reactive oxygen species (ROS) induced by a hyperglycemic environment, which results in an oxidative stress status [56]. We suggested that oxidative stress in rats led to a decreased PDX-1 activation on β-cells, which reduced insulin synthesis, causing hyperglycemia [38,56]. Thus, our study provides some information about pancreatic islet cell during early postnatal period, contributing for understanding the diabetes progression in rodents and human. Some limitations could be mentioned on this study, such as other co- localization for identification the relationship between pancreatic endocrine cells, and determination of other transcription factors as BMP-4 (acts with BMP- 2), Pax-4 (specific for β-cell differentiation), MafA (specific for β-cell), MaFB and Arx (specific for α-cell), FoxO-1 (β-cell survival and Ngn-3 relationship), and active caspase-3 (a specific marker for death cell for contributing to comprehend the relationship between death and proliferation cell). These immunohistochemical analyses are planned for future studies because there are still samples stored for these analyses, thus avoiding the use of other animals. This makes possible to reduce animals and materials during new experimental studies. In conclusion, both groups presented an activity for remodeling and adaptation to physiological stimuli, such as birth, breastfeeding and weaning. In control animals, β-cell presents functional and mature performance in order to adequately respond to the glycemic oscillations and, consequently, to maintain glycemic homeostasis. Nevertheless, the capacity for remodeling and adaptation is reduced in diabetic animals, since the greater proliferation and activation of transcription and growth factors (BMP-2, BMP-7 and Ngn-3) were not enough to recompose the population of β-cells, which led to functional impairment of these cells, contributing to hyperglycemia. 38 Figure 6. Flowchart. Summarized and connected results of comparison of adaptative response related to growth and transcription factor of the endocrine pancreatic cells at first month of life in (A) control (B) diabetic animals and (C) between both groups. 39 Funding This study was financed by the Coordination for the Improvement of Higher Education Personnel (CAPES) – Financial Code 001 (Process number 88882.432893/2019-01) for the fellowship to C.A. Miranda. Acknowledgments The authors thank Mr. Danilo Chaguri, the technician responsible for the Laboratory of Experimental Research in Gynecology and Obstetrics for the handling of the animals and assistance in the surgeries; Profa. Dra. Noeme Sousa Rocha for lending your laboratory and equipment for the histological processing of samples; Prof. Dr. Sérgio Luis Felisbino for reagents yelding for immunofluorescence; Mr Caio Cesar Damasceno Monção for assistance during immunofluorescence standardization; Profa. Dra. Luciane Alarcão Dias-Melício for enabling the use of confocal microscope; Mr. Leandro Alves dos Santos and Ms. Ana Paula Dória P. Cruz for assistance during image analysis obtained from confocal microscope; Prof. Dr. José Eduardo Corrente for design and statistical analysis. Declaration of competing interest The authors declare that there is no conflict interest. Author contributions C.A. Miranda, F.Q. Gallego, Y.K. Sinzato, and D.C. Damasceno designed the study. C.A. Miranda and F.Q. Gallego collected the data. C.A. Miranda and J. Varas performed the immunohistochemical image analyses. C.A. Miranda, F.Q. Gallego, Y.K. Sinzato, and D.C. Damasceno performed all statistical analyses and interpreted the data. C.A. Miranda, F.Q. Gallego, Y.K. Sinzato, R.H. Pando, S. SanMartín and D.C. Damasceno drafted the work and performed final revision of the intellectual content. All authors were responsible for critical revisions of the paper. All authors approved the final version of the manuscript. REFERENCES [1] Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreatic 40 lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 2002;129:2447–57. [2] Goulley J, Dahl U, Baeza N, Mishina Y, Edlund H. BMP4-BMPR1A signaling in beta cells is required for and augments glucose-stimulated insulin secretion. Cell Metab 2007;5:207–19. https://doi.org/10.1016/j.cmet.2007.01.009. [3] Ahnfelt-Rønne J, Ravassard P, Pardanaud-Glavieux C, Scharfmann R, Serup P. Mesenchymal bone morphogenetic protein signaling is required for normal pancreas development. Diabetes 2010;59:1948–56. https://doi.org/10.2337/db09-1010. [4] Chung W-S, Andersson O, Row R, Kimelman D, Stainier DYR. Suppression of Alk8-mediated Bmp signaling cell-autonomously induces pancreatic beta-cells in zebrafish. Proc Natl Acad Sci U S A 2010;107:1142–7. https://doi.org/10.1073/pnas.0910205107. [5] Nostro MC, Sarangi F, Ogawa S, Holtzinger A, Corneo B, Li X, et al. Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 2011;138:861–71. https://doi.org/10.1242/dev.055236. [6] Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, Asadi A, et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 2014;32:1121–33. https://doi.org/10.1038/nbt.3033. [7] Hebrok M, Kim SK, Melton DA. Notochord repression of endodermal sonic hedgehog permits pancreas development. Genes Dev 41 1998;12:1705–13. https://doi.org/10.1101/gad.12.11.1705. [8] Pictet RL, Clark WR, Williams RH, Rutter WJ. An ultrastructural analysis of the developing embryonic pancreas. Dev Biol 1972;29:436–67. https://doi.org/10.1016/0012-1606(72)90083-8. [9] Pan FC, Brissova M. Pancreas development in humans. Curr Opin Endocrinol Diabetes Obes 2014;21:77–82. https://doi.org/10.1097/MED.0000000000000047. [10] Shih HP, Wang A, Sander M. Pancreas Organogenesis: From Lineage Determination to Morphogenesis. Annu Rev Cell Dev Biol 2013;29:81– 105. https://doi.org/10.1146/annurev-cellbio-101512-122405. [11] Miralles F, Battelino T, Czernichow P, Scharfmann R. TGF-β plays a key role in morphogenesis of the pancreatic islets of langerhans by controlling the activity of the matrix metalloproteinase MMP-2. J Cell Biol 1998;143:827–36. https://doi.org/10.1083/jcb.143.3.827. [12] St-Onge L, Wehr R, Gruss P. Pancreas development and diabetes. Curr Opin Genet Dev 1999;9:295–300. https://doi.org/10.1016/S0959- 437X(99)80044-6. [13] Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H. beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes Dev 1998;12:1763–8. https://doi.org/10.1101/gad.12.12.1763. [14] Ahlgren U, Jonsson J, Edlund H. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development 1996;122:1409–16. [15] Kawaguchi Y, Cooper B, Gannon M, Ray M, MacDonald RJ, Wright CVE. 42 The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat Genet 2002;32:128–34. https://doi.org/10.1038/ng959. [16] Gradwohl G, Dierich A, LeMeur M, Guillemot F. neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A 2000;97:1607–11. https://doi.org/10.1073/pnas.97.4.1607. [17] Oliver-Krasinski JM, Kasner MT, Yang J, Crutchlow MF, Rustgi AK, Kaestner KH, et al. The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice. J Clin Invest 2009;119:1888–98. https://doi.org/10.1172/JCI37028. [18] Bakhti M, Böttcher A, Lickert H. Modelling the endocrine pancreas in health and disease. Nat Rev Endocrinol 2019;15:155–71. https://doi.org/10.1038/s41574-018-0132-z. [19] Scaglia L, Cahill CJ, Finegood DT. Apoptosis Participates in the Remodeling of the Endocrine Pancreas in the Neonatal Rat * 1997;138:1736–41. [20] Aguayo-Mazzucato C, Sanchez-Soto C, Godinez-Puig V, Gutiérrez- Ospina G, Hiriart M. Restructuring of Pancreatic Islets and Insulin Secretion in a Postnatal Critical Window. PLoS One 2006;1:e35. https://doi.org/10.1371/journal.pone.0000035. [21] Jermendy A, Toschi E, Aye T, Koh A, Aguayo-Mazzucato C, Sharma A, et al. Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells. Diabetologia 2011;54:594–604. https://doi.org/10.1007/s00125-010-2036-x. 43 [22] Blum B, Hrvatin S, Schuetz C, Bonal C, Rezania A, Melton DA. Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3. Nat Biotechnol 2012;30:261–4. https://doi.org/10.1038/nbt.2141. [23] Talchai C, Xuan S, Lin H V., Sussel L, Accili D. Pancreatic β cell dedifferentiation as a mechanism of diabetic β cell failure. Cell 2012;150:1223–34. https://doi.org/10.1016/j.cell.2012.07.029. [24] Lucas A. Programming by Early Nutrition: An Experimental Approach. J Nutr 1998;128:401S-406S. https://doi.org/10.1093/jn/128.2.401s. [25] Gittes GK. Developmental biology of the pancreas: A comprehensive review. Dev Biol 2009;326:4–35. https://doi.org/10.1016/j.ydbio.2008.10.024. [26] Jennings RE, Berry AA, Kirkwood-Wilson R, Roberts NA, Hearn T, Salisbury RJ, et al. Development of the human pancreas from foregut to endocrine commitment. Diabetes 2013;62:3514–22. https://doi.org/10.2337/db12-1479. [27] Bonner-Weir S, Sullivan BA, Weir GC. Human Islet Morphology Revisited: Human and Rodent Islets Are Not So Different After All. J Histochem Cytochem 2015;63:604–12. https://doi.org/10.1369/0022155415570969. [28] Workman P, Aboagye EO, Balkwill F, Balmain A, Bruder G, Chaplin DJ, et al. Guidelines for the welfare and use of animals in cancer research. Br J Cancer 2010;102:1555–77. https://doi.org/10.1038/sj.bjc.6605642. [29] Gallego FQ, Miranda CA, Sinzato YK, Iessi IL, Dallaqua B, Pando RH, et al. Temporal analysis of distribution pattern of islet cells and antioxidant enzymes for diabetes onset in postnatal critical development window in 44 rats. Life Sci 2019;226:57–67. https://doi.org/10.1016/J.LFS.2019.03.061. [30] Gallego FQFQ, Sinzato YKYK, Miranda CACA, Iessi ILIL, Dallaqua B, Volpato GTGT, et al. Pancreatic islet response to diabetes during pregnancy in rats. Life Sci 2018;214:1–10. [31] Santos TMM, Sinzato YK, Gallego FQ, Iessi IL, Volpato GT, Dallaqua B, et al. Extracellular HSP70 levels in diabetic environment in rats. Cell Stress Chaperones 2015;20:595–603. https://doi.org/10.1007/s12192- 015-0581-4. [32] Sinzato YK, Gelaleti RB, Volpato GT, Rudge MVC, Herrera E, Damasceno DC. Streptozotocin-induced leukocyte DNA damage in rats. Drug Chem Toxicol 2018;43:165–8. https://doi.org/10.1080/01480545.2018.1510956. [33] Nagasao J, Yoshioka K, Amasaki H, Tsujio M, Ogawa M, Taniguchi K, et al. Morphological changes in the rat endocrine pancreas within 12 h of intravenous streptozotocin administration. J Vet Med Ser C Anat Histol Embryol 2005;34:42–7. https://doi.org/10.1111/j.1439-0264.2004.00566.x. [34] Cubillos V, López C, Alberdi A. Estudio histopatológico e inmunohistoquímico de páncreas en perros diabéticos inducidos con aloxano. Arch Med Vet 2008;40:169–77. https://doi.org/10.4067/s0301- 732x2008000200009. [35] Bakir M, Geyikoglu F, Koc K, Cerig S. Therapeutic effects of oleuropein on cisplatin-induced pancreas injury in rats. J Cancer Res Ther 2018;14:671–8. https://doi.org/10.4103/jcrt.JCRT_1040_16. [36] Scholzen T, Gerdes J. The Ki-67 protein: From the known and the unknown. J Cell Physiol 2000;182:311–22. 45 https://doi.org/10.1002/(SICI)1097-4652(200003)182:3<311::AID- JCP1>3.0.CO;2-9. [37] Bruno S, Darzynkiewicz Z. Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells. Cell Prolif 1992;25:31–40. https://doi.org/10.1111/j.1365-2184.1992.tb01435.x. [38] McKenna B, Guo M, Reynolds A, Hara M, Stein R. Dynamic recruitment of functionally distinct Swi/Snf chromatin remodeling complexes modulates Pdx1 activity in Islet β cells. Cell Rep 2015;10:2032–42. https://doi.org/10.1016/j.celrep.2015.02.054. [39] Iñiguez G, Gallardo P, Castro JJ, Gonzalez R, Garcia M, Kakarieka E, et al. Klotho Gene and Protein in Human Placentas According to Birth Weight and Gestational Age. Front Endocrinol (Lausanne) 2018;9:797. https://doi.org/10.3389/fendo.2018.00797. [40] Cabrera O, Berman DM, Kenyon NS, Ricordi C, Berggren P-O, Caicedo A. The unique cytoarchitecture of human pancreatic islets has implications for islet cell function. Proc Natl Acad Sci U S A 2006;103:2334–9. https://doi.org/10.1073/pnas.0510790103. [41] Di Cairano ES, Davalli AM, Perego L, Sala S, Sacchi VF, La Rosa S, et al. The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death. J Biol Chem 2011;286:14007–18. https://doi.org/10.1074/jbc.M110.183517. [42] Di Cairano ES, Moretti S, Marciani P, Sacchi VF, Castagna M, Davalli A, et al. Neurotransmitters and Neuropeptides: New Players in the Control of Islet of Langerhans’ Cell Mass and Function. J Cell Physiol 2016;231:756–67. https://doi.org/10.1002/jcp.25176. 46 [43] Hellman B, Hellerstrom C, Petersson B. Postnatal growth of the endocrine and exocrine parts of the rat pancrease. Its relationship to the metabolism of DNA. Diabetes 1961;10:470–5. https://doi.org/10.2337/diab.10.6.470. [44] Nielsen JH, Haase TN, Jaksch C, Nalla A, Søstrup B, Nalla AA, et al. Impact of fetal and neonatal environment on beta cell function and development of diabetes. Acta Obstet Gynecol Scand 2014;93:1109–22. https://doi.org/10.1111/aogs.12504. [45] Kaung HC. Growth dynamics of pancreatic islet cell populations during fetal and neonatal development of the rat. Dev Dyn 1994;200:163–75. https://doi.org/10.1002/aja.1002000208. [46] Bruun C, Christensen GL, Jacobsen MLB, Kanstrup MB, Jensen PR, Fjordvang H, et al. Inhibition of beta cell growth and function by bone morphogenetic proteins. Diabetologia 2014;57:2546–54. https://doi.org/10.1007/s00125-014-3384-8. [47] Puri S, Roy N, Russ HA, Leonhardt L, French EK, Roy R, et al. Replication confers β cell immaturity. Nat Commun 2018;9:1–12. https://doi.org/10.1038/s41467-018-02939-0. [48] Wang S, Jensen JN, Seymour PA, Hsu W, Dor Y, Sander M, et al. Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function. Proc Natl Acad Sci U S A 2009;106:9715–20. https://doi.org/10.1073/pnas.0904247106. [49] Wang Z, York NW, Nichols CG, Remedi MS. Pancreatic β cell dedifferentiation in diabetes and redifferentiation following insulin therapy. Cell Metab 2014;19:872–82. https://doi.org/10.1016/j.cmet.2014.03.010. [50] Blum B, Roose AN, Barrandon O, Maehr R, Arvanites AC, Davidow LS, et 47 al. Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway. Elife 2014;3:e02809. https://doi.org/10.7554/eLife.02809. [51] Klein D, Álvarez-Cubela S, Lanzoni G, Vargas N, Prabakar KR, Boulina M, et al. BMP-7 induces adult human pancreatic exocrine-to-endocrine conversion. Diabetes 2015;64:4123–34. https://doi.org/10.2337/db15- 0688. [52] Qadir MMF, Álvarez-Cubela S, Klein D, Lanzoni G, García-Santana C, Montalvo A, et al. P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics. Cell Rep 2018;22:2408–20. https://doi.org/10.1016/j.celrep.2018.02.006. [53] Dutta S, Bonner-Weir S, Montminy M, Wright C. Regulatory factor linked to late-onset diabetes? Nature 1998;392:560. https://doi.org/10.1038/33311. [54] Zhu Y, Liu Q, Zhou Z, Ikeda Y. PDX1, Neurogenin-3, and MAFA: critical transcription regulators for beta cell development and regeneration. Stem Cell Res Ther 2017;8:240. https://doi.org/10.1186/s13287-017-0694-z. [55] Wang RN, Bouwens L, Klöppel G. Beta-cell proliferation in normal and streptozotocin-treated newborn rats: site, dynamics and capacity. Diabetologia 1994;37:1088–96. https://doi.org/10.1007/BF00418372. [56] Guo S, Dai C, Guo M, Taylor B, Harmon JS, Sander M, et al. Inactivation of specific β cell transcription factors in type 2 diabetes. J Clin Invest 2013;123:3305–16. https://doi.org/10.1172/JCI65390. 48 Capítulo 2 49 GROWTH AND TRANSCRIPTION FACTORS INVOLVED IN PANCREATIC ENDOCRINE CELL ADAPTATION OF MILDLY DIABETIC ADULT FEMALE RATS Carolina Abreu Miranda1, Franciane Quintanilha Gallego1, Yuri Karen Sinzato1, Juan Varas2, Rogélio Hernandez Pando3, Sebastian SanMartín2, Gustavo Tadeu Volpato4, Débora Cristina Damasceno1* 1Laboratory of Experimental research on Gynecology and Obstetrics, Postgraduate Course on Tocogynecology, Botucatu Medical School, São Paulo State University_UNESP, Botucatu, São Paulo State, Brazil. 2Biomedical Research Centre, Universidad de Valparaíso, Valparaíso, Chile. 3Department of Pathology, National Institute of Medical Sciences and Nutrition “Salvador Zubíran”, Mexico City, Mexico. 4Laboratory of System Physiology and Reproductive Toxicology, Federal University of Mato Grosso_UFMT, Barra do Garças, Mato Grosso State, Brazil. Corresponding author: *Débora Cristina Damasceno, PhD, Departamento de Ginecologia e Obstetrícia, Faculdade de Medicina de Botucatu, UNESP, Distrito de Rubião Júnior, s/n, CEP 18.618-970, Botucatu, São Paulo, Brasil E-mail: debora.damasceno@unesp.br _____________________________________________________________________________________ Este manuscrito será submetido à revista Diabetology &Metabolic Syndrome (Fator de Impacto = 2,466). 50 ABSTRACT Background: STZ-induced diabetic neonatal animals present pancreatic islet structure modifications, with functional impairment of endocrine cells. However, the responsible mechanisms for this injury are not clear. Thus, this study aimed to understand the condition existing in adulthood related to cell proliferation (Ki- 67), growth (BMP-2 and BMP-7) and transcription factors (PDX-1 and Ngn-3) in pancreatic islet cells of diabetic rats. Methods: The female newborns Wistar rats received streptozotocin at birth for diabetes induction, and control received citrate buffer. At reproductive age (85 days of life), the animals were challenged to an oral glucose tolerance test for glycemic assessment. At day 90 of life (D90), the female rats were euthanized, and blood samples were collected for insulin, glucagon and HbA1c determinations. Next, pancreas was collected and processed for immunohistochemical analysis of insulin, glucagon, somatostatin, PDX-1, Ki-67, Ngn-3, BMP-2 and -7 and co-localization for insulin and glucagon. Results: The diabetic animals presented hyperglucagonemia and high levels of HbA1c and a higher percentage of pancreatic endocrine cell proliferation, cytoplasmic staining for BMP-2 and -7, nuclear staining for Ngn-3 and PDX-1. Conclusion: During hyperglycemia status, the endocrine pancreatic cells try to reestablish the β-cell mass by dedifferentiation mechanism, but it is not enough to recover the β-cell structure and function, contributing to pancreatic injury, which leads to diabetes and is related to future complications at adulthood. Keywords: dedifferentiation, diabetes, islet cell, pre-pregnancy, rats. 51 INTRODUCTION The human pancreatic islets show few functional differences and are poorly understood, but these pancreatic islets present cellular arrangement similar to rodents [1]. The islet architecture contributes to the physiological role of pancreatic islet to adapt to metabolic challenges and maintenance of glucose homeostasis throughout life [2]. When the pancreatic β-cell fails to maintain the compensatory mechanism in response to increased metabolic challenge, these cells became dysfunctional, leading to diabetes because the body is not able to maintain glucose homeostasis [3,4]. Concerning to mild diabetes experimental models, the streptozotocin administration in the neonatal period of rats has been used [5–8]. STZ is a beta (β)-cytotoxic drug capable to cause pancreatic β-cell necrosis, leading to insulin deficiency [9,10]. STZ has short life and causes DNA damage within 24 hours after its administration, thus the repercussions observed after STZ administration in rats is due to hyperglycemia [11]. When this drug is administered in the neonatal period, the animals present glucose intolerance and declined insulin response in adult life [5,12], leading to a mild diabetes status. The diabetes progression has been associated with a reduction of β-cell mass in adult mammals, which arises due to increased apoptosis and possible impairment of β-cell proliferation [4,13]. However, it has been proposed that β- cell dedifferentiation against hyperglycemic status is related to loss of β-cell mass in mammals [4,13,14]. Dedifferentiation process is the regression of a mature endocrine cell to an endocrine progenitor-like stage. In relation to β-cell, it loses or decreases the gene expression related to cell identity (PDX-1, MafA, Glut2 and insulin), and a re-activation of endocrine progenitor genes occur such as Ngn-3 (neurogenin 3). Therefore, these alterations lead to the same cell to express more than one type of pancreatic hormone [4,14]. In addition, other factors that participate in the rising of pancreatic endocrine progenitors could affect the function of mature islet cells. BMP-7 (bone morphogenetic protein 7), a growth factor that belongs to the TGF-β (transforming growth factor – β) family, during the embryonic period is 52 responsible for determining the proportion of endocrine-exocrine cells [15], and can convert exocrine tissue into endocrine, specifically insulin-producing cells as demonstrated in vitro experiments [16]. Besides BMP-7 and BMP-2 can also act on mature islet cells, by inhibiting β-cell proliferation [17]. However, the studies involving growth factors, cell proliferation and other influences in pancreatic islet after and during pregnancy are unclear. Previous investigations have shown that rats with mild diabetes during pregnancy presented greater proliferative activity in pancreatic islet cells, as demonstrated by immunostaining with Ki-67 [18], a marker of cell proliferation [19]. However, these animals had reduced islet area and exacerbated glycemic values during the Oral Glucose Tolerance Test (OGTT). These findings indicate there is a functional impairment of pancreatic β-cells in diabetic pregnant rats, which was evidenced by decreased presence of nuclear PDX-1 during pregnancy [18]. When activated, the PDX-1 is translocated to the nuclei of β-cells and stimulates the transcription of the insulin gene for synthesis [20,21], besides acting in the maturation of β-cells [21]. In addition, these same animals showed a lower percentage of β-cells, followed by an higher percentage of α- and δ- cells during pregnancy [18]. However, it is already expected that the β-cells mass increases in response to physiological insulin resistance during pregnancy period in both human and rodent [22,23]. During pregnancy of rodent, an increased cell proliferation is one of the mechanisms that could be responsible for increasing β-cells mass [23,24]. Thus, it is not clear if the islet structural modification and function impairment of pancreatic endocrine cells occur due to physiological adaptation to pregnancy or if owing to diabetic status due to β-cell loss during neonatal period. Therefore, we intended to understanding the relation between cell proliferation (Ki-67) and growth (BMP-2 and BMP-7) and transcription factors (PDX-1 and Ngn-3) in pancreatic islet cells of diabetic rats before pregnancy period (90 days of life) that might be involved in the future complications for diabetic pregnancy. METHODS 53 Animals Wistar rats were used and kept in the vivarium in the Laboratory of Experimental Research in Gynecology and Obstetrics in controlled conditions of temperature (22 ± 2ºC), light (12h light/dark cycle) and relative humidity (50 ± 10%), with tap water and fed laboratory chow ad libitum. Female rats were mated with males, weighing approximately 220-260 g (ratio of three females and one male rat) to obtain female newborns (NB) for the composition of the non-diabetic (control) and diabetic groups. The Ethics Committee on Animal Use (CEUA) of Botucatu Medical School, Unesp, approved all the experimental procedures applied in this study (Protocol Number: 1219/2017). Diabetes Induction On day of birth (D0), for diabetes induction, streptozotocin (STZ - 100 mg/kg, subcutaneous route – sc., Sigma–Aldrich®, St. Louis, MO, USA) diluted in citrate buffer (0.10 M, pH 4.5, Sigma–Aldrich®, St. Louis, MO, USA) was injected in 17 female newborn (NB), weighing approximately 5±1 g [8]. Five control animals were injected only with citrate buffer (vehicle) under similar conditions to that of the diabetic group. Blood glucose level was used as inclusion criterion on day 5 of life (D5) for each NB. A small puncture at the distal end of the animal's tail was made to obtain a drop of blood, which was then used to determine the blood glucose using a conventional glucometer. Rats given STZ with glycemia level equal to or greater than 400 mg/dL on D5 were considered as diabetic (five animals attended the inclusion criteria), and female offspring that presented lower glycemia were discarded. For the control group, female offspring with glycemia lesser than 120 mg/dL [25] on D5 were included (all animals that received vehicle attended the inclusion criteria). The female newborns were maintained with the dams until weaning and then kept in polypropylene cages of a maximum of three rats/cage until the adult life (D90). Experimental Groups The experimental groups were distributed into: Control (n=5) – composed of non-diabetic female rats, and Diabetic (n=5) – composed of streptozotocin- 54 induced mildly diabetic female rats. Oral Glucose Tolerance Test On five days before sacrifice, the female rats were submitted to an oral glucose tolerance test (OGTT). To confirm the diabetic status and the development of abnormal glycemic metabolism, the animals were fasted for 6 hours and a drop of blood were collected from the tail of the rats for glycemic determination (time point 0). Then, the rats received for an intragastric route a glucose solution (0.2 g/mL) at a dose of 2.0 g/Kg body weight. At time points 30, 60 and 120 minutes (min) after glucose overload, the blood glucose levels were determined. These measurements were also used to estimate the total area under the curve (AUC) using a mathematically trapezoidal method [18,26]. Blood and pancreas sample collection At day 90 of life, after 6 hours of fasting, the female rats were anesthetized with sodium thiopental (Thiopentax®, Cristália, Brazil) intraperitoneal route – 120 mg/Kg according to Ethical Committee’s protocols) and sacrificed by decapitation. The blood samples were collected and used to determine HbA1c percentage, and serum insulin and glucagon levels. Then, the animals were submitted to laparotomy for the collection of the pancreas for histological and morphological analysis by HE staining, and immunolabeling to identify insulin, glucagon and somatostatin positive cells in pancreatic islet; Ki- 67 and PDX-1 for pancreatic proliferation of islet cells, Ngn-3, BMP-2 and BMP- 7 for pancreatic differentiation; and, insulin and glucagon labeling to verify if there was overlapping of these hormones in the same pancreatic cells. Serum insulin, glucagon and glycated hemoglobin determinations The whole blood samples were centrifuged at 1,575 x g for 10 minutes (min) at 4ºC, and the obtained serum was stored in a freezer at -80ºC until the measurement moment. The serum insulin (Crystal Chemical® - Code: 90060, USA) and glucagon (Sigma Aldrich® - Code: RAB0202) levels were determined 55 by a specific commercial kit according to manufacturer instructions. A fraction of total blood samples was collected in tubes containing EDTA and used to measure HbA1c by method of high-performance liquid chromatography in a specialized laboratory. Islet pancreatic cell morphology Histological analysis After dissection, the pancreas was weighed and fixed in 4% formaldehyde for 24 hours (h). The fragments were paraffin-embedded and cut into sections of 5 μm, using a rotary microtome and stained with hematoxylin and eosin for conventional morphological analysis of pancreatic tissue. The pancreatic islets were identified and the images were captured using the computerized image system (Software KS-300, version 3.0, Zeiss®, Germany), integrated with the digital camera image (CCD-IRIS/RGB, Sony®, China) microscope (DMR, Leica®, Brazil). The images were analyzed regarding to pancreatic islet size, atrophy, shape, and cell limit [27–29]. The islet area was performed using ImageJ® software (NIH – public access). Immunohistochemistry procedures The slides were rinsed and rehydrated through a grade ethanol series. In general, the immunohistochemical procedures included following steps: (1) tissue antigen retrieval; (2) blocking of endogenous peroxidase activity; (3) blocking non-specific proteins; (4) incubation with the primary antibody; (5) incubation with the secondary antibody; (6) peroxidase revelation using a specific chromogen; and (7) counterstaining with Harris’s Hematoxylin. Negative controls were performed for all antibodies by omitting the primary antibody step similar to described by Gallego et al. [8]. The Chart 1 (Additional file 1) described the stages of antigen retrieval, primary and secondary antibodies, dilutions, incubations, and chromogens used for all immunohistochemical procedures used in this study. The immunolabeled sections were quantitatively evaluated for insulin, 56 glucagon, and somatostatin for determination of the endocrine pancreatic cells and semi-quantitatively evaluated for Ki-67, PDX-1, Ngn3, BMP-2 and BMP-7 for cell proliferation, identity, and differentiation. For insulin, glucagon, somatostatin, Ki-67, PDX-1 and Ngn-3 immunostainings the images were captured through the computerized image system (Software KS-300, version 3.0, Zeiss®), which receives a digital camera image (CCD-IRIS/RGB, Sony®), coupled under a microscope (DMR, Leica®). For BMP-2 and BMP-7 the images were captured through the computerized image system (Software Olympus® DP MANAGER,), which receives a digital camera image (Olympus® DP71), coupled under a microscope (Olympus® CX21). The percentage of present cells in the pancreatic islet was performed by the ratio between the number of positive cells for each marker (insulin, glucagon, and somatostatin) and the number of total cells in the islet. The value obtained was multiplied by 100 [18,30]. The Ki-67 is a cellular marker for proliferation and is present during all active phases of the cell cycle but is absent in quiescent cells [19,31]. The previous analysis revealed Ki-67 staining in pancreatic islet cell nuclei. Therefore, the Ki-67-positive immunostained cell nuclei ratio were counted in 10 microscopic fields at a final magnification 400 x (10 x eyepiece and 40 x/0.65/∞/0.17 objective) from five different animals from each experimental group. The results are expressed as a Ki-67 ratio [(number of labeled nuclei/total number of cell in islet) / islet area)]. The counting was performed using ImageJ® software (NIH – public access). PDX-1 and Ngn3 have been present in the cytoplasm and nuclei of all endocrine pancreatic cells. For the purpose of this study, we considered only the staining in pancreatic nuclei, because the transcription factors were being activated in the cell nuclei [32]. The PDX-1 and Ngn3 immunostaining analyses were performed by ratio between nuclei counting and number of total cells for islet area, and it was presented as PDX-1 ratio and Ngn-3 ratio. The immunolabeling for BMP-2 and BMP-7 in nuclei was not observed, but only in the cytoplasm. Then, the true color image analysis Image Pro-Plus System software was applied to determine the integrated optical density (IOD) values for BMP-2 and BMP-7 [33]. In addition, the calculation of ratio between IOD and islet area (pixels²) for BMP-2 and BMP-7 was determined. The value 57 obtained was multiplied by 100. Immunofluorescence procedure for insulin and glucagon double staining Double immunostaining was performed in this experiment for insulin and glucagon co-localization. The slides were dehydrated, and antigen retrieval was performed as described above for 20 min. The sections were incubated in 0.01% sodium borohydride (vol/vol) for 10 min and 5% BSA in PBS for 1h at room temperature to block nonspecific binding. Then, for the primary antibody incubation, a monoclonal anti-insulin mouse antibody (1:10,000; Abcam®, Code: ab8304, Carlsbad, CA, USA) and a polyclonal anti-glucagon rabbit antibody (1:500; Abcam®, Code: ab8055, Carlsbad, CA, USA) were used overnight at 4º C. Alexa Fluor 568 goat anti-mouse IgG (red color) and Alexa Fluor 488 goat anti-rabbit IgG (green color) (1:500, Abcam®, Code: ab175473 and ab150077, Carlsbad, CA, USA, respectively) were used as secondary antibody for 1h in room temperature. The sections were mounted for fluorescence with antifade mounting media with DAPI (Vecta Shield®, Burlingame, CA, USA). Immunostaining was visualized using a Leica TCS SP8 Confocal microscope. For analysis of these parameters, the number of insulin and glucagon positive cells were counted in four microscopic fields at a final magnification (630 x) from 5 different animals from each experimental group. The results were presented as image demonstrating by the overlapped cells (yellow color) in both groups. Statistical analysis For the calculation of the sample size (n), a entirely randomized design with the factorial design performed by Research Support Office (EAP) of Botucatu Medical School was used. For immunohistochemical analysis, the calculation was estimated in 5 animals/group, with a minimum of 10 islets/animal/pancreas. Poison’s Distribution was used for comparison of percentage of immunostained for insulin-, glucagon- and somatostatin-positive cells, and the ratio of IOD/islet area of BMP-2 and BMP-7. For the OGTT, islet area, serum insulin concentration, Ki-67 ratio, PDX-1 ratio and Ngn-3 ratio, 58 Gamma Distribution Test was used. The glucagon levels, glycemia obtained by area under curve (AUC) and HbA1c percentage were analyzed by Student t test. For all comparison, p<0.05 was considered as significant limit. 59 Chart 1. Description of the primary and secondary antibodies, stages of antigen retrieval, dilution, incubation and chromogen 60 RESULTS The oral glucose tolerance test (OGTT) (Fig.1A), area under curve analysis (AUC) (Fig.1B) and HbA1c levels were used to confirm the diabetic status. During the OGTT, the animals that received STZ had increased serum glucose levels during fasting, at 30 and 60 min after glucose overload compared to control animals. Besides, an elevated circulating glucose levels per minute in AUC calculation during OGTT in diabetic animals was observed. The HbA1c percentage were higher in diabetic group (9.08±0.93%) compared to non- diabetic (control) group (2.98±0.24%) (data not shown). In adulthood (D90), the serum insulin levels presented no statistically significant difference (p = 0.06) between the experimental groups (Fig.1C), while the glucagon levels were higher in diabetic group (p<0.05) compared with the control rats (Fig.1D). In addition, the AUC values and serum glucagon levels showed a positive correlation with the HbA1c levels (p = 0.0059 and p = 0.020, respectively). Figure 2 presents the morphological analyses of pancreatic islet observed by hematoxylin and eosin (H&E). The islets showed a rounded shape and well-defined limits in both groups. However, the diabetic animals presented smaller islets as observed by the islet area analyses (Fig.2A). There was a lower percentage of positive-insulin cells (Fig.2B) and a higher percentage of positive-glucagon (Fig.2C) and positive-somatostatin cells (Fig.2D) in the diabetic rats in relation to the control rats. The figure 3 shows the results related to ratio of proliferation, differentiation, and cellular identity. The staining ratio of cell proliferation marker (Ki-67) (Fig.3A) was significantly increased in islet cells nuclei in diabetic group, with predominance of staining in periphery cells in both groups. The diabetic animals presented increased immunostaining intensity for Ngn-3 ratio (Fig.3B) as well as the nuclear labeling for PDX-1 ratio (Fig.3C). Figure 4 represent the BMP-2 and -7 images. The diabetic animals presented increased immunostaining intensity for BMP-2 (Fig.4A) and BMP-7 (Fig.4B). The figure 5 shows representative images of glucagon (green) and insulin (red). No co-localization was observed at adulthood in both groups. The 61 figure 6 is a representative diagram to summary the major results found in this study. Figure 1. Glucose tolerance test for control and diabetic animals. (A) Oral Glucose Tolerance Test (OGTT) at 85 days of life. (B) Total area under curve at 85 days of life. (C) Serum insulin levels (ng/mL). (D) Serum glucagon levels (ng/mL) of the control and diabetic animals at 90 days of life Data expressed as mean ± standard deviation (n = 5 animals/group/moment). *p<0.05 – compared with the control group (Gamma Distribution for OGTT; Student t test for AUC data, insulin, and glucagon). 62 Figure 2. Pancreatic islet morphology. (Right panel) Representative photomicrographs of pancreatic islets of control and diabetic rats at 90 days of life showing Hematoxylin & Eosin staining, anti-insulin, anti-glucagon, and anti-somatostatin immunostaining. Scale bar: 50μm. Magnification: 40x. (A) Pancreatic islet area. (B) Comparison between the percentage of cells immunolabeled for insulin; (C) Comparison between the percentages of cells immunolabeled for glucagon; (D) Comparison between the percentages of cells immunolabeled for somatostatin for both experimental groups. Data expressed as mean ± standard deviation (n = 5 animals/group/moment and 50 islets/group). *p<0.05 – compared with the control group (Gamma Distribution for pancreatic islet area and Poisson Distribution for insulin-, glucagon- and somatostatin-positive cells) 63 Figure 3. Pancreatic islet of control and diabetic animals at 90 days of life. (Right panel). Representative photomicrographs of pancreatic islets showing Ki-67, Ngn-3, and PDX-1 immunostaining. Magnification: 40x. Scale bar: 50μm. (A) Ki-67 ratio (x10-6); (B) Ngn-3 ratio (x10-6); (C) PDX-1 ratio (x10-6), in pancreatic islet for both groups. Data expressed as mean ± standard deviation (n = 5 animals/group/moment and 50 islets/group). *p<0.05 – compared with the control group (Poisson Distribution) 64 Figure 4. Pancreatic islet of control and diabetic animals at 90 days of life. (Right panel). Representative photomicrographs of pancreatic islets showing BMP-2 and BMP-7, immunostaining. Magnification: 40x. Scale bar: 50μm. (A) IOD/islet area of BMP-2; (B) IOD/islet area of BMP-7 in pancreatic islet for both groups. Data expressed as mean ± standard deviation (n = 5 animals/group/moment and 50 islets/group). *p<0.05 – compared with the control group (Poisson Distribution) 65 Figure 5. Representative images of nuclei staining (DAPI-blue), glucagon (green), insulin (red) and merged images for control and diabetic groups at 90 days of life. Total magnification: 63x. Scale bar: 50μm. 66 Figure 6. Flowchart. Summarized and connected results of comparison of adaptative response related to growth and transcription factor of the endocrine pancreatic cells at adulthood between both groups. 67 DISCUSSION During the diabetes progression, there is a gradual decrease in β-cell mass and their function [13,34]. Studies carried out using pancreas of individuals with Type 2 diabetes during human autopsy revealed a reduced β- cell mass about 50% lower compared to non-diabetic individuals [35–38]. Nevertheless, the mechanisms involved in β-cell reduction are not well understood. In our experimental model, we observed a reduction in the percentage of insulin-positive cells and an increase in glucagon- and somatostatin-positive cells. In addition, the islet area was reduced, which corroborates the human findings about β-cell reduction in diabetic individuals. Thus, this model was capable to develop glycemic changes and morphological alterations in pancreatic islet of diabetic animals, which supports better comprehend the pathophysiological mechanisms involved in diabetic syndrome. Throughout first month of postnatal life and during pregnancy, the diabetic animals showed greater proliferative activity [8,18]. These periods are considered critical for pancreatic development and lead to adaptations of this micro-organ [8,18,39,40]. Thus, the pancreatic endocrine cells of diabetic animals show greater proliferative activity throughout all life, including during adulthood (D90), as evidenced in our findings by Ki-67 marker. However, this is not sufficient to recover the islet structure in diabetic group. For better understanding of mechanisms related to cell proliferation in pancreatic islet, some important growth and transcription factors for pancreatic embryonic development and for endocrine cell specification and differentiation were evaluated. The proportion of BMP-2 immunostaining by the islet area was higher in diabetic animals. The BMP-2 are described as responsible by inhibiting the proliferation of β-cell [17]. Thus, we can infer that possibly the role of this growth factor in adulthood is to prevent the functional capacity of β-cell in these animals. Besides, the non-beta endocrine cells are likely proliferating in the diabetic animals. Considering the Ngn-3 evaluation, this factor is not typical in adult animals, but the increased Ngn-3 immunostaining in mature islet cells from diabetic animals could be responsible for β-cell dysfunction [14,41]. During 68 embryonic pancreatic development, the Ngn-3 is a marker for immature cells which is essential for β-cell maturation process (TALCHAI et al., 2012). However, at adult age it is not observed any overlapped cells in both groups. Thus, the presence of Ngn-3 indicates that these cells are possible undergoing a dedifferentiation process. During this process, the cells have no well-defined cell identity and became insulin- and/or glucagon- and/or somatostatin-positive cells [4], as a compensatory mechanism against hyperglycemia. This explains the difference in proportion of cell types in pancreatic islets and higher serum glucagon concentrations in the diabetic animals. Besides, the hyperglucagonemia is a result of an imbalance of regulatory mechanisms for pancreatic adaptation due to hyperglycemia and the presence of insulin resistance [4,42]. Besides the dedifferentiation processes that occur in islet cells, the presence of BMP-7 appears to be an indicator showing non β-cells are differentiating into insulin-producing cells. The BMP-7 immunostaining in pancreatic cells after birth is not well understood. Our research group appears to be the first to show the presence of BMP-7 in pancreatic islet cells, both in neonatal (Miranda et al., 2020 – manuscript in preparation) and adulthood in control and diabetic animals. A study using cell culture of non-endocrine human pancreatic tissue (hNEPTS) showed that after stimulation with BMP-7, these cells were able to synthesize insulin [16,43]. Then, we suggest that this growth factor can act probably on cell differentiation in non β-cells population. Nevertheless, it is not yet clear what role this growth factor plays on pancreatic islet cells after birth and it is necessary more studies for better understanding. The nuclear immunolabeling for PDX-1 is increased in diabetic animals at D90, unlike the first month of life (Miranda et al., 2020 - manuscript preparation) and pregnancy [18] of these animals when this marker were decreased for diabetic group. The increase of nuclear PDX-1 might be related to a compensatory mechanism for hyperglycemia, as confirmed by OGTT and HbA1c in diabetic animals. For PDX-1 be activated, elevated levels of serum glucose are necessary, so PDX-1 is translocated to the nuclei and activation of insulin synthesis [20,21,32]. Thus, the functional capability of the some β-cells, which are complete mature and functional, is slightly greater. These findings 69 can be evidenced by the increased presence of PDX-1 and BMP-2 in D90, since the serum insulin levels did not differ between groups. Perhaps other transcription factors related to survival of β-cells, as TGF-β/STAT3/FoxO1 signaling pathway. These factors might be linked to maintenance of other transcription factors responsible for the identity of β-cells, such as PDX-1 and MafA [14,44]. It is important to note the immunostaining for BMP-2 and BMP-7 was still not studied in control or diabetic rats during pregnancy. However, our research team has also been analyzing these growth factors in this period. In adulthood, the pancreatic islet cells of diabetic animals showed a higher proliferative (Ki-67 and BMP-2) capability and an increased presence of dedifferentiation markers (Ngn-3 and BMP-7) followed by hyperglycemia. However, even with a decrease in the beta cell population, they were able to synthesize and secrete insulin during fasting. In addition, the increase in the percentage of alpha and delta cells it is related with the presence of hyperglucagonemia. Some limitations could be mentioned on this study, such as the analysis of other co-localization for identification of relationship among endocrine cells of pancreas, and determination of other transcription factors as BMP-4 (because acts with BMP-2), Pax-4 (specific for β-cell differentiation), MaFA (specific for mature β-cell), MaFB and Arx (specific for mature α-cell), FoxO-1 (β-cell survival and Ngn-3 relationship), and active caspase-3 (a specific marker for cell death and might contribute for better understanding the relationship between death and proliferation cell). These immunohistochemical analyses are planned for future studies in our laboratory because there are still samples stored for these analyses, thus avoiding the use of other animals. This makes it possible to reuse samples and reduce animals and materials during new experimental studies. CONCLUSION In view of our findings, the pancreatic islet cells are capable to activate some transcription (Ngn-3) and growth (BMP-2 and BMP-7) factors responsible to β-cell mass recovery at adulthood. However, this activation does not seem 70 enough to reestablish the morphological structure and function of insulin- producing cells after massive loss of these cells during neonatal period. Thus, the impaired repercussions on endocrine pancreatic cells are related to deficient restoration in endocrine cells throughout life. Declarations Ethics approval and consent to participate The Ethics Committee on Animal Use (CEUA) of Botucatu Medical School, Unesp, approved all the experimental procedures applied in this study (Protocol Number: 1219/2017). Consent for publication Not applicable Availability of data and materials The datasets during and/or analysed during the current study available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was financed by the Coordination for the Improvement of Higher Education Personnel (CAPES) – Financial Code 001 (Process number 88882.432893/2019-01) for the fellowship to C.A. Miranda. Authors’ contributions CAM, FQG, YKS, and DCD designed the study. CAM and FQG collected the data. CAM and JV performed the immunohistochemical image analyses. CAM, FQG, YKS, and DCD performed all statistical analyses and interpreted the data. CAM, FQG, YKS, RHP, SS, GTV and DCD drafted the work and performed final revision of the intellectual content. All authors were responsible for critical revisions of the paper. All authors read and approved the manuscript. 71 Acknowledgments The authors thank Mr. Danilo Chaguri, the technician responsible for the Laboratory of Experimental Research in Gynecology and Obstetrics for the handling of the animals and assistance in the surgeries; Profa. Dra. Noeme Sousa Rocha for lending your laboratory and equipment for the histological processing of samples; Prof. Dr. Sérgio Luis Felisbino for reagents yelding for immunofluorescence; Mr Caio Cesar Damasceno Monção for assistance during immunofluorescence standardization; Profa. Dra. Luciane Alarcão Dias-Melício for enabling the use of confocal microscope; Mr. Leandro Alves dos Santos and Ms. Ana Paula Dória P. Cruz for assistance during image analysis obtained from confocal microscope; Prof. Dr. José Eduardo Corrente for design and statistical analysis. REFERENCES 1. Bonner-Weir S, Sullivan BA, Weir GC. Human Islet Morphology Revisited: Human and Rodent Islets Are Not So Different After All. J Histochem Cytochem. Histochemical Society Inc.; 2015;63:604–12. 2. Arrojo e Drigo R, Ali Y, Diez J, Srinivasan DK, Berggren PO, Boehm BO. New insights into the architecture of the islet of Langerhans: a focused cross-species assessment. Diabetologia. Springer Verlag; 2015. p. 2218– 28. 3. Prentki M, Nolan CJ. Islet β cell failure in type 2 diabetes. J. Clin. Invest. 2006. p. 1802–12. 4. Dor Y, Glaser B. Beta-cell dedifferentiation and type 2 diabetes. N Engl J Med. 2013;368:572–3. 5. Portha B, Levacher C, Picon L, Rosselin G. Diabetogenic effect of streptozotocin in the rat during the perinatal period. Diabetes. 1974;23:889–95. 6. Bonner-Weir S, Trent DF, Honey RN, Weir GC. Responses of neonatal rat islets to streptozotocin: limited B-cell regeneration and hyperglycemia. Diabetes; 1981 [cited 2019 Nov 19];30:64–9. 7. White V, Jawerbaum A, Sinner D, Pustovrh C, Capobianco E, González E. 72 Oxidative stress and altered prostanoid production in the placenta of streptozotocin-induced diabetic rats. Reprod Fertil Dev; 2002;14:117–23. 8. Gallego FQ, Miranda CA, Sinzato YK, Iessi IL, Dallaqua B, Pando RH, et al. Temporal analysis of distribution pattern of islet cells and antioxidant enzymes for diabetes onset in postnatal critical development window in rats. Life Sci; 2019;226:57–67. 9. Lenzen S. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia. 2008;51:216–26. 10. Rakieten N, Rakieten ML, Nadkarni MR. Studies on the diabetogenic action of streptozotocin (NSC-37917). Cancer Chemother Rep; 1963;29:91–8. 11. Sinzato YK, Gelaleti RB, Volpato GT, Rudge MVC, Herrera E, Damasceno DC. Streptozotocin-induced leukocyte DNA damage in rats. Drug Chem Toxicol; 2018;43:165–8. 12. Triadou N, Portha B, Picon L, Rosselin G. Experimental chemical diabetes and pregnancy in the rat. Evolution of glucose tolerance and insulin response. Diabetes; 1982;31:75–9. 13. Weir GC, Bonner-Weir S. Islet β cell mass in diabetes and how it relates to function, birth, and death. Ann N Y Acad Sci. Blackwell Publishing Inc.; 2013;1281:92–105. 14. Talchai C, Xuan S, Lin H V., Sussel L, Accili D. Pancreatic β cell dedifferentiation as a mechanism of diabetic β cell failure. Cell. 2012;150:1223–34. 15. St-Onge L, Wehr R, Gruss P. Pancreas development and diabetes. Curr Opin Genet Dev; 1999;9:295–300. 16. Klein D, Álvarez-Cubela S, Lanzoni G, Vargas N, Prabakar KR, Boulina M, et al. BMP-7 induces adult human pancreatic exocrine-to-endocrine conversion. Diabetes. 2015;64:4123–34. 17. Bruun C, Christensen GL, Jacobsen MLB, Kanstrup MB, Jensen PR, Fjordvang H, et al. Inhibition of beta cell growth and function by bone morphogenetic proteins. Diabetologia; 2014;57:2546–54. 18. Gallego FQ, Sinzato YK, Miranda CA, Iessi IL, Dallaqua B, Volpato GT, et al. Pancreatic islet response to diabetes during pregnancy in rats. Life Sci; 2018;214:1–10. 73 19. Scholzen T, Gerdes J. The Ki-67 protein: From the known and the unknown. J Cell Physiol; 2000;182:311–22. 20. Dutta S, Bonner-Weir S, Montminy M, Wright C. Regulatory factor linked to late-onset diabetes? Nature. 1998;392:560. 21. Zhu Y, Liu Q, Zhou Z, Ikeda Y. PDX1, Neurogenin-3, and MAFA: critical transcription regulators for beta cell development and regeneration. Stem Cell Res Ther; 2017;8:240. 22. Van Assche FA, Aerts L, Prins F De. A morphological study of the endocrine pancreas in human pregnancy. Br J Obstet Gynaecol; 1978;85:818–20. 23. Parsons JA, Brelje TC, Sorenson RL. Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion. Endocrinology; 1992;130:1459–66. 24. Sorenson R, Brelje T. Adaptation of Islets of Langerhans to Pregnancy: β- Cell Growth, Enhanced Insulin Secretion and the Role of Lactogenic Hormones. Horm Metab Res; 1997;29:301–7. 25. Santos TMM, Sinzato YK, Gallego FQ, Iessi IL, Volpato GT, Dallaqua B, et al. Extracellular HSP70 levels in diabetic environment in rats. Cell Stress and Chaperones; 2015;20:595–603. 26. Tai MM. A mathematical model for the determination of total area under glucose tolerance and other metabolic curves. Diabetes Care.; 1994;17:152–4. 27. Nagasao J, Yoshioka K, Amasaki H, Tsujio M, Ogawa M, Taniguchi K, et al. Morphological changes in the rat endocrine pancreas within 12 h of intravenous streptozotocin administration. J Vet Med Ser C Anat Hist