1 
 

UNIVERSIDADE ESTADUAL PAULISTA 
“JÚLIO DE MESQUITA FILHO” 

FACULDADE DE MEDICINA 
 
 
 
 
 
 
 

Eduardo Klöppel 
 
 
 
 
 
 

Impacto transgeracional do diabete materno sobre a morfofisiologia 
mitocondrial e expressão proteica do  

músculo esquelético de ratas fêmeas adultas 
 
 
 
 
 
 
 
 

Tese apresentada à Faculdade de 
Medicina, Universidade Estadual 
Paulista “Júlio de Mesquita Filho”, 
Câmpus de Botucatu, para obtenção do 
título de Doutor(a) em Tocoginecologia 
(Especialidade: Bioquímica). 

 
 
 
 
 
 
 

Orientadora: Profa. Dra. Débora Cristina Damasceno 

 
 

Botucatu 
2021 

 
 

 
 



2 
 

Eduardo Klöppel 
 
 
 
 
 
 
 
 
 
 
 

Impacto transgeracional do diabete materno sobre a 
morfofisiologia mitocondrial e expressão proteica do 

músculo esquelético de ratas fêmeas adultas 
 
 
 
 
 
 
 
 
 
 

Tese apresentada à Faculdade de Medicina, 
Universidade Estadual Paulista “Júlio de 
Mesquita Filho”, Campus de Botucatu, para 
obtenção do título de Doutor em 
Tocoginecologia (Especialidade: 
Bioquímica).  

  
 
 
 
 
 
 
 
 
 

Orientadora: Profa. Dra. Débora Cristina Damasceno 
 
 
 

Botucatu 
2021 

 
 
 
 



3 
 

 
 
 
 
 

  



4 
 

UNIVERSIDADE 
ESTADUAL PAULISTA 

Câmpus de Botucatu 
 

ATA DA DEFESA PÚBLICA DA TESE DE DOUTORADO DE EDUARDO 
KLOPPEL, DISCENTE DO PROGRAMA DE PÓS-GRADUAÇÃO EM 
TOCOGINECOLOGIA, DA FACULDADE DE MEDICINA - CÂMPUS DE 
BOTUCATU. 
 

Aos 18 dias do mês de outubro do ano de 2021, às 14:30 horas, por meio de 

Videoconferência, realizou-se a defesa de TESE DE DOUTORADO de EDUARDO 

KLOPPEL, intitulada Impacto transgeracional do diabete materno sobre a 

morfofisiologia mitocondrial e expressão proteica do músculo esquelético de ratas 

fêmeas adultas. A Comissão Examinadora foi constituida pelos seguintes membros: 

Profa. Dra. DEBORA CRISTINA DAMASCENO MEIRELLES DOS SANTOS 

(Orientador(a) - Participação Virtual) do(a) Depto. de Ginecologia e Obstetrícia / 

FM/Botucatu - Unesp, Profa. Dra. DANIELA CARVALHO DOS SANTOS 

(Participação Virtual) do (a) Depto de Biologia Estrutural e Funcional / IB/Botucatu - 

Unesp, Profa Dra CAMILA RENATA CORREA CAMACHO (Participação Virtual) 

do(a) Unidade de Pesquisa Experimental / FM/Botucatu - Unesp, Prof. Dr.TIAGO 

RODRIGUES (Participação Virtual) do(a) CCNH/Mogi das Cruzes / Universidade 

Federal do ABC, Profa. Dra, ANA IZABEL SILVA BALBIN VILLAVERDE 

(Participação Virtual) do(a) Universidade do Contestado - Campus de Mafra/SC. Após 

a exposição pelo doutorando e arguição pelos membros da Comissão Examinadora que 

participaram do ato, de forma presencial e/ou virtual, o discente recebeu o conceito final 

APROVADO. Nada mais havendo, foi lavrada a presente ata que, após lida e aprovada, 

foi assinada pelo(a) Presidente(a) da Comissão Examinadora. 

 
Profa. Dra. DEBORA CRISTINA DAMASCENO MEIRELLES DOS SANTOS 

 
 

Faculdade de Medicina - Câmpus de Botucatu - 
Av.: Prof. Mário Rubens 
Guimarães Montenegro, 

s/nº, 18618687 
https://www.fmb.unesp.br/

ggtocoCNPJ: 
48.031.918/0019-53.  

http://www.fmb.unesp.br/pggtocoCNPJ
http://www.fmb.unesp.br/pggtocoCNPJ


5 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

EPÍGRAFE 
  



6 
 

 
 

 
 
 
 
 
 
 
 
 

“Não se conhece completamente 

uma ciência enquanto não se 

souber da sua história”. 

 
Auguste Comte  



7 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

DEDICATÓRIAS 
 
 
  



8 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Dedico essa tese ao processo das coisas, 

processo da vida.  

Como diria o mestre Paulo freire:  

“Não há transição que não implique um ponto 

de partida, um processo e um ponto de chegada. 

Todo amanhã se cria num ontem através de um 

hoje.  

De modo que o nosso futuro se baseia num 

passado e se corporifica num presente.  

Temos de saber o que fomos e o que somos para 

sabermos o que seremos”.  



9 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
AGRADECIMENTOS 

 
 
 
 
 
  



10 
 

Ao Criador, YHWH (transliterado do hebraico - הוהי), por sua eterna misericórdia, e 

que apesar de eu não merecer, me concede todo o bem. 

 

Aos meus familiares, meus pais Sr. Agostinho Klöppel e Sra. Soraia da Silva, meus 

irmãos Caio Alex Klöppel e Murilo Klöppel pelo apoio e por sempre desejarem o meu 

sucesso. 

 

Aos meus amigos que me apoiam, Andrieli Hauschildt, Beatriz Magalhães, João 

Pedro Maia, Juliana de Carvalho, Carolina Abreu Miranda, Oto Schönholzer, 

Tatiele Schönholzer, Lucas Gabriel Venturini, Renato Henrique Ferreira, Raquel 

Cristina, Rosa Jacinto Volpato, Carolina Saullo, Thierres Hernani e Thamires 

Ballarini Gratão. 

 

Aos bons amigos que fiz durante o período de doutorado sanduiche e que me ensinaram 

e compartilharam momentos felizes e difíceis em meio a uma pandemia tão intensa e 

difícil como a que vivemos, especialmente a Nathnael Mulatu, Joanna Pabichu, 

Maria Zielińska, Domenico Marrocu, Lara Sahin, Marcia Roquette, Bernardo 

Ferrara, Vu Phv, Halef Spencer, Anastasia Fanduberina e muitos outras pessoas 

corajosas e incríveis as quais tive o prazer de conhecer. 

 

À Evdokia Papadopoulou, pelo carinho, companheirismo e parceria que faz de nossos 

dias melhores e mais felizes. 

 

Aos meus grandes amigos de tatame, presentes do Jiu-jitsu para a vida, ao meu querido 

Mestre Felipe Bianchi, Helena Bianchi, Giovanna Rochel, Laís Hernandes, 

Jefferson (Jeff), Arthur Pafetti, João Correa, João Tinti, Suelen Rodrigues, Josué 

Santana e Caroline Geraldini. 

 

À 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 nas análises estatísticas do estudo. 

 



11 
 

Ao Programa de Pós-graduação em Tocoginecologia da Faculdade de Medicina de 

Botucatu, pela oportunidade de realizar o experimento, realizar a dissertação e utilizar a 

estrutura da Instituição como um todo para minha formação. 

 

Aos funcionários da Seção de Pós-graduação da Faculdade de Medicina de Botucatu, 

em particular à secretária do Programa de Pós-graduação em Tocoginecologia, Sra. 

Solange Sako Cagliari. 

 

Aos funcionários da biblioteca da UNESP, pela confecção da ficha catalográfica. 

 

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 Lima, José Márcio Cândido (Ari) e Jurandir Antonio, 

pela manutenção dos biotérios, limpeza e cuidados com os animais. 

 

À minha colega de doutorado Larissa Lopes, e equipe do Prof. Dr Luís Antônio 

Justulin, pelo auxílio e desenvolvimento da técnica de Western Blotting. 

 

Ao Adam Eckhardt, Phd e Profa Dra. Ana Izabel Villaverde, pelo auxílio na 

interpretação inicial dos dados de proteômica. 

 

À Profa Dra. Daniela Carvalho dos Santos, pelo auxílio na interpretação das fotos de 

microscopia eletrônica de transmissão. 

 

Aos colegas do Laboratório de Pesquisa Experimental de Ginecologia e Obstetrícia, 

Dra. Yuri Karen Sinzato, Prof. Dr. Gustavo Tadeu Volpato, Dra. Franciane Q. 

Gallego Souza, Mestra Larissa Lopes da Cruz, Vinícius S. Barco e Mestra 

Verônyca Gonçalves Paula, pela amizade, boa convivência e por toda a ajuda durante 

esses anos de doutorado. 

 

Aos participantes da banca de qualificação e de defesa de Doutorado, Profa Dra. Ana 

Izabel Villaverde, Profa Dra. Daniela Carvalho dos Santos, Prof. Dr. Tiago 

Rodrigues, Profa Dra Camila Correa e aos suplentes Prof. Dr. Gustavo Tadeu 



12 
 

Volpato, Dra. Franciane Q. Gallego Souza e Prof. Dr. Kleber Eduardo Campos 

pela disponibilidade, críticas e sugestões. 

 

À minha orientadora, Profa. Dra. Débora Cristina Damasceno, primeiramente, pela 

orientação e confiança, mas também por toda paciência, pelos inúmeros ensinamentos e 

exemplo diário na liderança e condução de um grupo de pesquisa. Muito obrigado por 

todo suporte e ajuda, por acreditar nas minhas capacidades, por todas as oportunidades o 

qual a senhora se empenhou para que eu tivesse acesso e por toda a sua a valiosa e 

contínua contribuição para a minha formação profissional e pessoal. 

 

Aos meus supervisores e colaboradores da Segunda Faculdade de Medicina da Karlova 

Univerzita em Praga e da Academia Tcheca de Ciências, no Instituto de Fisiologia e 

Departamento de Metabolismo Translacional, pelo suporte, ajuda em momentos difíceis 

e por todo aprendizado científico que me permitiram crescer muito como pesquisador e 

ser humano durante essa experiência no exterior. Gostaria de destacar especialmente os 

Prof. Václav Hampl PhD, Adam Eckhardt PhD, Prof. Premysl Jiruska PhD, 

Barbora Kaftanová PhD, Prof. Olga Vajnerová PhD and Marcela Minariková 

Msc. 

 

À CAPES, pela concessão das bolsas no país (DS) e no exterior (PDSE/CAPES-

PrInt/Unesp), permitindo total dedicação à execução desse estudo. 

 

Aos animais, que involuntariamente participaram desse estudo em prol da ciência.  



13 
 

 
 
 
 
 
 
 
 

CAPÍTULO I 
 

CHAPTER 1 
 
Este manuscrito será formatado para submissão à revista International Journal of 

Molecular Sciences (Fator de Impacto = 5,923) 

 

This manuscript will be formatted for submission to the International Journal of 

Molecular Sciences (Impact factor = 5.923) 



14 
 

TRANSGENERATIONAL EFFECTS OF MATERNAL HYPERGLYCEMIA ON 

INSULIN RESISTANCE AND ULTRASTRUCTURAL MORPHOLOGY OF 

SOLEUS MUSCLE IN ADULT FEMALE RATS 

 

 

Eduardo Klöppel1, Larissa L. Cruz1, Franciane Q. Gallego1, Isabela L. Iessi1, Rafael B. 

Gelaleti1, Rafaianne Q. Moraes-Souza1,2, José E. Corrente3, Daniela C. dos Santos4, 

Luis A. Justilin4, Tiago Rodrigues5, Gustavo T. Volpato2, Débora C. Damasceno1* 

 
1Laboratory of Experimental Research on Gynecology and Obstetrics, Postgraduate 

Course on Tocogynecology, Botucatu Medical School, Sao Paulo State University 

(UNESP), Botucatu, 18618-689, São Paulo State, Brazil. 
2Laboratory of System Physiology and Reproductive Toxicology, Institute of Biological 

and Health Sciences, Federal University of Mato Grosso (UFMT), Barra do Garças, 

78600-000, Mato Grosso State, Brazil. 
3Research Support Office, Botucatu Medical School, Sao Paulo State University 

(UNESP), 18618-689, São Paulo State, Brazil. 
4Department of Structural and Functional Biology, Institute of Biosciences, Sao Paulo 

State University (UNESP), 18618-689, São Paulo State, Brazil. 
5Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC 

(UFABC), Santo André, 09210-580, São Paulo State, Brazil. 

 

 

 

*Correspondence to:  Profa. Dra. Débora Cristina Damasceno 

 Unidade de Pesquisa experimental - UNIPEX 

 Faculdade de Medicina de Botucatu - UNESP 

 Distrito Rubião Júnior s/n 

 CEP. 18618-970 -  Botucatu/SP, Brasil 

 Phone/Fax: (55 14) 38801630  

 debora.damasceno@unesp.br 

  

mailto:damascenofmb@gmail.com


15 
 

ABSTRACT 

 

Maternal diabetes has a negative impact on offspring phenotype, which contributes to 

an impaired metabolism in adulthood. Nonetheless, the mechanisms under these 

outcomes are still unclear. In this study by utilizing a rat model, we aimed to investigate 

the impact of maternal diabetes on the metabolism of adult female offspring in relation 

to mitochondrial dynamics, and ultrastructure of skeletal muscle. For this, female 

offspring coming from diabetic mothers (FOD) were obtained through pregnancy of 

Sprague-Dawley rats with streptozotocin neonatally-induced diabetes. Control mothers 

received citrate buffer during their neonatal life. At adulthood, FOD and Control rats 

were submitted to oral glucose tolerance test (OGTT) for inclusion criteria in the 

groups. At 150 days of life, the FOD and Control rats were sacrificed for collection of 

blood and muscle samples. The FOD rats presented a higher insulin and triglyceride 

levels, and abnormal OGTT. These animals also showed decreased activation of 

molecular markers of insulin signaling, and impaired mitochondrial function and 

ultrastructure in soleus muscle. Thus, the transgenerational impact of maternal diabetes 

caused fetal plasticity, which led to insulin resistance and glucose intolerance at 

adulthood, and the mitochondrial network of skeletal muscle is shown to have an 

important role during the prediabetic state. 

 

Keywords:  Skeletal muscle; insulin resistance; hyperglycemia; mitochondrial 

dynamics; AMPK; DOHaD; rat. 

  



16 
 

1. INTRODUCTION  

 

Evidence has proved that environmental exposure in early life to different 

nutritional conditions is related to the incidence of multiple diseases in adulthood, 

which was recognized into a new arm of the scientific knowledge, called as the 

developmental origins of health and disease (DOHaD) [1]. There are experimental, 

clinical, and epidemiological studies demonstrating the consequences of programmed 

changes induced by diseases like diabetes, dyslipidemia, and insulin resistance leading 

to an impaired fetal metabolism, which plays a pivotal role in the fetal origin of 

adulthood diseases [2–5].  

In fact, it is estimated that every year about 21.3 million of births worldwide are 

affected by abnormal blood glucose level during pregnancy [6]. In this matter, it has 

been explained for decades that the “thrifty” genotype of rapid insulin trigger which 

allows primitive man to be able to store ingested food and survive a low-nutrient 

environment, is now one of those responsible for the worldwide diabetic pandemic, also 

when associated with the diabetic intrauterine environment [7].  This “thrifty” genotype 

is now associated it to a sedentary lifestyle with a higher caloric intake and increased 

longevity of modern man [8]. There disturbances may interfere in the metabolism of the 

offspring during the whole developmental period, leading to an impaired metabolic 

adaptation which increases the risks of diabetes and other diseases of adulthood. 

Moreover, studies has been focused on the role of mitochondrial metabolism and 

dynamics among the emerging mechanisms involved in the etiology and 

pathophysiological progression of insulin resistance and diabetes [9, 10]. The imbalance 

in mitochondrial metabolic processes has been pointed as having an important role 

diabetes, mainly when associated to the mitochondrial fission and fusion through outer 

membrane-bound proteins like Mitofusin 2 (MFN2) and mitochondrial fission factor 

(MFF) and to post-translational modification (PTM) effectors which controls 

mitochondrial homeostasis and mitophagy, through adenosine monophosphate-activated 

protein kinase (AMPK) [11–14]. Supplementary, AMPK is presented as a 

heterotrimeric holoenzyme, responsible for regulation of many biological processes like 

oxidative stress, mitochondrial function, and cell survival [15]. In the other hand, 

oxidative stress and insulin resistance can affect the downstream subtracts involved in 

cell survival and metabolism, such as MAPK (Mitogen Activated Protein Kinases) 

which are enzymes highly conserved among eukaryotes, and involved in diverse 



17 
 

metabolic processes of controlling proliferation and cell death. There are four classical 

subfamilies of MAPK in humans, including ERK1 and ERK2 (MAPK 3/1) [16]. 

Additionally, the Phosphatidylinositol 3-kinase (PI3K), is a critical effector on insulin 

action and plays key regulatory function on cell metabolism and insulin response [17].  

Besides mitochondrion function on energetic metabolism and oxidative stress, 

emerging evidence points the especial role of skeletal muscle and its mitochondrial 

network on diabetes etiology and development mainly due to its high glycolytic and 

oxidative activity [18–22]. Equivalently, some authors showed that the soleus muscle is 

an adequate tissue to access these alterations due its high mitochondrial activity and 

network [20, 23]. 

 Our research team has been studied the influence of the maternal diabetes on the 

offspring at different periods of life in order to better understand the effects of maternal 

diabetes on fetal programming/plasticity. It was verified that the diabetic mothers 

presented higher rates of embryo fetal losses at day 18 of pregnancy, which was 

associated changes in the endocrine pancreas [24]. Our laboratory have performed 

several studies to evaluate the diabetic repercussions in animals models, and one of 

these studies shows the negative correlation between the maternal 

hyperglycemia/oxidative stress levels and the number of fetal pancreatic islets in fetuses 

from mildly diabetic rats . Another one has demonstrated oxidative stress in the liver 

and blood in the perinatal period (at postnatal days 5 and 15 of life) of pups coming 

from diabetic mothers. Additionally, the female adult offspring (120 days of life) 

presented glucose intolerance, impaired function of the β-cells and a low-grade 

inflammatory in the blood as response to a hyperglycemic intrauterine environment. 

However, insights about the mechanistic ways of these outcomes remains unclear and 

needs to be elucidated. To our knowledge, our study is the first to investigate the effects 

of the maternal hyperglycemia in adult offspring’s skeletal muscle and mitochondrial 

metabolism despite the organelle being widely involved in the diabetes-induced 

consequences. Thus, this study aimed to investigate if diabetic intrauterine environment 

during embryo fetal period increased the prevalence of insulin resistance in adult female 

rats (150 postnatal days) and if the skeletal muscle, more specifically the structural and 

molecular dynamics of mitochondrial network of soleus muscle, could play a role on 

this phenotype.  

 

2. RESULTS 



18 
 

 

2.1 Criteria of inclusion in the experimental groups 

From criteria inclusion by OGTT in the parenteral generation, all rats (future 

mothers) that received STZ presented glycemic levels higher than 200 mg/dL during 

OGTT and were considered as diabetic, while all control rats presented glycemia 

inferior to 140 mg/dL, characterizing a normoglycemic status (data not shown). In 

relation to female pups from control and diabetic dams at adulthood, all (100%) control 

pups were considered glucose tolerant. For FOD group, 66.6% of the rats had glycemia 

greater than 140 mg/dL during OGTT being considered glucose intolerant and included 

in the experimental group (data not shown). 

 

2.2 Biochemical analyses – at adulthood 

 

2.2.1 OGTT and AUC 

The adult FOD rats presented higher glycemic levels at time point 30 minutes of 

OGTT when compared to the control female pups (Figure 1A). The AUC levels were 

also statistically higher in FOD group (Figure 1B). 

 

 
Figure 1. Blood glucose levels by oral glucose tolerance test (OGTT) (A), and Area 

under de curve (AUC) (B) of the female adult pups from control and diabetic mother 

(FOD) rats. 

Values expressed as mean ± standard deviation (n=10 animals/group). 

* p<0.05 - compared to the Control group (Gamma Distribution test). 

 

2.2.2 Insulin metabolism and HOMA measurements 



19 
 

The FOD group presented increased serum insulin concentration and HOMA-IR 

level (Figure 2A and 2C), but not in HOMA-beta and fasting blood glucose 

concentration (Figure 2B and 2D, respectively). 

 

 
Figure 2. Serum fasting glucose (a) and insulin (B) concentrations, HOMA-IR (C) and 

HOMA-beta (D) of female adult pups from control and diabetic mother (FOD) rats. 

Values expressed as mean ± standard deviation (n=10 animals/group). 

* p<0.05 - compared to the Control group (Gamma Distribution test). 

 

2.2.3 Body weight, TyG index and lipid profile 

The FOD rats had  increase in body weight (Figure 3A), serum triglyceride 

concentration (Figure 3B) and TyG index (Figure 3D). However, the serum cholesterol 



20 
 

concentration presented no significant differences between the groups analyzed(Figure 

3C). 

 

2.2.4 Oxidative stress biomarkers 

The levels of TBARS and reduced thiol groups (-SH) were not changed in the 

experimental groups (Figure 4A and 4B). 

 

 
Figure 3. Body weight (A), Triglyceride (B), Cholesterol (C) and Triglyceride-glucose 

index (TyG) (D) of female adult pups from control and diabetic mother (FOD) rats.  

Values expressed as mean ± standard deviation (n=10 animals/group). 



21 
 

* p<0.05 - compared to the Control group (Student's t-test for body weight and 

Cholesterol concentrations, and Gamma Distribution test for Triglyceride 

concentrations and TyG Index). 

 

 
Figure 4. Thiobarbituric acid reactive substance (TBARS) (A) and reduced thiol group 

(-SH) concentrations (B) of the female adult pups from control and diabetic mother 

(FOD) rats. 

Values expressed as mean ± standard deviation (n=10 animals/group). 

p>0.05 – not statistically significant difference. 

 

2.3 Western blotting analyses 

 

The FOD group had statistically lower protein expression of PI3K 

(phosphatidylinositol 3-kinase – p110- subunit) (Figure 5A), MAPK (mitogen activated 

protein kinases – ERK1/2) (Figure 5B), AMPK (adenosine monophosphate-activated 

protein kinase) (Figure 5C), and mitochondrial fission factor (MFF) (Figure 5D) in the 

soleus muscle when compared to the control adult pups. However, the levels of Mfn2 

(mitofusin 2) (Figure 5E) presented no difference between experimental groups. 

 

2.4 Structural and Ultrastructural Morphological Analyses 

 

2.4.1 Central nuclei analysis in the soleus muscle fibers and quantification of 

type of slow and fast fibers in the soleus muscle 



22 
 

The morphological analysis demonstrated that the percentage of centrally 

located nuclei counting in the muscle fibers of the soleus muscle was higher in FOD 

adult pups than the control one. The experimental groups were not statistically different 

regarding the percentage of fast or slow type fibers in soleus muscle (Figure 6). 

 

 
Figure 5. Immunoblot analyses of protein expression of PI3K-α (A), MAPK (ERK-

1/ERK-2) (B), AMPK (C), mitochondrial fission factor (MFF) (D) and mitofisun-2 

(Mnf-2) (E) in soleus muscle samples of female adult pups from control and diabetic 

mother (FOD) rats. 

Values expressed as mean ± standard deviation (n = 6 animals/group). 

* p<0.05 - compared to the Control group (Gamma Distribution test) 

 

2.4.2 Ultrastructural analysis of soleus muscle 

The figure 7 shows the descriptive ultrastructural analysis performed in the 

soleus muscle of control (Figures 7A, 7B, and 7C) and FOD (Figures 7D, 7E, and 7F) 

female offspring. In Control group, the transmission electron microscopy showed 

integral muscle cells, with myofibrils organized in sarcomeres aligned in a regular 

pattern, oval or elongated intermyofibrillar mitochondria with a moderately electron-

dense matrix and well-preserved parallel cristae. In the region of the T tubules, the 



23 
 

cisterns of sarcoplasmic reticulum forming the triads were also seen close to the 

mitochondria with rare lipid drops. In contrast, the FOD rats presented some small 

modification in the cellular ultrastructure when compared to the characteristics 

described in Control group. The sarcomeres were slightly disorganized amidst the 

intermyofibrillar mitochondria. The presence of mitochondria with aberrant morphology 

from the rarefaction of the matrix to the destruction of the mitochondrial cristae was 

observed in FOD rats. Despite the intact ultrastructure, the T tubules and Z lines region 

presents an irregular pattern when compared to the Control group. The FOD group also 

showed the cisterns of sarcoplasmic reticulum with a mild dilation close to the 

mitochondria. 

 

 
Figure 6. Histological sections of the soleus muscle stained with hematoxylin and eosin 

(A and B) and immunohistochemistry of fast (D and E) and slow (G and H) fibers. 

Graphs of the percentages of central nuclei (C), fast (F) and slow type (I) fiber number 

in the female adult pups from control and diabetic mother (FOD) rats. 



24 
 

Values expressed as mean ± standard deviation (n=1 sample/animal, 4 counted 

randomly fields of each animal, and 6 animals per group). 

* p<0.05 - compared to the Control group (Gamma Distribution). 

 

 

 
Figure 7. Transmission electronic microscopy of soleus muscle showing mitochondrial 

cristae conformation (MIT), sarcoplasmic reticulum (SR) cistern size, Z lines and T-

tubes organization of the female adult pups from control (A, B and C) and diabetic 

mother (FOD) (D, E and F) rats.  

Scale bars: 500 nm (A, B, D, E); 1 µm (C, F). 

  



25 
 

3. DISCUSSION  

 

The impaired beta (β)-cell function is recognized as a major cause of diabetes. 

However, the mechanisms of diabetes development are not fully understood [25]. In 

order to access these mechanisms, preclinical studies have committed on experimental 

models of diabetes development that could give tools for human clinical practice [26]. 

Based on DOHaD concept of fetal programming and adaptation to an inadequate 

intrauterine environment [27], studies about the diabetes-induced transgenerational 

effects on adult offspring are ongoing in our laboratory. In these investigations we 

verified that an unfavorable intrauterine environment induced by maternal 

hyperglycemia led to alterations in pancreatic beta (β)-cells in embryos, fetuses, 

neonates, and adult daughters. These (β)-cells showed an impairment on insulin 

synthesis homeostasis, which caused the glucose intolerant state of these animals. 

However, the periphery pathophysiological mechanisms involved are not clear so far, 

especially the role of proteins linked to the insulin signaling pathway and its 

repercussions on skeletal muscle cells in the face of this glycemic change at 150 days of 

life. Thus, the present study was carried out for a better understanding and relationship 

between these proteins and also to highlight the role of certain enzymes that have been 

studied in the onset of diabetes and metabolic diseases. 

According to the results obtained in this study, we were able to confirm that fetal 

programming induced by maternal diabetes caused the emergence of diseases in adult 

life in female offspring of rats, confirmed by hyperlipidemia, insulin resistance, and 

glucose intolerance at 150 days postnatal. The increase in maternal glucose 

concentration during pregnancy leads to fetal hyperglycemia, which stimulates insulin 

synthesis, causing fetal hyperinsulinemia [28]. Early embryos exposed to elevated 

glucose levels experienced higher tricarboxylic acid (TCA) metabolites, changing 

alterations in mitochondrial physiology [29]. Diabetes models confirmed that 

mitochondrial bioenergetics and function are impaired in various tissues [30]. Then, a 

study about mitochondrial status in adult offspring of diabetic dams may bring us 

mechanistical insights on the fetal programmed insulin resistance in adult female rats. 

This study showed that even daughters which mothers have mild hyperglycemia 

presented hypertriglyceridemia associated to insulin resistance. Moreover, insulin 

resistance in skeletal muscle is manifested long before the hyperglycemia becomes 

evident [31,32]. Furthermore, adult daughters showed reduced expression of different 



26 
 

proteins (PI3K, AMPK, ERK1/2 and MFF). These factors are directly or indirectly 

related to a damaged mitochondrial fusion causing morphological changes in the 

mitochondria-endoplasmic reticulum associated membrane (MAM) connection, 

possibly leading to ER stress. Thus, the association between reduced expression of 

insulin signaling pathway proteins, MAM disturbance and structural abnormalities of 

myofiber nuclei contributed to the impairment of mitochondrial and cellular 

homeostasis in the skeletal muscle, outlining the resistance insulin state. In normal 

conditions, the binding of insulin to the α-subunits of its receptor stimulates the tyrosine 

activity of the β-subunit that phosphorylates several intracellular proteins leading to the 

activation of PI3K and MAPK (ERK1\2) signaling pathways [32,33]. In order to 

address the major catalytic subunit of PI3K downstream after insulin stimulation, the 

p110 subunit was accessed once its lower expression is related to impaired insulin 

signaling, as evidenced in our study by its diminished protein expression, which might 

partially explain the hyperinsulinemia and glucose intolerance in the experimental 

group. Li et al. [34] demonstrated that p110α is the dominant catalytic isoform of PI3K 

with effector activity in the muscle’s mass control and insulin sensitivity, in conjunction 

with major role in mitochondrial homeostasis in the skeletal muscle. In its turn, the 

mitochondrion is a very mobile organelle that is able to divide (fission) or fuses (fusion) 

itself in response to a wide range of favorable or adverse conditions. [35]. The literature 

shows  that the loss of p110α subunit of PI3K in the muscles causes a decrease in the 

mitochondrial fission and an increase on mitochondrial fusion , leading to larger and 

more elongated mitochondria. In addition, the loss of p110α reduced insulin signaling 

due to a decreased PI3K activity, which turned to an enhanced mitochondrial oxidative 

capacity with increased muscle NADH content, and higher muscle free radical 

production [34]. Therefore, the reduced PI3K p110 subunit expression suggests it’s 

relation to lower MFF expression, compromising mitochondrial dynamics, as observed 

in the elongated mitochondrial pattern in skeletal muscles in our study. 

Furthermore, there was a decrease in ERK 1/2 expression. ERK1/2 are members 

of MAPK family, which is mainly linked to proliferation and cellular differentiation 

[36]. The tight regulation of these proteins is crucial for determining of survival or death 

cell [37]. Likewise, ERK-MFF/FIS1/Drp1 axis is a mitochondrial fission controller and 

is essential for the reprogramming process to cell [38].The reduction in the protein 

expression of ERK1/2 and MFF can be related to a diminished metabolic ability of the 

cell to self-adapt and in activate the mitochondrial fission process of the muscle fibers 



27 
 

of the studied rats, further supporting the alteration in the mitochondrial dynamics of the 

muscle tissue.  

Our results demonstrated AMPK, known as the guardian of mitochondrial 

homeostasis [39], presented a lower protein expression, confirming the abnormal 

signaling pathway and compromised mitochondrial health in the soleus muscle of 

female offspring of diabetic dams. Besides, AMPK is also a protector against insulin 

resistance by endoplasmic reticulum (ER) stress [40]. ER stress is associated to insulin 

resistance in skeletal muscle, as it was observed in our study. Because AMPK is a redox 

stress sensor, it phosphorylates substrates, increases catabolism to generate more ATP, 

and stimulates mitophagy and biogenesis [41]. This rapid AMPK-activated 

mitochondrial fission serves as an adaptive response to external and internal insults 

aiming to maintain their quality and function [42]. In mammals, loss of AMPK results 

in a defective mitophagy [43].  Different AMPK isoforms are capable to sense the 

cellular energetic in vivo and control the mitochondria-ER interaction during 

mitophagy, and for that reason, the discovery of a mitochondrial AMPK pool has local 

importance for the quality and control of the mitochondrial network This fact highlights 

the importance of targeting mitochondrial plasticity for the treatment of conditions like 

diabetes [44]. Toyama et al. [45] found that MFF phosphorylation by AMPK is a 

critical step for mitochondrial fission in response to both cellular and mitochondrial 

stress. This finding corroborates our data since AMPK and MFF protein expression 

were reduced and consequently caused abnormal mitochondrial fission. This might be 

related to the others morphological and functional mitochondrial abnormalities. 

The mitochondria and the ER present some independent biological functions, but 

are not independent structures. These organelles are not only related to lipid synthesis 

and transport, Ca2+ signaling, ER stress, mitochondria shape, motility, bioenergetics, 

mitophagy, and cell death, but also to the molecular plasticity in regulation of metabolic 

mechanisms at the ER–mitochondria interface. It has been found that the physical 

connection between the ER and mitochondria MAM ([46],has several proteins that are 

directly involved in mitophagy and are related to ER and mitochondria linkage, as such 

as the dynamic and complex subcellular signaling, regulating a variety of cellular 

processes [47]. In the present study, the ultrastructural analysis demonstrated a slight 

change in the ER-mitochondria connection, suggesting an ER stress, which might be 

also related to reduced AMPK expression leading to alteration of insulin signaling in the 

muscle fiber. In addition, the disrupted organelle crosstalk at MAM interfaces also may 



28 
 

alter insulin signaling [48], contributing for insulin resistance in the experimental group. 

In relation to nuclei positioning, a higher number of cells with central nuclei is another 

morphological alteration verified in the skeletal muscle of these rats at day 150 of life. 

This characteristic differs of the muscle fiber morphology, since those are 

multinucleated and the position of their nuclei is peripheral, close to the plasma 

membrane [49]. 

There is a report on the prevalence of centrally positioned nuclei in the 

myofibers of patients suffering from different muscle diseases [50]. However, the 

importance of nuclear positioning for disease pathogenesis and muscle weakness is 

unclear. As such, there are few studies on the role of nuclear positioning in muscle 

function or disease. This is explained in part by the prevailing hypothesis that is used to 

explain centrally positioned nuclei: central nuclei are considered merely a marker of 

ongoing myofiber repair. Several mechanisms of nuclear movement or nuclear 

positioning are attributed to a cytoskeletal network and myofibril organization. In this 

way, new research paths arise considering the questions of where the different 

organelles are located and why they are located in this way, how they become located 

and if the organization of different organelles is connected. It is relevant to consider 

centrally located nuclei as more than merely a marker of ongoing muscle repair and as a 

phenotype that can influence muscle function and health [50]. Abnormal nuclear 

positioning has previously been linked to muscle dysfunction [51]. Then, our results 

supports that this finding could also be an indicator of muscular degeneration related to 

several molecular and morphological alterations in the skeletal muscle. Literature 

suggests that both satellite cells and muscle-resident mesenchymal progenitors present 

roles in maintaining skeletal muscle homeostasis and defects in either cell population 

could contribute to the pathogenesis of muscle diseases [52,53]. 

Is now well recognized that the best way to prevent and treat insulin resistance is 

with diet control and regular physical exercise, which can change lipid deposition and 

activate intracellular pathways involved in energy expenditure and mitochondrial 

homeostasis during muscle contraction. This leads to a more favorable distribution in 

adipose and muscular tissues. But is also good to remember that there are still many 

issues to be determined on the most appropriate training intensity to cause these changes 

before exercise can be faithfully prescribed as "precision medicine" to combat the IR 

and its progression for Type 2 diabetes [54]. 



29 
 

 Besides all the efforts to help the patients living with diabetes, to determine the 

pathophysiological mechanisms and etiology of diabetes is still a very complex puzzle 

in many aspects. In order to reach some of these mechanisms, this is the first study to 

experimentally demonstrate that the adaptive responses caused by hyperglycemia in the 

intrauterine environment is involved to the insulin resistance and mitochondrial 

impairment on skeletal muscle of adult female offspring. Further, we highlighted the 

importance of AMPK, PI3K and MAPK protein levels during the regulation of 

mitochondrial dynamic (fission/fusion), and the connection between its molecular and 

ultrastructural muscular fiber compounds. In the other hand, there are some limitations 

to be pointed out; it is necessary to understand how the downregulation of these proteins 

effectively modify the downstream pathways involved in the metabolic control of 

energy expenditure and cell adaptation. And also if these mitochondrial adaptive 

changes associated to maternal hyperglycemia are reversible. This could help develop 

future therapeutic strategies by the elucidation of other proteins. The understanding of 

these mechanisms could not only help clarify the important regulators of the 

mitochondrial homeostasis in skeletal muscle, but also might help to provide more 

specific targets that could be used to better clarify some of the pathophysiological 

mechanisms of diabetic syndrome. 

 In summary, this study demonstrated the negative transgenerational effects of 

maternal hyperglycemia on insulin resistance, mitochondrial homeostasis and 

ultrastructural morphology of soleus muscle in adult female offspring in rat model. 

These data add a new understanding of the mechanisms of mitochondrial adaptation to 

the hyperglycemic environment and insulin sensitivity on diabetes etiology. 

 

4. METHODS AND MATERIALS  

 

4.1 Animals and experimental design 

 

All procedures in this study were approved by the Animal Experimentation 

Ethics Committee of our institution under the register number: 1326/2019. Sprague-

Dawley rats (one month of life) were acquired by CEMIB/Unicamp-Brazil and 

maintained in controlled conditions in our vivarium until mating. The sequence of the 

experimental design is shown in the figure 8. Our experimental sequence was performed 

in two steps: five days after birth, the female pups were injected with 70 mg/Kg of 



30 
 

streptozotocin (beta-cytotoxic drug) by subcutaneous (s.c.) route for composition of 

future mothers. The nondiabetic (Control) rats received citrate buffer (vehicle) within 

the same conditions as the diabetic group. The non-used male pups were anaesthetized 

(Thiopentax®, 120 mg/kg dosage) and sacrificed by decapitation. The female rats were 

maintained in polypropylene cages of a maximum of three rats/cage under controlled 

lighting (lights on from 7 am to 7 pm at 22–23°C). Oral glucose tolerance test (OGTT) 

was used as the inclusion criterion at adulthood (75 days of life). Rats given STZ that 

presented glycemic levels equal to or greater than 200 mg/dL in one or more time points 

were considered as diabetic, while the animals that presented lower glycemia were not 

used in this study. For the Control group, female pups with glycemic levels lower than 

140 mg/dL during OGTT were included [55]. The included female rats were mated with 

nondiabetic male rats (3 females: 1 male) for obtaining of their female pups for vaginal 

delivery, and composition of two experimental groups: Female offspring from 

nondiabetic mothers (Control) and female from diabetic mothers (FOD). All the female 

pups were maintained with their mother until the weaning (day 30 of postnatal life) in a 

maximum of 8 pups per mother, equivalent to functional nipple numbers. The female 

pups were housed and kept in polypropylene cages of a maximum of three rats/cage 

under controlled lighting (lights on from 7 am to 7 pm at 22–23°C), and the male pups 

were analyzed for another researcher team. 

At adult life (145 postnatal days), the female rats were submitted to OGTT for 

inclusion criteria similarly to their mothers. For Control group, female rats with 

glycemic levels lower than 140 mg/dL during OGTT were used. For FOD, were 

included those rats that presented glycemic levels equal to or greater than 140 mg/dL in 

one or more time points during OGTT, being considered rats with glucose intolerance, 

and those with glycemia greater 200 mg/dL as diabetic rats. At day 150 of life, all the 

included animals were weighted, anesthetized with Thiopentax® (120 mg/kg, 

intraperitoneal route) and killed by decapitation. One part of the blood samples was 

collected in dry tubes and centrifuged at 1,600 ×g for 10 min at 4°C. After, the serum 

was obtained and stored in a freezer at −80 °C and processed for biochemical analysis. 

Another aliquot of total blood was processed to obtain washed erythrocytes as 

previously described for de Souza [56]. Soleus muscle samples from the right paw were 

collected, weighted and immediately frozen in liquid nitrogen for Western Blotting and 

frozen tissue morphology. And samples from the left paw were collected in 

glutaraldehyde for ultrastructure analysis.  



31 
 

 

 

  



32 
 

4.2 Biochemical determinations 

 

4.2.1 Oral glucose tolerance test - OGTT 

All female rats were submitted to the OGTT according to Gallego [57] while 

glycemic values obtained during the two hours test were calculated in order to obtain 

the total area under the curve (AUC) [58]. 

 

4.2.2 Insulin measurement 

Serum insulin levels were measured by spectrophotometry using a commercial 

kit from Cayman® following the manufacturer instructions. 

 

4.2.3 Lipid profile 

Triglyceride and total cholesterol levels were measured in the serum by 

spectrophotometry - using commercial kits from Wiener® and following the instructions 

of the manufacturer. 

 

4.2.4 HOMA-IR, HOMA-beta and Triglyceride-Glucose index (TyG) 

calculations 

HOMA-IR and HOMA-beta calculations were made as follows by Matthews et 

al. [59] and Wallace et al. [60], respectively: 

HOMA-IR: [fasting insulin (μIU/ml) × fasting glucose (mmol/ml)]/22.5 

HOMA-beta: [20 × fasting insulin (μIU/ml)]/[fasting glucose (mmol/ml) − 3.5]  

 

In order to obtain the TyG (triglyceride-glucose index) values, the formula 

proposed by Simental-Mendía et al. [61]: 

TyG = [fasting triglycerides (mg/dL) × fasting glucose (mg/dL)]/2 

 

4.2.5 Oxidative stress biomarkers 

 Thiobarbituric acid (TBARS) and Reduced thiol groups (-SH) concentrations 

were measured in washed erythrocytes samples following procedures modified from de 

Souza et al.  [56]. 

 

4.3 Western blotting 



33 
 

 

 The samples of frozen muscle tissue were homogenized in RIPA Lysis Buffer 

extraction (Millipore®, USA) added with protease inhibitor (Sigma Chemical 

Company®, USA). Subsequently, they were centrifuged at 4000 rpm at 4°C for 20 min 

to remove the insoluble material. The determination of protein concentration was 

performed by method of Bradford [62]. Aliquots containing 70 μg of total proteins 

samples (n=6 animals per group) were separated on polyacrylamide gel (SDS-PAGE). 

Following the electrophoresis, the proteins were transferred to nitrocellulose membrane 

(Millipore, USA). The nonspecific blinding of proteins was blocked by incubation the 

membrane with 5% non-fat milk diluted in TBS-T buffer for 1h at room temperature.  

The membranes were incubated for 16 hours at 4°C with primary antibodies: PI3K 

(Santa Cruz®, USA, #sc-134766, 1:500), MAPK (Erk1/2) (CellSignaling®, USA, 

#4695, 1:800), AMPK (Abcam®, USA, #ab32047, 1:800), MFF (CellSignaling®, USA, 

#84580, 1:800), MFN-2 (Abcam®, USA, #ab56889, 1:800), and β-Actin (Abcam®, 

USA, #ab8224, 1:800). After washing in TBS-T, the membranes were incubated for 90 

min with an HRP-conjugated secondary antibody and specific for each primary 

antibodies (CellSignaling®, USA, #7074, #7076, 1:10,000). The reaction was 

developed by an ECL reagent kit (Bio-Rad®, USA) and the images of the bands were 

captured using a CCD camera (a G: BOX system (Syngene®, Cambridge, UK). The 

integrated optical densities (IODs) of the targeted protein bands were measured using 

ImageJ software (National Institutes of Health, USA). Expression levels were 

normalized by the values obtained from the internal control (β-actin) and the results 

were normalized from fold change and expressed as mean ± SD. 

 

4.4 Morphological analyses 

 

4.4.1 Central nuclei number in analysis of the soleus muscle fibers 

The samples of frozen muscle (n=6 animals per group) were cut into 10-mm 

thick cross-sections in a cryostat (Leica CM 1800, Germany). The cross-sections were 

fixed for 10 min on glass slides for microscopy with cold acetone and then stained with 

hematoxylin and eosin (H&E) (n=6 animals samples per group). The slides were 

examined using a light microscopy and subsequently photographed (DMR, Leica 

coupled with digital camera CCD-IRIS/RGB, Sony, Germany) for the analysis of 

centrally located nuclei [63]. 



34 
 

 

4.4.2 Immunohistochemical analysis 

The frozen sections of muscle (n=6 animals per group) were hydrated for 10 min 

followed by endogenous peroxidase block using a 3% H2O2 solution embedded in 

methanol. After washing, the sections were incubated with 3% of fetal bovine serum 

solution at 37°C. Then, the slides were incubated with antibodies against WB-myosin 

heavy chain fast (WB-MHCf, Novocastra, 1:120) or WB-MHC slow (WB-MHCs, 

Novocastra, 1:180) overnight at 4°C. Next day, the samples were washed three times 

with 0.01 M PBS and incubated with 3.3-diaminobenzidine tetrahydrochloride (DAB) 

(Sigma-Aldrich) for 5 minutes, washed with 0.01 M PBS for 10 minutes and stained 

with hematoxylin for 1 minute. The visualization of fast and slow fibers was done using 

a light microscopy (DMR, Leica coupled with a CCD-IRIS/RGB digital camera, Sony), 

and the fiber type were counted and quantified using the image processing and analysis 

Java software ImageJ (National Institutes of Health, USA) [64]. 

 

4.4.3 Ultrastructural analysis of muscle samples 

For TEM, the samples (n=6 animals per group) were fixed in 2% glutaraldehyde 

solution in 0.1 M phosphate buffer (pH 7.4) for 24 h at room temperature and then 

postfixed in 1% osmium tetroxide in the same buffer for 2 h. After being washed with 

distilled water, the samples were contrasted with an aqueous solution of 0.5% uranyl 

acetate for 2 h at room temperature, dehydrated in a graded acetone series (50%, 70%, 

90% and 100%), and embedded in Araldite resin. Ultrathin sections were contrasted 

with uranyl acetate and lead citrate and were then observed and photographed with a 

Jeol JEM – 100CXII accoupled to Hamamatsu ORCA-HR digital camera.  

 

 

4.5 Statistical analysis 

 

The effect size was determined considering the variability of our biomarkers and 

the two experimental groups based on previous experiments conducted in our 

laboratory. Based on this effect size, the sample calculation was 10 rats per group for 

biochemical analysis and OGTT, while it was 6 rats per group for morphological 

analysis and Western Blotting quantifications. The calculation of the sample size 

defined by a specialist in Biostatistics in order to obtain results in animal research 



35 
 

compatible with the aimed translational sampling. All data are presented as means ± 

standard deviation (SD) and were analyzed by the Biostatistics from Research Support 

Office of our Institution. The minimum significance limit of 5% (p<0.05) was 

considered. For the comparisons between glycemic means by oral glucose tolerance test 

(OGTT), the area under the curve (AUC) during OGTT, serum insulin and triglyceride 

concentrations, and central nuclei quantification, the Gamma Distribution test was used 

in view of the asymmetric distribution of the data. For TBARS, immunohistochemical 

and Western blotting analysis, reduced thiol Groups and total cholesterol, the Student’s 

t-test was used. 

 

Author Contributions: Conceptualization - E.K., and D.C.D.; performance of 

methodology: E.K., L.L.C., F.Q.G., Statistical analysis and interpretation – J.E.C., 

D.C.D.; E.K.; Ultrastructural analysis: E.K., D.C.S.; Data discussion: E.K., L.L.C., 

F.Q.G., D.C.S., L.A.J., G.T.V., and D.C.D., Writing, Review and Editing of manuscript 

- E.K., L.L.C., D.C.S., G.T.V., and D.C.D.; Funding Acquisition, D.C.D. All authors 

have read and agreed to the published version of the manuscript. 

 

Funding: This study was supported by São Paulo Research Foundation- FAPESP - 

Grant number 2016/25207-5) coordinated by Dr. Débora Cristina Damasceno. 

 

Institutional Review Board Statement: Not applicable.  

 

Informed Consent Statement: Not applicable. 

 

Acknowledgments: The authors thank Mr. Danilo Chaguri, Mr. Jurandir Antonio, and 

Mr. Carlos Roberto G. Lima (Academic Support Assistant – ASA, UNIPEX). Authors 

thank Mr. Lucas Gabriel Rodrigues Venturini (USP) by providing the antibodies for 

western blotting analyzes, and Barshana Karki, (Massachusetts, United States), for 

improving the writing of the English text. We thank also to Coordination of Superior 

Level Staff Improvement (CAPES) for the Eduardo Klöppel’s scholarship. 

 

Conflicts of Interest: The authors declare no conflicts of interest. 

 

 



36 
 

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431x20177035. 

  



43 
 

 
 
 
 
 
 
 
 
 

CAPÍTULO II 
 

CHAPTER 2 
 
 
 

  



44 
 

PROHIBITIN-MODULATED MITOCHONDRIAL METABOLISM IN 

YOUNG AND ADULT OFFSPRING AS ADAPTATIVE RESPONSE AFTER 

INTRAUTERINE DIABETIC EXPOSURE: A PROTEOMIC STUDY IN 

LIVER OF FEMALE RATS. 

Eduardo Klöppel1, Olga Vajnerova2, Barbora Kaftanová2, Adam Eckhardt3, Gustavo 

T. Volpato4, Václav Hampl2, Débora C. Damasceno1* 

 
1Laboratory of Experimental Research on Gynecology and Obstetrics, Postgraduate 

Course on Tocogynecology, Botucatu Medical School, Sao Paulo State University 

(UNESP), Botucatu, 18618-689, São Paulo State, Brazil. 
2Department of Physiology, Second Faculty of Medicine, Charles University, 

Prague, Czech Republic. 
3Department of Translational Metabolism. Institute of Physiology of the Czech 

Academy of Sciences, Prague, Czech Republic. 
4Laboratory of System’s Physiology and Reproductive Toxicology, Institute of 

Biological and Health Sciences, Federal University of Mato Grosso (UFMT), Barra 

do Garças, 78600-000, Mato Grosso State, Brazil. 

 

 

 

*Correspondence to:  Profa. Dra. Débora Cristina Damasceno 

 Unidade de Pesquisa experimental - UNIPEX 

 Faculdade de Medicina de Botucatu - UNESP 

 Distrito Rubião Júnior s/n 

 CEP. 18618-970 -  Botucatu/SP, Brasil 

 Phone/Fax: (55 14) 38801630  

 debora.damasceno@unesp.br 

  

mailto:damascenofmb@gmail.com


45 
 

ABSTRACT 

 

The concept of intrauterine plasticity presupposes those physiological adaptations of 

offspring in early life can predict a phenotype for long-term. Maternal diabetes is 

one condition that can cause maladaptive response in the fetus and increase the risk 

of insulin resistance and metabolic disease in the offspring. However, the 

mechanism underlying these fetal adaptations remains obscure. In this way, we 

aimed to investigate the global expression of proteins in the mitochondrial content 

of female rats coming from diabetic mothers at weaning and adulthood. Female 

offspring coming from mothers with diabetes (FOMD) were obtained through 

pregnancy of Wistar rats with streptozotocin neonatally-induced diabetes. At 30 and 

110 days of life, FOMD rats were killed, and blood and liver were collected. Blood 

was used for insulin analysis and liver was processed for obtaining the fresh 

mitochondrial content. Proteomics analysis were performed in the mitochondrial 

tissue by nLC-MS/MS mass spectrometry. A total of 399 proteins were identified in 

the liver mitochondria and 54 and 51 proteins were differentially expressed, 

respectively at 30 and 110 days of life. In both ages the FOMD rats presented an 

increase mitochondrial metabolism and biogenesis in addition to increased beta-

oxidation and endoplasmic reticulum stress. At 110 days of life the FOMD rats 

presented also increased response to oxidative stress. Furthermore, prohibitin is 

upregulated in both ages and appears to have a pivotal role in theses adaptative 

responses found in the FOMD rats. 

 

Keywords: Mitochondria; insulin resistance; prohibitin; proteomics; fetal 

programming; rat. 

  



46 
 

INTRODUCTION 

 

The developmental plasticity processes change tissular structure and function by 

effects on anatomy or microanatomy or on cellular activity in these tissues. Early life 

can change the physiological configurations of offspring so that the response to an 

environmental challenge will be different in long-term. This process evolves as an 

adaptive phenomenon to help the individual to the environment, but that may be 

maladaptive if the environmental prediction is incorrect because of faulty signal 

transduction, maternal illness, maternal consumption of a non-repair diet (or even 

dieting to lose weight) or unpredictable environmental changes [1]. 

Evidence indicates that children of women with pre-gestational Diabetes 

mellitus (type 1 and type 2 DM) and gestational diabetes had a higher prevalence of 

glucose intolerance and insulin resistance compared to controls [2]. Pettitt et al. (1991) 

also confirmed this effect when they verified abnormal glucose tolerance in the 

offspring of Pima indigenous women during pregnancy [3]. The authors correlated 

metabolic abnormalities that occur in diabetic pregnancy with states of insulin 

resistance, hypertension, obesity and diabetes in the offspring. Therefore, the 

mechanisms by which maternal hyperglycemia increases metabolic risk in offspring 

have not been still fully elucidated. 

Pre-clinical studies are very important to analyze pathophysiological 

mechanisms and to advance our knowledge regarding the discovery of new treatments 

for different types of diseases, including Diabetes mellitus. In our laboratory, several 

experimental models of diabetes have been developed in female rats with the aim of 

associating the diabetic syndrome and pregnancy for a better understanding of the 

outcomes in the offspring [4–7]. A more recent study about to be published by our 

research group (Paula et al., 2021 - in press) showed that daughters coming from 

diabetic rats had impaired glucose tolerance and dyslipidemia in young adulthood. This 

confirms the harmful repercussions of maternal diabetes on successive generation. 

 To verify the mechanisms involved in the transgenerational outcomes induced 

by maternal diabetes, Klöppel et al. (2021 – in press) in our laboratory showed that 

mature adult female rats from diabetic dams presented mitochondrial alterations; 

reduced protein expression of transcription factors implicated in the insulin signaling; 

abnormal relationship between mitochondrial and endoplasmic reticulum (ER) leading 

to ER stress. Taken together, these findings contributed to insulin resistance in the adult 



47 
 

rats (150 days of life). 

Insulin resistance is a major metabolic feature in obesity and a key factor in the 

pathogenesis of type 2 DM. The underlying molecular mechanisms are complex and 

still incompletely understood. Insulin signaling is impaired at several levels, but 

whether these changes are primary or secondary to the metabolic changes remains 

unclear. In general, insulin resistance is considered “harmful” and is related to Type 2 

DM. Therefore, it is necessary to maintain insulin sensitivity as an integral component 

of normal metabolic physiology [8]. 

 In other hand, liver is a tissue with a high adaptative and metabolic rate and rich 

in mitochondria. Works as a metabolic sensor and is involved in glycogenesis, 

glycogenolysis, lipolysis, ketogenesis and in the production of ATP for other tissues, 

being, therefore, highly responsive to changes related to insulin and increased serum 

glucose levels [9,10].  

Considering the previous results from our laboratory on the evaluation of mature 

adult offspring (150 days old) from diabetic mothers, there is evidence that the 

mitochondrial imbalance induced by the intrauterine hyperglycemic insult leads to the 

adaptive responses present in these animals. Thus, there is a need to develop 

mechanistic studies to better understand the relationship between insulin resistance, 

glucose intolerance and plasticity between mitochondria and endoplasmic reticulum in 

different moments of postnatal life: after weaning (30 days of life) and in young 

adulthood (110 days of life). life). Thus, the aim of this study was to investigate how 

maternal diabetes influences protein expression related to metabolism and 

mitochondrial function in the offspring's liver at weaning and whether this change 

remains in adulthood. Furthermore, we intend to verify if this dysfunction altered the 

expression of specific proteins of mitochondrial energy metabolism induced by adaptive 

plasticity since intrauterine life. 

 

MATERIALS AND METHOD 

 

All procedures in this study were approved by the Animal Experimentation 

Ethics Committee of our institution. Wistar female rats at five days after birth were 

injected with 70 mg/Kg of streptozotocin (beta-cytotoxic drug) by subcutaneous (s.c.) 

route for composition of future diabetic mothers. The control rats received only citrate 

buffer (vehicle) in the same conditions as the diabetic group. All the male pups were not 



48 
 

used and anaesthetized (Thiopentax®, 120 mg/kg dosage) and killed. The female rats 

were kept in polypropylene cages of a maximum of three rats/cage under controlled 

conditions of light and temperature (7 AM to 7 PM at 22–23°C). Oral glucose tolerance 

test (OGTT) was used as the inclusion criterion at adulthood (75 days of life). The rats 

that received STZ and presented glycemic levels equal to or greater than 200 mg/dL in 

one or more time points were considered as diabetic, while the animals that presented 

lower glycemia were discarded. In the Control group all female pups with glycemic 

levels lower than 140 mg/dL during OGTT were used as control mothers [55]. In 

sequence all included female rats were mated with normal and healthy male rats (3 

females: 1 male). The female pups were obtained for vaginal delivery, and composition 

of two experimental groups: Female offspring from healthy mothers (Control) and 

female from mothers with diabetes (FOD). All the female pups were maintained with 

their mother until the weaning (day 30 of postnatal life) in a maximum of 8 pups per 

mother, equivalent to functional nipple numbers. At 30 days of life each litter of female 

rats were separated in two: One to compose the group with 30 days old rats, and the 

other were housed and kept in polypropylene cages of a maximum of three rats/cage 

under controlled lighting (lights on from 7 am to 7 pm at 22–23°C) until 110 days of 

life. 

 At 30 and 110 days of life, according to each experimental group, Control and 

FOMD female rats were killed and blood for serum obtaining and liver for 

mitochondrial content extraction were collected. 

 

Insulin measurement 

 

Serum insulin levels will still be measured by spectrophotometry using a commercial kit 

from Cayman® following the manufacturer instructions. 

 

Chemicals 

 

All chemical reagents used in this experiment were purchased from Sigma-Aldrich®. 

 

Mitochondrial isolation 

 

The mitochondrial isolation was performed with fresh liver tissue. The liver 



49 
 

samples were homogenized in 220mM D-Manitol 70mM sucrose 2mM hepes pH 7.2 

solution, using a Glass/Teflon Potter Elvehjem homogenizer for 10 seconds. Then, the 

samples were centrifugated at 600rpm for 10 minutes at 4ºC. The supernatant was 

collected and filtered using a teflon net. The supernatant was again centrifuged at 

15.000rpm for 10 minutes at 4ºC. The supernatant was then discarded and the pellet 

with all mitochondrial content was frozen for further analysis. 

 

Mitochondrial protein extration 

 

Around ~30mg of each sample was add to 10-fold volume/weight 6M GuHCl 1mM 

EDTA 10nM Dithiothreitol (DTT) 0.01% NaN3 with Protease Inhibitor Cocktail. The 

samples were then homogenized using a T 10 basic ULTRA-TURRAX® - IKA for 10 

seconds. The samples were heated for 5 minutes as 95ºC, again homogenized and then 

and kept during 48 hours in a shaker at 4ºC room. After 48 hours, the samples were 

again heated for 30 minutes at 56ºC. After, a 30mM iodoacetamide (IAA) was add and 

each sample was kept in dark room at 25ºC shaking for 45 minutes. Further, the samples 

were centrifugated at 15.000rpm for 15 min at 4ºC. The supernatant was collected and 

add to a 4-fold volume of cold acetone (-20ºC) overnight. The samples were again 

centrifugated at 15.000rpm for 15 min at 4ºC, followed by trypsin (Sigma-Aldrich®) 

addition in 1/25 volume proportion overnight at 37ºC, shaking. By the last, the peptides 

concentration was measured by commercial kit. 

 

Peptide quantification 

 

The peptides were quantified in order to access the exact amount into to Nano-liquid 

chromatography equipment. The quantification was done using a commercial kit of 

quantitative colorimetric peptide assay from Thermo Scientific® 

 

Purification of samples 

 

Purification of samples was performed by extracting tryptic peptides using Stage Tips 

[11]. 

 

 



50 
 

Mass specrometry analysis 

 

The Nano-liquid chromatography procedure, mass spectrometry (MS), and tandem MS 

(MS/MS) analyses were performed as described in our previous studies [12]. Injection 

of each sample was 0,2 ug. Database searches were carried out using the Uniprot 

databases (uniprot.org) as described in [13] with the taxonomy restricted to Rattus 

norvegicus. Only significant hits (MASCOT score ≥80 for proteins (at least two 

peptides for protein) and ≥25 for peptides, http://www.matrixscience.com) were 

accepted.  

 

Statistics 

 

The suitable software (Profile Analysis version 2.1, Bruker Daltonik) was used to 

evaluate differences in the protein composition between groups by means of MS label-

free quantification. The peptides under consideration had to be found in at least 40% of 

all samples, regardless of the group, and had to be found in at least 50% in one group.  

The p-value was returned by two-sample t-tests (and corrected for multiple-testing by 

false discovery rate rate (FDR) based on frequency histogram) (adjusted p-value 

threshold 0.05). 

 
RESULTS AND DISCUSSION 

 

To date, this is the first study to report a proteomic evaluation of the 

transgenerational effects of maternal diabetes on the hepatic mitochondrial content. No 

studies have reached what this plasticity period causes to the pattern of protein 

expression during the weaning period and later in adulthood. From our analysis, 399 

proteins were identified in the liver mitochondria of rats (Control and FOMD groups) at 

30 days of life (Supplementary Table S1) and 110 days of life (Supplementary Table 

S2) in which 54 and 51 proteins were differentially expressed, respectively. After 

analysis of these proteins of the FOMD group at weaning and adulthood periods, 

respectively, we verified that three and two of them were downregulated, whereas 51 

and 49 proteins were upregulated (Tables 1 and 3). 

36 KEGG and nine Reactome pathways at 30 days of life, and 31 KEGG and 19 

Reactome pathways at 110 days of life were verified. 124 biological processes were 



51 
 

enriched in the protein list of the FOMD group at 30 days of life. Considering the same 

experimental group at 110 days of life, 162 biological process terms were enriched in 

the protein list of the FOMD group. 

Since of the list of the enriched pathways and processes, we mostly found those 

related to the lipid metabolism (fatty acid degradation and oxidation), amino acid and 

mitochondrial metabolism and endoplasmic reticulum stress in both ages while the 

antioxidant enzymes and mitochondrial biogenesis were increased at 110 days of life 

(Tables 2 and 4). 

 

Insulin level measurement  

 

The data will be presented and discussed later. This is due to delay in the kit 

arrival in Brazil, which impaired to conclude this analysis. 

 

Protein Folding and Endoplasmic Reticulum (ER) Stress 

 

The protein enrichment showed the biological process “Response to 

endoplasmic reticulum stress (ER Stress)” in FOMD at days 30 and 110 of life. On 

postnatal day 30, the daughters from diabetic rats presented “Positive regulation of 

protein folding”, and at day 110 “Protein refolding” processes were identified. In these 

matters, at 30 days of life, the FOMD group presented upregulation of the enriched 

proteins Calreticulin (P18418), Endoplasmin (Q66HD0), and Protein disulfide-

isomerase (P11598 and G3V6T7). At 110 days of life, FOMD rats showed 

overexpression of mitochondrial heat shock 10-kDa protein (P26772) and mitochondrial 

heat shock 70-kDa protein (F1M953) (Table 2), indicating ER stress as well as 

mitochondrial remodeling and biogenesis [14,15]. Moreover, endoplasmic reticulum 

chaperone (BiP) was also overexpressed in FOMD groups in both ages (P06761) 

(Tables 2 and 4). BiP is a hallmark with the pivotal role as a chaperone, modulating the 

unfolded protein response (UPR) under ER accumulation of misfolded protein and 

stress [16,17], showing the relationship among these proteins involved with ER stress 

and mitochondrial biogenesis in the female rats from diabetic dams. 

In our laboratory team, has been observed that adult female rats (150 days of 

life) coming from diabetic mothers presented insulin resistance (IR) and glucose 

intolerance (Klöppel et al. 2021– in press). which activates several other stress kinases 



52 
 

that eventually might impair the insulin signaling pathway. According to Klöppel et al. 

(2021 - in press) the mature adult female rats presented lower protein expression of 

AMPK/ERK1/2 and other transcription factors, leading to damaged insulin signaling 

pathway. Besides the fact that the accumulation of misfolded or unfolded proteins 

results in ER stress or UPR, it may be a mechanism related to IR once UPR interacts 

with AMPK/ERK1/2 [18]. The activation of ER stress-induced kinases impairs the 

insulin signaling pathway and promotes lipid accumulation through the synthesis of 

proinflammatory cytokines, which also negatively affect insulin signaling [19]. 

Moreover, the high glucose concentration in the intrauterine environment might be the 

cause of excessive mitochondrial respiration as an adaptative response that disturbs 

mitochondrial homeostasis and dynamics in the young and adult offspring. Then, it is 

very conceivable that the mechanism behind this ER stress state is a response to an 

increased mitochondrial metabolism leading to mitochondrial biogenesis. 

 

Lipid Metabolism 

 

“Fatty acid beta-oxidation” biological process and “Fatty acid degradation” and 

“Mitochondrial Fatty Acid Beta-Oxidation” pathways were strongly enriched in the 

differentially expressed protein list at the days 30 and 110 of life of the FOMD group. 

Among these differentially expressed proteins, we highlight the Mitochondrial 3-

hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) (P22791), long-chain 

acyl-CoA dehydrogenase (P15650), Electron transfer flavoprotein (Q68FU3), Enoyl-

CoA hydratase (P14604), and mitochondrial long-chain trifunctional enzyme (MTP) 

(Q64428), which were all upregulated at day 30 and 110 of life of the FOMD group 

(Tables 2 and 4). These proteins are involved in the complete fatty acid β-oxidation 

process [20–24] and their increased long-life might be related to a ketogenic pathway 

phenotype in this group, as observed at the 110 days of life. This ketogenic state can be 

represented by both acetyl-CoA acetyltransferase (P17764) and HMG-CoA synthase 

upregulation, which is one of the enzymes also involved in the last step of the 

mitochondrial beta-oxidation (thiolases) and first irreversible step of the ketogenesis 

pathway, respectively, playing a major role in ketogenic liver metabolism [25,26]. 

Moreover, the MTP complex is recognized as responsible for catalyzing the 

three last steps of the β-oxidation [27], and its upregulation among the life of the FOMD 

rats might be explained by an adaptation to the maternal hyperglycemic state, since the 



53 
 

excessive glucose concentrations increase the production of TCA cycle products, such 

as acetyl-CoA and NADH [28]. In addition, the increase of both acetyl-CoA and NADH 

levels induces the acetylation of mitochondrial proteins [29], including the MTP 

complex. There are studies involving MTP overexpression and deacetylation as a 

protective novel for reducing insulin resistance [20]. This evidence suggests that the 

MTP might have a pivotal role in the adaptive response to maternal hyperglycemia 

during the fetal developmental and postnatal periods, which remained present during the 

adulthood of the FOMD rats related to insulin resistance. It is still pertinent to be 

elucidated if the increased MTP is just a consequence of an increased mitochondrial β-

oxidation or an adaptive mechanism to protect the cell against lipotoxicity during 

mitochondrial respiration and biogenesis in liver. In this matter, more studies are needed 

to better understand the MTP's role on glucose metabolism, insulin resistance, and 

diabetes onset in offspring from diabetic mothers. 

 

Mitochondrial metabolism, biogenesis, and Oxidative stress 

 

The enrichment shows an upregulation in the pathways of “Oxaloacetate 

metabolic process”, “Citrate cycle (TCA cycle)”, “Pyruvate metabolism” and 

“Respiratory electron transport” at both studied ages in FOMD group (Tables 2 and 4). 

The maternal hyperglycemia during pregnancy caused a long-life (30 and 110 days of 

life) upregulation in the offspring’s expression of Electron transfer flavoprotein 

(Q68FU3), Pyruvate carboxylase, mitochondrial (P52873), Pyruvate dehydrogenase E1 

(P49432) and Prohibitin (PHB) (P67779). At 30 days of life, the FOMD rats presented 

upregulation of Alcohol dehydrogenase 1 (P06757), Malate dehydrogenase (O88989), 

Pyruvate dehydrogenase (P49432) and Cytochrome b-c1 complex (P32551), while at 

110 days of life the rats presented upregulation of Malate dehydrogenase 2 (P04636), 

and Succinate dehydrogenase (Q920L2). In other hand, the Reactome pathways of 

“Transcriptional activation of mitochondrial biogenesis”, “Mitochondrial biogenesis” 

and “Cristae formation” was present only in FOMD rats at 110 days of life (Table 4). 

The increase of these pathways is highlighted by the upregulations in the proteins 

related to mitochondrial cristae respiration machinery, such as Isocitrate dehydrogenase 

[NADP] (P56574), Mitochondrial membrane ATP synthase F(1)F(0) (G3V6D3), ATP 

synthase subunit O (Q06647), ATP synthase subunit f (D3ZAF6), ADP/ATP translocase 

2 (Q09073), Prohibitin (PHB) (P67779). The adult FOMD rats presented an increased 



54 
 

expression of the mitochondrial respiratory chain proteins, and this might be related to 

its driver PHB [30], which was possibly caused by a chronic demand of exacerbated 

oxidation and degradation of lipid as showed in our results. PHB is considered as a 

mitochondrial chaperone [31], and has been observed to be related to mitochondrial 

respiratory chain subunit degradation, association and activity of the oxidative 

phosphorylation system, mitochondrial biogenesis and networks, and apoptosis, and 

mitophagy [32]. Then, it is suggested that overexpression of PHB in the weaning and 

young adult phase of rat daughters coming from diabetic dams might be implicated to 

plasticity of the offspring involving several mitochondrial alterations, such as 

mitophagy, found at 150 days of life (Klöppel et al, 2021), confirming an abnormal 

cellular machinery on the mitochondrial biogenesis, oxidative stress, and PHB 

expression/levels induced by hyperglycemia since intrauterine life. 

The biological process “Reactive oxygen species metabolic process” and the 

pathways “Glutathione metabolism” and “Detoxification of Reactive Oxygen Species” 

were enriched in the FOMD groups at 110 days of life (Table 4). The enzymes 

Glutathione S-transferase (B6DYP8), Glutathione peroxidase (Q6PDW8), Carbamoyl-

phosphate synthase (P07756), Superoxide dismutase [Mn], mitochondrial (P07895), 

Catalase (P04762) and Isocitrate dehydrogenase [NADP], mitochondrial (P56574) were 

upregulated in this group (Table 3), which represents a complete machinery against 

reactive oxygen species (ROS) formation. To date, there was not studies reporting 

transgenerational effects, leading to increased antioxidant defenses of mitochondrial 

network on the offspring at adulthood. It is recognized that the increased activity in the 

mitochondrial respiratory chain is the main cause of higher production of ROS in 

pathologic and adaptive conditions [33,34]. Therefore, it looks to be mostly a 

consequence of the increased global metabolism in the liver tissue of FOMD rats.  

In addition of energetic function, mitochondria are also a major source of 

reactive oxygen species (ROS) [35]. The energy produced is used by ATP synthetase to 

transform ADP into ATP. However, mitochondrial respiration also results in the 

formation of ROS such as superoxide and hydrogen peroxide. For prevention of an 

excessive ROS production, the lowering the mitochondrial proton gradiente (uncoupling 

substrate oxidation from ATP production) has been performed, resulting in a 

pronounced lowering in mitochondrial production of ROS [36]. Evidence shows that 

prohibitin acts against oxidative stress, Muraguchi et al. (2010) verified overexpressed 

PHB prevented mitochondrial membrane depolarization and cause an inhibition of cell 



55 
 

death induced by hypoxia in H9c2 cardiomyocytes [37]. PHB overexpression protected 

mitochondria aginst hydrogen peroxide-induced oxidative stress, reduced apoptosis by 

mitochondrial pathway, and diminished mitochondrial membrane permeability 

compared with untransfected cardiomyocytes [31]. 

Mattox et al. (2021) reported that prohibitin caused an upregulation of 

glutathione reductase enzyme in the circulation [38]. Besides, PHB is associated to 

regulation of genes with GSH homeostasis, such as glutamylcysteine synthetase, 

glutathione peroxidase, and glutathione reductase [39]. Taken together, these evidences 

indicates that the prohibitin might be a perspective to drible the diabetes or insulin 

resistance-induced oxidative stress, contributing to decrease the damages from 

hyperglycemia in dams and successive generation. The upregulated expression of 

enzymes related to oxidative stress might be implicated to prevent the oxidative stress 

complications, confirming the protective measurement. 

 

Prohibitin as a metabolism sensor 

 

Prohibitin (PHB) (P67779) was strongly upregulated and enriched in both 30 

and 110 days of life in FOMD (Tables 1-4), participating in pathways related to 

mitochondrial metabolism and biogenesis. PHB complex comprises two subunits (1 and 

2) in mitochondria. It is an evolutionary conserved, ubiquitously expressed protein with 

pleiotropic functions [40,41]. For this reason, PHB shows to be able to participate in 

different biological processes and pathways related to mitochondrial metabolism and 

biogenesis [41]. PHB is known to control mitochondrial-encoded subunits of the 

respiratory in cristae formation and function [42,43] as well as prevents ROS by 

modulating the complexes 1-4 in the mitochondrial respiratory chain [44]. PBH also 

influence mitochondrial mitophagy when it binds the autophagosomal membrane-

associated protein LC3 [45] and activate the mitochondrial fusion trough the dynamin-

like GTPase OPA1 [46]. Some studies have reported a controversial role for PHB in 

cancer [47,48], while PHB appears to be protective against liver diseases [49]. In animal 

model, PHB1 inhibition caused alteration on the mitochondrial respiratory chain 

functioning, leading to an enhanced the production of oxidants [50]. Despite the fact 

that PHB is now also associated to the insulin signaling trough the MAPK/ERK 

pathway [51], a mystery of the PHB complex mechanisms behind the different 

protective/adaptative functions still remains. In the present study, the FOMD rats 



56 
 

showed PHB-induced overexpressed pathways on mitochondrial oxidative capacity, 

which implies that PHB might have also a pivotal role in adaptative responses to 

maternal hyperglycemia and long-life phenotype. In this sense, although PHB has been 

studied for decades, there is still a mechanistic gap in the systemic and local effects of 

PHB on in vivo metabolism. Thus, the prohibitin might be the hallmark for better 

mechanistic knowledge between impaired glycemic control and mitochondrial 

adaptations, contributing for its understanding in the diabetic syndrome and possible 

therapies. 

 

Conclusions 

 

In conclusion, we have shown that the mitochondrial content in liver of newly 

weaned and young adult females rats coming from diabetic mothers undergone to a fetal 

plasticity that remained for life. These adaptations are associated with different 

expression of multiple proteins and the abundance of oxidative enzymes in 

mitochondrial network appears to be part of its phenotype. In addition, our data suggest 

that this is accompanied by a shift in the glucose oxidative metabolism to a lipid 

oxidation and degradation. Although of a universal increase of mitochondrial 

metabolism and ER stress response, the adaptive response to hyperglycemia is largely 

related to many other mechanisms, such as decreased insulin signaling events that were 

detected by the current method. The changes in overexpressed protein in these multiple 

pathways add information on how these interactions happened in the diabetic 

intrauterine environment. Viewed in this way, the current evidence suggest that PHB 

can have a key role on adaptative responses related to mitochondrial function through 

life, although of the minor available knowledge of its biological functions and pathways 

need more clarification. Therefore, the role and exact pathways in several of the 

regulated differentially proteins remain to be elucidated.  

 

Acknowledgements  

We acknowledge Mr. Armenuhi Kirakosjanová and Ing. Statis Pataridis for 

skilled technical assistance, and to Coordination of Superior Level Staff Improvement 

(CAPES) for the Eduardo Klöppel’s scholarship (88887.473269/2020-00) in Prague for 

12 months. 



57 
 

Funding  

This study was supported by São Paulo Research Foundation- FAPESP Grant 

number 2016/25207-5 - coordinated by Dr. Débora Cristina Damasceno), and CAPES-

PRINT (PRINT - PROGRAMA INSTITUCIONAL DE INTERNACIONALIZAÇÃO) 

Contribution statement  

EK, DCD, VH, BK and AE conceived and designed the study. EK, BK and AE 

performed the experiments. EK, DCD, and AE analyzed and interpreted the data. EK 

and DCD wrote the manuscript, and all authors will check the manuscript for 

intellectual content and approved the final version.  

 

Duality of interest  

All authors declare that there is no duality of interest associated with this 

manuscript. 

 

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