New generation antidiabetics: what are the implications on spermatogenesis nutritional support?

Universidade do Minho Escola de Ciências João Pedro Cardoso Ribeiro New Generation Antidiabetics: what are the implications on spermatogenesis nutritional support? setembro de 2019 New Generation Antidiabetics: what are the implications on spermatogenesis nutritional support? João Pedro Cardoso Ribeiro UMinho | 2019 Instituição de Acolhimento: Unit for Multidisciplinary Research in Biomedicine (Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto) Universidade do Minho Escola de Ciências setembro de 2019 João Pedro Cardoso Ribeiro New Generation Antidiabetics: what are the implications on spermatogenesis nutritional support? Dissertação de Mestrado Bioquímica Aplicada Especialização em Biomedicina Trabalho efetuado sob a orientação do Professor Doutor Pedro Fontes Oliveira ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iii AGRADECIMENTOS A realização desta dissertação de mestrado foi suportada por contribuições e incentivos fundamentais sem os quais não seria possível a conclusão desta etapa académica e de crescimento pessoal. Assim sendo, tenho todo o gosto em expressar os meus sinceros agradecimentos a todos os que contribuíram direta ou indiretamente para a elaboração deste trabalho. Ao meu orientador, Professor Doutor Pedro Fontes Oliveira, por me ter acolhido no seu grupo de trabalho, financiamento, conhecimento científico, paciência e disponibilidade demonstrada, bem como o acompanhamento e conselhos dados na duração deste percurso. Agradecer também todas as críticas, correções e sugestões que contribuíram para o melhoramento deste trabalho. Sem a sua orientação este trabalho não seria possível. À minha coorientadora, Professora Sandra Paiva, pela total disponibilidade que demonstrou e por me ter guiado para o fantástico grupo de trabalho do Professor Doutor Pedro Oliveira e Professor Doutor Marco Alves. Ao Professor Doutor Marco Alves pelo conhecimento científico, disponibilidade e motivação que me forneceu durante todo o percurso. Aos colegas de laboratório que me ensinaram as rotinas do laboratório bem como as técnicas que foram realizadas neste trabalho, nomeadamente: Ana Martins, David Carrageta, Doutora Raquel Bernardino, Doutora Ivana Jarak, Ana Maria Silva e Doutor Romeu Videira. Obrigado pela simpatia, paciência e conhecimentos transmitidos. Aos restantes colegas do grupo de trabalho: Doutor David Martín-Hidalgo, Doutor Tito Jesus, Susana Almeida, Luís Crisóstomo, Bruno Moreira, Sara Pereira, Cláudia Peixoto, Anette Veiga, Patrícia Braga e Cassandra Santos que me integraram no grupo e se mostraram sempre disponíveis para ajudar ao longo da realização da dissertação de mestrado. A todos os meus amigos, em especial o Henrique, SóFres e Maazou que me motivam e me fazem rir mesmo nos piores momentos. À Rita Peixoto pela paciência que tem comigo, por me motivar e me fazer melhorar todos os dias que passamos juntos. A toda a minha família, em especial os meus pais, irmã, e avós por me motivarem e sustentarem para tornar este trabalho possível e acima de tudo, por me tornarem no que sou hoje. iv DECLARAÇÃO DE INTEGRIDADE Declaro ter atuado com integridade na elaboração do presente trabalho académico e confirmo que não recorri à prática de plágio nem a qualquer forma de utilização indevida ou falsificação de informações ou resultados em nenhuma das etapas conducente à sua elaboração. Mais declaro que conheço e que respeitei o Código de Conduta Ética da Universidade do Minho. v ANTIDIABÉTICOS DE NOVA GERAÇÃO: QUAIS SÃO AS IMPLICAÇÕES NO SUPORTE NUTRICIONAL DA ESPERMATOGÉNESE? A incidência de doenças metabólicas como Diabetes mellitus tipo 2 (TD2M) e obesidade tem aumentado entre crianças e jovens adultos. Estas doenças estão intimamente ligadas, portanto, novos agentes farmacológicos têm como objetivo controlar a glicémia enquanto provocam perda de peso. A recentemente implementada terapia combinada de dapagliflozina (inibidor do cotransportador de sódio-glucose) e exenatida (análogo do glucagon-like peptide 1) tem sido prescrita para tratar T2DM. Este tratamento influencia o metabolismo da glucose em todo o corpo, portanto, também podem afetar o metabolismo dos tecidos responsáveis pela fertilidade masculina. A espermatogénese é altamente dependente da cooperação metabólica estabelecida entre as células germinativas em desenvolvimento e as células de Sertoli. Estas usam glucose extracelular para produzir lactato, que as células germinativas usam como combustível. Neste trabalho, avaliamos os efeitos da dapagliflozina e exenatida no metabolismo das células de Sertoli. Para tal, células de Sertoli TM4 de murganho foram tratadas na ausência (controlo) ou na presença de concentrações sub-farmacológicas, farmacológicas e supra-farmacológicas de dapagliflozina (50; 500; 5000 nM, respectivamente) ou exenatida (2,5; 25; 250 pM, respectivamente) ou ainda, com uma combinação das concentrações farmacológicas de ambas as drogas, durante 24 horas. A citotoxicidade destes tratamentos para as células de Sertoli foi avaliada através de ensaios de MTT, liberação da lactato desidrogenase (LDH) e SRB. O perfil glicolítico das células de Sertoli também foi determinado (espectroscopia de 1H-RMN), bem como os níveis de expressão dos principais transportadores e enzimas como a LDH, transportadores de glucose 2 (GLUT2) e transportador de monocarboxilatos 4 (MCT4). Além disso, foi quantificado o conteúdo em glicogênio e as reservas lipídicas nas células de Sertoli TM4. A concentração farmacológica de dapagliflozina mostrou um leve efeito citotóxico que também foi observado no tratamento combinado. Além disso, a concentração farmacológica de dapagliflozina mostrou aumentar a eficiência glicolítica e aumentar a secreção de lactato pelas células de Sertoli, que é visto como um potencializador da espermatogénese. O tratamento combinado demonstrou que os diferentes fármacos parecem ter um efeito sinérgico para manter a homeostase metabólica das células de Sertoli. Assim, os nossos resultados sugerem que o tratamento com dapagliflozina, exenatida e o tratamento combinado de ambos os fármacos em concentrações farmacológicas podem ser adequados para homens na idade reprodutiva. Palavras-chave: Células de Sertoli, Dapagliflozina, Doenças metabólicas, Exenatida, Infertilidade. vi NEW GENERATION ANTIDIABETICS: WHAT ARE THE IMPLICATIONS ON SPERMATOGENESIS NUTRITIONAL SUPPORT? The incidence of metabolic diseases such as type 2 Diabetes mellitus (T2DM) and obesity has been increasing among children and young adults. Both conditions are tightly linked thus new pharmacological agents aim to control glycemia while provoking weight loss. Recently implemented combined therapy of dapagliflozin (sodium-glucose cotransporter inhibitor) and exenatide (glucagon-like peptide 1 analogue) has been prescribed against T2DM. This treatment influences whole-body glucose metabolism, and thus can also impact the metabolism of tissues responsible for male fertility. Spermatogenesis is highly dependent on the metabolic cooperation established between Sertoli cells and germ cells. The former uses extracellular glucose to produce lactate, which germ cells use as fuel. In this work, we evaluated the effects of dapagliflozin and exenatide on Sertoli cells metabolism. For this purpose, mice TM4 Sertoli cells were treated in the absence (control) or presence of sub-pharmacologic, pharmacologic and supra-pharmacologic concentrations of dapagliflozin (50; 500; 5000 nM, respectively), or exenatide (2,5; 25; 250 pM, respectively), or with a combination of the pharmacological concentrations of both drugs, during 24 hours. The cytotoxicity of these compounds on Sertoli cells was evaluated by MTT, lactate dehydrogenase (LDH) release, and SRB assays. The glycolytic profile of SCs was also determined (1H-NMR spectroscopy), as well as the expression levels of key metabolite transporters and enzymes, such as LDH, glucose transporters 2 (GLUT2), and monocarboxylate transporter 4 (MCT4). To further pursue our aim, glycogen storage and lipid reserves were quantified in TM4 Sertoli cells. Pharmacological concentration of dapagliflozin showed a mild cytotoxic effect that was also seen in the combined treatment. In addition, the pharmacological concentration of dapagliflozin showed to enhance the glycolytic efficiency and increase lactate secretion by Sertoli cells, which is seen as a positive enhancer of spermatogenesis. The combined treatment demonstrated that the different drugs seem to have a synergic effect to maintain the metabolic homeostasis of Sertoli cells. Thus, our results suggest that treatment with dapagliflozin, exenatide and the combined treatment of both drugs at pharmacological concentrations may be suitable for males in reproductive age. Keywords: Dapagliflozin, Exenatide, Infertility, Metabolic diseases, Sertoli cells. vii TABLE OF CONTENTS RESUMO ............................................................................................................................ v ABSTRACT ........................................................................................................................ vi LIST OF ABBREVIATIONS ................................................................................................... x LIST OF FIGURES .............................................................................................................. xi LIST OF TABLES .............................................................................................................. xiii 1. INTRODUCTION .......................................................................................................... 14 1.1. Diabetes mellitus ................................................................................................. 14 1.1.1. Type 2 Diabetes mellitus .................................................................................. 15 1.2. Obesity and its role in the origin of T2DM ............................................................. 15 1.3. Sodium-glucose cotransporter 2 inhibitors ............................................................ 16 1.4. Glucagon-like peptide-1 receptor agonist .............................................................. 20 1.5. Combined therapy of exenatide plus dapagliflozin ................................................. 23 1.6. Male reproductive function and spermatogenesis ................................................. 25 1.6.1. Sertoli cells and Spermatogenesis .................................................................... 29 1.7. Negative influence of metabolic diseases on male fertility ..................................... 32 1.8. Exenatide and Dapagliflozin impact on male fertility .............................................. 34 2. OBJECTIVES ............................................................................................................... 37 3. METHODS .................................................................................................................. 39 3.1. Chemicals ........................................................................................................... 39 3.2. Mouse Sertoli cell line TM4 culture ...................................................................... 39 3.3. Experimental groups ............................................................................................ 39 3.4. Evaluation of cytotoxic profile of the compounds ................................................... 40 3.4.1. SRB cytotoxic assay ......................................................................................... 40 3.4.2. MTT cytotoxic assay ......................................................................................... 41 3.4.3. LDH release assay ........................................................................................... 41 3.5. Protein extraction and quantification .................................................................... 42 13 Chapter I INTRODUCTION 14 1. INTRODUCTION 1.1. Diabetes mellitus Diabetes mellitus (DM) is a chronic disease that has reached pandemic proportions and its incidence has been increasing dramatically over the last decades. Moreover, it is expected a growing trend in the number of individuals suffering from this metabolic disease. It has been estimated that a worrisome 693 million people will develop DM by 2045 (1). DM consists of multiple conditions related to deficient carbohydrate, lipid and protein metabolism. This dysregulation impacts multiple organs and systems such as the kidney, retina, circulatory system, and nervous system (2). Hence, acute complications in long-term DM patients can result in blindness, macrovascular and microvascular complications, and sexual dysfunction, among others (3). Moreover, DM increases the chance of strokes and coronary artery disease in undiagnosed or untreated individuals (4), which could result in death. Impaired glucose homeostasis results in hyperglycemia which is caused by hypoinsulinemia or insulin resistance (5). Insulin is a hormone that is secreted into circulation after a meal rich in carbohydrates. Glucose levels are sensed with the participation of specific glucose transporters together with neural stimuli and incretin signaling. This leads to the activation of insulin secretion on β-cells in the pancreas. Insulin will act on other cells increasing their glucose uptake and decreasing glycemia levels (6). Besides, insulin also mediates meal-time and has a role in cell growth and differentiation (7). The plasmatic insulin is insufficient to maintain glucose homeostasis in DM patients, which can be caused by autoimmune β-cell elimination, known as type 1 diabetes mellitus (T1DM); or gradual loss of insulin sensitivity, classified as type 2 diabetes mellitus (T2DM) (8). Patients with T1DM are increasing among children and has been reported that this growth in incidence does not only depend on genetic factors (9) but also in environmental factors (10) such as enterovirus infection or nutritional factors (e.g. vitamin D deficiency or premature contact with cow’s milk proteins) (11). These factors can lead to the destruction of insulin-producing β-cells by T lymphocytes in an autoimmune manner. Additionally, nearly one-third of patients with T1DM suffer from diabetic ketoacidosis (8). This condition is a consequence of diminished insulin/glucagon ratio presence in circulation that activates ketogenic machinery in liver cells to produce glucose, but also ketones from long-chain fatty acids (12). Ketones such as acetoacetate and β-hydroxybutyrate increase in plasma and this result in metabolic acidosis (13) that in severe cases can lead to coma (14). Thus, to work around this condition the patients suffering from T1DM will depend on exogenous insulin for life (15), forcing them to continuous health care. 15 1.1.1. Type 2 Diabetes mellitus T2DM is the most prevalent type of DM accounting approximately 90% of all occurrences (16) and is tightly linked with obesity, sedentary lifestyle (17), but also genetic factors (18). Usually, the risk of developing this condition increases with age (19). However, this epidemy is affecting an increasing number of individuals from younger group ages, which intensifies the health, sociologic, and economic problems associated with T2DM (20). This type of chronic hyperglycemia results from diminished insulin bioactivity, which can be due to anomalous insulin secretion, decreased insulin sensitivity, or a combination of both (21, 22). In a scenario with high glucose intake, a healthy individual would secrete proportional insulin to normalize glycemia levels. However, diabetic patients insulin secretion do not compensate the subnormal hormone effect on its receptors of skeletal muscle, pancreas, adipose tissue, and liver (23), promoting the increase of glucose levels on plasma thus, causing hyperglycemia. Lifestyle modifications are essential to counter the effects of T2DM, including changing diet and exercise habits. Multiple drugs were also developed to counteract hyperglycemia and its associated comorbidities. However, a complex study is needed to find the most suitable antidiabetic therapy for each patient situation (20). 1.2. Obesity and its role in the origin of T2DM According to the World Health Organization, a person is considered overweight when its Body Mass Index (BMI) score is above 25 and is considered obese when the score is superior or equal to 30. BMI is a crude measure for studying obesity in a population and can be calculated by dividing a person weight (kilograms) with the square of the person height (meters) (24). Through the years, BMI has increased in developed countries possibly by their easier access to high caloric meals. This excessive caloric intake is contributing to pandemic obesity never seen before. Most victims suffering from obesity also develop T2DM, since obesity already induces some degree of insulin resistance (25). In obese humans, the bioactivity of the circulating insulin is diminished by the excessive abdominal fat. Thus, hyperglycemia will aggravate and this causes more insulin secretion, increasing its concentration on plasma which worsens insulin resistance and promotes high blood pressure (26). Furthermore, fat cells can penetrate the pancreatic islets and influence insulin secretion in more advanced obese patients (26). Obesity is also known to dysregulate the secretion of hormones responsible for the all-body energy balance, mainly ghrelin, leptin, glucagonlike peptide (GLP-1), and insulin, being the former already discussed above. Ghrelin is a hormone 16 secreted by the endocrine cells of the gastrointestinal tract and it is known as the hunger hormone since it is related to the origin of appetite. Ghrelin levels in obese people plasma are reduced in relation to the ones of a healthy individual (27). Leptin, however, is released by white adipose tissue and its levels are increased in obese persons when compared with healthy individuals. It is believed that leptin plays a role in reducing appetite and together with ghrelin they help in the regulation of energy homeostasis, glucose metabolism, and reproductive function (28). Weight-loss should be the main goal to control glycemia in obese T2DM individuals, even though this can be the most challenging aspect of the treatment (29). In 2015, Whitmore stated that managing weight in T2DM patients is comparable to a “juggling act” with balanced energy intake/output, glycemic control, hypoglycemia, medication and the negative sides that those factors can generate (30). DM treatment is constantly evolving, and new approaches are being developed to improve the health of the patients and their lifestyle (30). Some of the antidiabetic drugs used to regulate T2DM also have anti-obesogenic properties which provoke weight-loss in the patient, namely: low affinity/high capacity sodium-glucose cotransporter 2 (SGLT2) inhibitors (e.g. dapagliflozin) and GLP-1 receptor agonist (e.g. exenatide) (20). Both these pharmacological agents were already used separately or in combination with other drugs. Combined therapy of dapagliflozin (10 mg once daily) plus exenatide (2 mg injection once weekly) is being studied in the form of phase III clinical trial to compare the results of this novel therapy with more standardized therapies (31). 1.3. Sodium-glucose cotransporter 2 inhibitors Kidneys role in glucose homeostasis starts when Malpighi glomerulus filters plasmatic glucose without restrains, followed by its reabsorption in the proximal tubule into circulation by specific glucose transport proteins. In individuals with chronic hyperglycemia, the renal threshold (180 mg/dL) is not enough to reabsorb all plasmatic glucose, forcing the remaining glucose to be excreted through the urine (32). This event can be explained by the SGLT2 activity and expression in the proximal tubule. This transporter is responsible for glucose resorption and its activity is insulin-dependent (33). In addition, kidneys are also involved in glucose metabolism homeostasis by performing gluconeogenesis, when humans are fasted for more than 14 hours, contributing with around 20% to 25% of total plasmatic glucose after that period (34). SGLT2 is mainly found in the initial part of renal convoluted proximal tubules, more specifically in the apical membrane. This transporter is responsible for the reabsorption of approximately 90% 17 of glucose in the kidneys (35). The reabsorption is established when SGLT2 imports one sodium ion and one glucose molecule into the cytosol finally, glucose is then transported into the bloodstream through the action of glucose transporter 2 (GLUT2) (36) (Figure 1). Interestingly, patients with T2DM have increased expression levels of SGLT2 in proximal tubules cells. This fact suggests that T2DM patients have an above normal renal threshold (37). Thus, more glucose is reabsorbed into the bloodstream which will only aggravate hyperglycemia (32). Considering this, compounds capable of inhibiting SGLT2 could be a convenient way of controlling glycemic levels, blocking the reabsorption of this hexose into circulation, increasing its excretion through the urine, and diminishing the overall caloric intake of the individual. Dapagliflozin favors this management of glucose homeostasis. Thus, the European Union approved dapagliflozin for the treatment of T2DM in 2012 (38). Dapagliflozin is chemically described as (2S,3R,4R,5S,6R)-2-[4-chloro 3-(4-ethoxybenzyl) phenyl]-6-(hydroxymethyl) tetrahydro-2H-pyran-3,4,5-triol). Its molecular formula is C21H25ClO6 which translates in a molecular Figure 1: Schematic representation of glucose reabsorption by sodium-glucose cotransporter 2 (SGLT2) in the proximal tubule. Glucose and sodium ion are transported from the lumen of the tubule to the cytoplasm through the SGLT2. After this, glucose transporters 2 (GLUT2) transport the glucose into the circulation. 18 weight of 408.87 g/mol (38). Dapagliflozin structure is organized in one glucose molecule which is linked to an aglycone component through a carbon-carbon bond with the purpose of increasing its metabolic stability against glucosidase enzymes (39). This fact allows dapagliflozin to be prescribed for oral administration and can be found in tablets of 5 mg or 10 mg (39). It needs only approximately 1 hour ( tmax ) after the first dosage of 5 mg or 10 mg dapagliflozin to be absorbed. Dapagliflozin is also rapidly absorbed even when the treatment is made after a meal, due to its high aqueous solubility and great intestinal absorption (40). Its maximal plasma concentration can reach up to approximately 70 ng/ml (5 mg treatment) and 200 ng/ml (10 mg treatment) (38, 41) moreover, dapagliflozin half-life time (t1/2) is approximately 17 hours (42, 43). The efficiency and safety of dapagliflozin 10 mg once-daily therapy for obese with T2DM patients has already been studied in multiple clinical trials. DURATION-8 was a 28-week long phase III clinical trial in adults with inadequate glycemic control (HbA1c 8-12%). During this clinical trial, 19% dapagliflozin treated group had a decreased on HbA1c score by 7%, 20% of the group lost more than 5% of their weight, and systolic blood pressure decreased 1,8 mm Hg in relation to the baseline values at the beginning of the trial (44). In another phase III clinical trial with similar dosage, dapagliflozin showed to improve HbA1c score and weight loss by a difference of -0,90% and -1.38 Kg, respectively, in comparison to individuals from the placebo group at the end of the 24-week trial duration (45). Moreover, 10 mg once daily therapy of dapagliflozin can be also used as a complementary agent to exogenous insulin treatment in patients with T1DM (46). In relation to the placebo group, patients treated with dapagliflozin improved their HbA1c score by -0,45% in the first 4 weeks of the trial, maintaining that difference in the remaining time of the study. In addition, dapagliflozin (10 mg/day) treatment allowed the reduction of the exogenous insulin dosage by 9,8% per kilogram of bodyweight at week 24 of the study. Similarly, body weight also decreased by 3.72% in relation to the individuals from the placebo group, most of this reduction occurred in the first 8 weeks of the trial and was maintained until the end of the experiment (46). As above demonstrated, dapagliflozin decreases hyperglycemia, body weight and blood pressure in DM patients, being that these are the key factors responsible for the most deleterious effects of DM, making this pharmacological agent a promising drug for the treatment of this condition and increasing the life quality of the user. However, like any drug in the market, dapagliflozin entails some negative side effects to the user. The main concern about this drug is its influence on renal and cardiovascular function of treated DM and obese patients. T2DM patients with normal or moderately impaired renal function were subjected to dapagliflozin therapy. Their 19 estimated glomerular filtration rate was calculated to access the implications that dapagliflozin could generate to patients’ renal function. Kohan and coworkers analyzed twelve clinical trials and stated that dapagliflozin (10 mg/day) decreased estimated glomerular filtration rate by -4,13 mL/min/1,73m2 in relation to the baseline value in the first week. Nevertheless, this value returned to baseline by week 24 and remained stable until the end of the experiment (47). Blood urea nitrogen values were significantly increased in relation to placebo at the end of the experiment which is justified by the osmotic diuretic effect that the drug entails (47) which also allows the reduction of blood pressure (48). Regarding cardiovascular function, a randomized clinical trial with 28 weeks of duration was performed. Dapagliflozin (10mg/day) was administered to patients with a high risk of cardiovascular problems and the results showed a greater occurrence of hypotension and dehydration in relation to the placebo group, with the usual improvement in glycemic levels and body weight (49). These results could be justified by a large number of plus 65 years old patients that participated in this study and by the fact that the patients already had high risk of cardiovascular problems. However, another study described that dapagliflozin treatment increased plasmatic levels of high-density lipoprotein 2-cholesterol, which are regarded as an advantageous cardiometabolic marker (50). On the other hand, low-density lipoproteins, which are considered a strong marker of possible cardiovascular complications, did not change between the dapagliflozin treated group and the placebo group (50). Moreover, SGLT2 inhibitors such as dapagliflozin have been also associated with risks of inducing diabetic ketoacidosis. This could be due to the increase of glucagon/insulin ratio levels in circulation which promote hepatic ketogenesis when in dapagliflozin therapy. Furthermore, treatment for T2DM with SGLT2 inhibitor combined with exogenous insulin increases the chances of developing diabetic ketoacidosis due to the need of lowering exogenous insulin dosage to prevent hypoglycemia, which only aggravates the glucagon/insulin ratio. This happens due to enhanced lipolysis caused by the lack of insulin, increasing ketones levels on plasma (51). SGLT2 inhibitors, such as dapagliflozin, are also linked with genitourinary tract infections caused by the enhanced glucose concentration in the urine (52). Besides that, studies suggest that dapagliflozin is a relatively safe pharmacological agent and even entails some advantages to the patient, such as the ability to protect pancreatic β-cells of hyperglycemia adverse effect, conserving their function (53). Moreover, this drug is associated with weight loss which results from the enhanced glucose excretion, maximizing caloric loss yet, this fact can originate an increase in appetite (54). In addition, a study stated that dapagliflozin (5mg/day) treatment for six months decreased total body weight, without significantly changing 20 skeletal muscle mass and soft lean mass in relation to the placebo group (55). Dapagliflozin also minimizes the chances of hypoglycemia since SGLT2 inhibitors do not interfere directly with insulin secretion (56), unlike other antidiabetic drugs. Thus, dapagliflozin can be complemented and used simultaneously with other antihyperglycemic drugs capable of modulating insulin secretion (44), such as exenatide which belongs in the GLP-1 receptor agonist class of antidiabetic drugs. 1.4. Glucagon-like peptide-1 receptor agonist GLP-1 is an incretin derived from a different path of post-transcriptional modification of proglucagon in enteroendocrine L-cells, which can be found along the gastrointestinal tract with greater abundance in the distal gut, ileum, and colon (57). Oral ingestion of nutrients is the major physiologic stimuli for the secretion of GLP-1 by L-cells, although its concentration in the bloodstream depends on the composition of the meal. Meals richer in carbohydrates and fats stimulate greater GLP-1 secretion than meals rich in proteins (58). It is believed that GLP-1 secretion is mediated by direct and indirect sensing since GLP-1 levels rise in circulation within minutes. Normally, this time is insufficient for the food to reach the L-cells in the distal gut. Thus, it is believed that some of these cells are situated in the upper gut to sense the nutrients ingested and share that information with the richest L-cells regions for a rapid and efficient GLP-1 release into circulation (57). After this, and when it binds to its G-protein coupled receptors on pancreatic β-cells, GLP-1 stimulates insulin secretion by activating the second messenger cyclic adenosine monophosphate (cAMP) which, in its turn, activates the insulin effectors protein kinase A (PKA) (59) (Figure 2). Interestingly, as the incretin effects are proportional to the glucose intake, also insulin secretion will be mediated by the ingested glucose (57). The ability of such peptide to modulate insulin secretion and suppress excessive glucagon secretion (reducing hepatic gluconeogenesis) (60), shows that GLP-1 receptors agonists could be an interesting approach to control glycemia in individuals with T2DM (61). Exenatide is an example of one GLP-1 receptor agonist composed of 39 amino acids with a molar mass of 4186.6 g/mol (62), originally extracted from the salivary excretion of the Heloderma suspectum (Gila monster) and commonly named exadin-4 (63). When compared with GLP-1, exenatide shares 53% of sequence homology (64), has a 20 to 30-fold optimized half-life and 5500 times greater potency in decreasing glycemic levels in T2DM patients (65). This significant difference in half-life time is due to the enzyme DPP-4 present in circulation, which cleaves GLP-1 in the second amino acid residue (alanine). In exenatide, this alanine was altered to a glycine 21 residue (Figure 3) to enhance the biodisponibility of the drug (58). This GLP-1 receptor agonist is administered by subcutaneous injection in dosages of 5 µg or 10 µg of exenatide twice daily (66) or 2 mg once weekly and has a maximal concentration found in plasma around 90 pg/ml (for dosage of 5 µg) or 210 pg/ml (for dosage of 10 µg and 2mg) (67-69). Moreover, when is prescribed the long action exenatide microspheres, the drug diffusion lasts for approximately 2 weeks and drug clearance around 7 weeks since the injection (67). Figure 2: Scheme of the mechanism of glucagon-like peptide 1 (GLP-1) release from L-cells and insulin secretion pathway activation. In the presence of different nutrients, L-cells secrete GLP-1 through different metabolic pathways. After, entering in circulation, GLP-1 binds to its receptor on pancreatic β -cells and activate the pathway of insulin secretion. FFA, free fatty acids; AA, amino acids; PKC Ϛ, protein kinase CϚ ; cAMP, cyclic adenine monophosphate; PKA, protein kinase A. 22 When comparing the two major dosage schedules of exenatide treatment (exenatide 10 µL twice daily or exenatide 2 mg once weekly), a study referred that exenatide 2 mg once weekly demonstrated the best results in lowering glycemic levels, and a significantly lower incidence in adverse effects in relation to the twice-daily dose in a 28-week long study (70). With this in mind, and together with a more convenient dosage schedule, we prioritized studies that evaluated the impact of the treatment of exenatide 2 mg once weekly. DURATION-8 clinical trial studied the effect of exenatide 2 mg once weekly on glycemic control, weight, and blood pressure in a similar manner to dapagliflozin treatment. The results showed that exenatide decreased the baseline score of HbA1c by 7% in 27% of the individuals. Some patients (14%) loss more than 5% of their total body mass and also decreased the systolic blood pressure by -1,2 mm Hg in the 28 weeks of the trial in the individuals treated with exenatide (44). In another study, exenatide 2 mg once weekly therapy was compared to a therapy of another GLP-1 receptor agonist (liraglutide) in a single daily administration of 1.8 mg. Exenatide was less efficient in regulating HbA1c score, weight loss and systolic blood pressure (-0,36 %; -0,90 kg; and -0,87 mm Hg, respectively), when compared with liraglutide therapy. Still, the occurrence of adverse effects on exenatide treated group was less common in almost all categories (71). Metabolic diseases, such as DM and obesity, already entail cardiovascular complications and have been described that some antidiabetic drugs could enhance the probability of those complications (20). However, studies have suggested that this is not the case for exenatide. In fact, exenatide showed to decrease arterial stiffness in T2DM patients averaging 64 years of age and improved diastolic function in relation to the placebo group. Unfortunately, this did not result in a significant increase in exercise capacity of the patients. Still, exenatide showed to have a protective Figure 3: GLP-1 amino acid sequence and the differences in the sequence (amino acids in yellow) that allow exenatide increased half-life time. 29 during maturation. Ectoplasmic specializations are also found together with gap junctions, tight junctions, and desmosomes-like junctions to create the already mentioned immune free environment unique to BTB (114). For the occurrence of the different phases of spermatogenesis, the BTB structure must suffer some changes to allow the developing germ cells to transit from basal to apical sections, to be released as viable spermatozoa (110). It will also need the decoupling of BTB in the spermiation (115). All these processes take place without compromising the structure and function of the BTB (110). BTB is also impermeable to several drugs, preventing their action in the developing germ cells. Thus, possibly interfering in the treatment of some infertility conditions, decreasing drug efficiency and detain the development of contraceptive pills for men. 1.6.1. Sertoli cells and spermatogenesis Spermatogenesis depends on Sertoli cells which occupy up to 40% of the volume of seminiferous epithelium in humans (116). Hence, a subnormal number of Sertoli cells is related to subnormal testis size and a diminished function and efficiency of Sertoli cells will result in subnormal number of viable germ cells (117). Sertoli cells are responsible for the support and nourish of all the cells during the processes of spermatogenesis. One man at reproductive age has about 800 to 1200 million of Sertoli cells (118). These cells are also involved in the transportation of spermatids into the lumen of seminiferous tubules and are the mediators and producers of endocrine and paracrine factors, which modulate the development of germ cells. Sertoli cells are able to supply the germ cells with their nutritional requirements for proper development. In the different stages of spermatogenesis, the metabolism of the developing germ cells will differ (119). In more detail, the main pathway utilized by spermatogonia to obtain energy is glycolysis and pentose phosphate pathway. Contrastingly, spermatocytes and spermatids obtain their energy mostly from the substrates like lactate and pyruvate. Finally, spermatozoa utilizes glucose/fructose to produce ATP in a similar manner as spermatogonia (120). Hence, for spermatids to develop properly, Sertoli cells metabolism need to perform a “Warburg-like” metabolism to supply the developing germ cells with their required substrates (121), establishing what has been named as the testicular metabolic cooperation. These somatic cells use a less cost-effective metabolism by using the glycolytic pathway which transforms a glucose molecule into 4 ATPs molecules and lactate, instead of directing the resultant pyruvate through the TCA cycle which would result into a max of 36 ATPs molecules (121). Thus, β-oxidation 30 of lipids followed by TCA cycle is the main metabolic pathway used by Sertoli cells to produce ATP to itself once almost all glucose is used to produce lactate, which is the preferred substrate for energy synthesis in developing germ cells (119). To internalize the glucose, cells rely on two different major families of membrane glucose transporters, GLUTs, and SGLTs. SGLTs expression has already been identified on testes tissue (122) and multiple isoforms of GLUTs were already described on Sertoli cells, namely: GLUT1 (123), GLUT2 (124), and GLUT3 (125). Once the glucose molecule enters Sertoli cell cytoplasm, the majority of it metabolized into two molecules of pyruvate through glycolysis (119), followed by the metabolization (approximately 95%) of pyruvate into lactate for the developing germ cells. The lactate is secreted through specific monocarboxylate transporters (MCTs) to germ cells that use it as their main energy source. However, Sertoli cells can utilize other substrates in a glucose-free environment to continue the production of lactate, energy, and ATP (119). In fact, to maintain the high levels of ATP and lactate needed to ensure spermatogenesis in the absence of glucose, Sertoli cells can metabolize lipids and glycogen (119). Fatty acids and amino acids are metabolized into energy by the TCA cycle, and the majority of the resulting ketone bodies are also transformed into energy by this metabolic pathway (112). Moreover, glycogen can also be mobilized and suffer glycolysis originating pyruvate, which is used to produce ATP through TCA cycle (119) or is metabolized into lactate by lactate dehydrogenase (LDH) (126). The developing germ cells consume the lactate with specialized transporters in the plasmatic membrane (MCT 2), which internalize the lactate into the cytoplasm. Therein, a specific LDH isoform (LDH-C4) metabolizes lactate into pyruvate to be transferred to the mitochondrion to start a new TCA cycle (via acetyl coenzyme A) (Figure 6) to produce ATP (127). Besides being the preferred energy source for the developing germ cells, lactate can also modulate gene expression (126), modify the expression of important proteins for spermiogenesis in spermatids (128), and has been reported to have antiapoptotic properties for the developing germ cells (129). To support, nourish, and facilitate the cooperation with all the developing germ cells, Sertoli cells cytoplasm is stretched enough to reach fully developed germ cells. This allows direct communication and better development management of germ cell development (130). Sertoli cells also produce and secret glycoproteins with different functionalities. The ones with the highest secretion rate are transporting enzymes, such as transferrin and androgen binding protein. Proteases and proteases inhibitors, important for the morphologic changes of germ cells, as well as insulin-like growth factor (131). This cellular type also secretes into circulation inhibin B 31 (132). This protein is a marker of Sertoli cell activity and is important in the regulation of gonadotrophic hormones. Spermatogenesis and Sertoli cell metabolism are tightly regulated by the hypothalamuspituitary-testis axis. Specialized neurons in the hypothalamus area create secretion impulses of gonadotropin-releasing hormone (GnRH), which influences the anterior pituitary to produce folliclestimulating hormone (FSH) and luteinizing hormone (LH) (133). Both hormones are secreted by the pituitary and have an impact on spermatogenesis (134). Sertoli cells are regulated by FSH and Leydig cells by LH (135) nevertheless, both cell types regulate each other activity (136). LH influences the secretion of androgens by Leydig cells to control spermatogenesis (135). Without testosterone or androgen receptors in Sertoli cells, spermatogenesis is not possible due to the inability of sperm cells develop past the meiosis phase, unsuccessful spermiation, and BTB structural problems (137). Moreover, testosterone can be also converted into estrogens by aromatase present in Leydig cells. It has been shown that this hormone has an important role in the regulation of testosterone and LH secretion (138). On the other hand, FSH plays a role in Sertoli cells proliferation during fetus development and also regulates Sertoli cells differentiation Figure 6: Mechanisms of Sertoli cells glucose metabolism and its metabolic cooperation with the germ cells. Sertoli cells can use multiple substrates to produce energy, but the main subtract used is glucose. Glycolysis uses glucose to produce pyruvate that can be transformed in lactate, alanine or in acetyl-CoA to start the tricarboxylic acid (TCA) cycle. Lactate and acetate are transported out of Sertoli cells and into the germ cells cytoplasm by monocarboxylate transporters (MCT4 and MCT2, respectively). Thicker arrow indicates prioritized pathway by Sertoli cells and germ cells. 32 after puberty (139). Androgens and estrogens, such as 5α-dihydrotestosterone and 17β-estradiol, respectively, also affect Sertoli cells metabolism. It has been reported that these hormones affect the glycolytic flux and the production of acetate (via acetyl coenzyme A) in a different manner. While 5α-dihydrotestosterone decreases acetate production, 17β-estradiol increases acetate production (140). Acetate is an important substrate for Sertoli cells once it is described that is involved in the formation of lipids by the Sertoli cells for the formation of new sperm cells (141). In sum, Sertoli cell metabolism is tightly regulated by endocrine, paracrine and autocrine factors, and these will contribute ultimately to the formation of new sperm cells and hence to the establishment of male reproductive potential. Metabolic diseases such as obesity and DM and what they entail are able to dysregulate the balance of this system and impact male fertility. In the next chapter, the main factors that negatively influence male fertility will be further discussed. 1.7. Negative influence of metabolic diseases on male fertility Infertility can be defined ha a disease of the reproductive system which prevents a couple of achieving clinical pregnancy and can be diagnosed after one year of regular sexual intercourse without contraceptive measures (142). Around the globe, approximately one in six couples encounter some form of infertility (143). In western countries, about half of those cases, the hurdle comes from the male part, either by himself or in combination with the female partner (144). Alterations in three major features of the semen are frequently associated with male infertility: the concentration (145), the motility, and the morphology of spermatozoa (146). Obese males are more susceptible to these alterations due to anatomical features, dysfunctional metabolism and impaired hormone secretion (147, 148). An obese male has more fat deposits in suprapubic and upper thigh regions which can difficult testicular cooling and so, obese male testis are more likely to have a superior scrotal temperature when compared with a male with normal BMI (148). This factor can cause heat stress on the testis and influence gene expression of germ cells as well as DNA repair (149), production of free reactive oxygen species (ROS), and apoptosis (150). The changes in gene expression profile and DNA repair can ultimately lead to a different proteomic profile in the spermatocyte. Some of the up-regulated genes are translated into proteins with specific roles in cellular apoptosis. However, a study described that this up-regulation stops after the testes reach normal temperature for a few hours to prevent the elimination of all sperm cells, in mice models (151). In a different manner, the increased ROS levels also increase apoptosis risk 33 by oxidation of DNA and lipids (150). Moreover, DNA polymerase beta and DNA ligase III have reduced expression in hyperthermia, which compromises DNA repair (150). In another perspective, obese males excessive adipose tissue transforms androgens into estrogens via aromatization at a higher rate than lean males. This process, similar to the one that occurs in the testis, often results in free testosterone reduction and the dysregulation of the hypothalamic-pituitary negative feedback, which compromises spermatogenesis (152). Thus, testosterone deficiency can result in diminished testes volume and functionality (147). In addition, the continuous formation of new adipose tissue characteristic of obese males enhances the release of cytokines (ILs and TNF-α), resulting in a constant inflammation status, which recruits leucocytes that enhance the production of ROS (148). This chronic inflammatory status and the secretion of TNFα observed on long term obese males also has a negative impact on rodent Leydig cells steroidogenesis (153). White adipose tissue in excess will secrete an abnormal quantity of leptin, which is the principal hormone secreted by white adipose tissue (154). It was described that this hormone plays a role in the regulation of appetite, glucose metabolism, and has an impact on reproductive and immune systems (155). Reports described that leptin is associated with increased LDH activity and GLUT2 expression levels in human Sertoli cells (156). This metabolic effect was corroborated by another study that stated that leptin can modulate Sertoli cells metabolism by conditioning cellular glucose transport (141) and the production of acetate in human Sertoli cells (141). In addition, the excess of adipocytes and consequent increase of leptin secretion was described to diminish free androgen levels (157) and consequently increase the apoptosis in germ cells (158). The dysfunctional androgen response is due to a subnormal transition of 17-OH-progesterone to testosterone (157) and the increase in apoptosis is due to the production of ROS and TNFα. In fact, increased ROS levels can still be found in seminal fluid (152). Ghrelin is another hormone which secretions are impacted by the BMI of the person. A higher BMI usually indicates diminished ghrelin levels. This 28-amino acid peptide is secreted by the stomach and is known to promote food consumption in humans. Its secretion is more accentuated in the fasting state when the person is in food deprivation. In fact, ghrelin has an important role in regulating energy expenditure, which directly and indirectly modulates Sertoli cell metabolism and spermatogenesis (159). Tena-Sempere and co-workers studied the effect of ghrelin on testosterone secretion by rat Leydig cells. This group of work discovered that ghrelin at concentrations of 10-7 M had an inhibitory role on testosterone secretion, by decreasing the gene expression of proteins 34 responsible for steroidogenesis (160). A distinct study described that ghrelin could upregulate apoptosis in germ cells. This increase in apoptosis rates is associated with the increased translation of apoptosis regulator Bcl-2-associated X protein and decrease in the expression of the proliferating cell nuclear antigen (161). On the other hand, a defective insulin secretion also impacts Sertoli cell metabolism (162). Oliveira and co-workers reported that Sertoli cells deprived of insulin showed decreased glucose and pyruvate consumption. They also described that when these cells were subjected to longer periods of insulin deprivation, lactate production was also affected (163). These results were explained by the ability of insulin to enhance some GLUTs and MCTs activity, modulating glucose and lactate transport, respectively. In a different study performed by the same group, insulin deprivation diminished acetate production by Sertoli cells. Thus, insulin deprivation affects Sertoli cells metabolism and energy synthesis, which could ultimately influence spermatogenesis (140). Furthermore, Sertoli cells apoptotic pathways dependent on caspases. This class of proteins is enhanced under insulin absence, promoting apoptosis of Sertoli cells (164) and concurrently diminishing germ cell numbers (132). 1.8. Exenatide and Dapagliflozin impact on male fertility Exenatide and dapagliflozin, like the other pharmacological agents prescribed for the treatment of DM and obesity, modulate energy expenditure and the glucose metabolism in multiple tissues and cellular systems. Still, almost no data is available regarding the effects of each of these two drugs on male fertility. In addition, no study has been made to evaluate the effect of the combined therapy of exenatide plus dapagliflozin on male reproductive function. Unfortunately, not only the impact of exenatide and GLP-1 receptors agonists on male fertility have been overlooked, but also that of the incretin GLP-1. One of the few studies available indicated that GLP-1 interfered with the hypothalamic-pituitary-gonadal axis, which resulted in decreased testosterone secretion (165). This reduced testosterone production can result in arrested germ cell development. However, in a more recent study, exenatide treatment (1 nmol/kg) showed to protect spermatogenesis from ROS by increasing antioxidant enzyme activity (166). Furthermore, exenatide showed the ability to increase testosterone levels in aging mice, expanding their fertile period. In another study, exenatide influence on testis inflammation of induced obese mice was accessed (167). In this study, exenatide seemed to decrease the expression of pro-inflammatory cytokines. However, these results were attributed to the ability of exenatide to reduce weight, hence 35 reducing inflammation (167). Another example of this indirect effect was described by Topyildiz and coworkers. In their 2016 study, Topyildiz workgroup described that exenatide decreased ghrelin levels in circulation in individuals with T2DM to values near those seen in healthy individuals after a meal (27, 168). In regarding Sertoli cells metabolism, a study published by our laboratory described that, when these cells were exposed to 1 nM of GLP-1, glucose consumption was decreased. An increase in lactate production was also noticed when the Sertoli cells were exposed to either 0.01 or 1 nM of GLP-1 (61). Being lactate a critical substrate for the proper progress of spermatogenesis and having anti-apoptotic properties for the developing germ cells (129), it is possible that this metabolic change provoked by GLP-1 enhanced spermatogenesis. In addition, 1 nM of GLP-1 also increased acetate secretion that has been described as an important substrate for the formation of lipids for the developing germ cells (141). In the case of SGLT2 inhibitors and dapagliflozin, the available published work accessing the effect of this class of antidiabetic drug on any type of tissue is scarce, even more addressing its effect on male fertility. This could be explained by the fact that dapagliflozin and the inhibition of the SGLT2 is a novel therapy and the fact that is also considered safe. Nevertheless, the more common side effect in dapagliflozin treatment is the urinary tract infection caused by the excess of glucose in urine (52). This factor can compromise male fertility by pathogenic infection present on semen (169), namely Candida (170). Interestingly, individuals subjected to treatment with dapagliflozin (10mg per day) have increased values of ketones in circulation (171). In fact, increased seminal plasma lipid oxidation is considered to be an indicator for sperm malfunction and subfertility (172). However, to the best of our knowledge, no data exists that links the circulating plasmatic and the seminal plasmatic fluid ketone levels. Hopefully, this study will bring more insight into the effect of this class of drugs on Sertoli cells and on their impact on male fertility. It will be interesting to comprehend if the blockage of SGLT2 will alter glucose metabolism by Sertoli cells. In fact, understanding the influence that both exenatide and dapagliflozin will have on these cells, if any, will make us understand more about Sertoli cell glucose metabolism and the way of action of the pharmacological agents. 36 Chapter II OBJECTIVES 37 2. OBJECTIVES The treatment of metabolic diseases, such as T2DM and obesity, involves the administration of drugs, like dapagliflozin and exenatide, that are prescribed to assist in the regulation of glucose homeodynamics and to lessen the comorbidities associated with these pathological conditions. Glucose metabolism is essential for the normal development of spermatogenesis. However, no data is available on the impact of the antidiabetic drugs dapagliflozin and exenatide on male fertility and how they influence Sertoli cells metabolism and their function in nursing and supporting the spermatogenesis. Furthermore, the influence of the combined therapy of dapagliflozin and exenatide on the male reproductive tract and particularly on Sertoli cells is even more scarce. The aim of this project is to study the influence of the exposure of Sertoli cells to dapagliflozin and/or exenatide, with a particular focus on their metabolism and viability. The target of the study will be the glucose metabolism in Sertoli cells when the antidiabetic and anti-obesogenic drugs are present in the medium. Therefore, we will focus on the following objectives: 1. Determine the expression of SGLT2 and GLP-1 receptor in murine Sertoli cells (TM4 cell line); 2. Evaluation of the cytotoxic profile of dapagliflozin and/or exenatide in TM4 cell line; 3. Determine Sertoli cells (TM4 cell line) glycolytic profile after the exposure to dapagliflozin and/or exenatide; 4. Evaluate of the expression of key metabolic enzymes and transporters in Sertoli cells (TM4 cell line) after the exposure to dapagliflozin and/or exenatide; 5. Evaluate the oxidative status of Sertoli cells (TM4 cell line) after the exposure to dapagliflozin and/or exenatide; 6. Determine Sertoli cells (TM4 cell line) glycogenic and lipidic reserves after the exposure to dapagliflozin and/or exenatide. 38 Chapter III METHODS 45 of 80 µL of phosphoric acid 10%. Following this, the samples were centrifuged at 16000 g for 15 minutes at 4 ºC. Then, 250 µL of the supernatant was collected to a new tube, where was added 250 µL of ethanol 100% and incubated overnight at 4 ºC. In the next day, the samples were centrifuged at 16000 g for 15 minutes at 4 ºC and the supernatant was discarded. The resultant glycogen pellet was dissolved in 100 µL of worm water. After dissolution, the samples were treated with 100 µL of phenol at 6,5% followed by 500 µL of concentrated sulfuric acid. The samples were left at room temperature until they cooled and reached room temperature. Finally, the calibration line was calculated by using different glycogen concentrations and the absorbance of the samples was read at 490 nm using BioTek Synergy HT (BioTek, Winooski, VT, U.S.A). Results were normalized in relation to protein concentration of the sample and the results were presented in nmol of glycogen per mg of protein. 3.11. Oil Red O Staining Oil Red O staining technique allows the detection and quantification of lipid droplets in cultured cells (182). The red dye will accumulate in cells lipid droplets that can be removed with isopropanol 100% for quantification. With this in mind, we developed this technique based on the protocol used by Xie Ge et al (183) in TM4 Sertoli cells however, with some modifications. In brief, cells were seeded and treated with the treatments for 24 hours in 12-wells plates with coverslips previous placed in the bottom. After this, cells were washed with PBS with Ca2+ and Mg2+ and then fixated with a buffered solution of formalin 10%, for 60 minutes. Cells were washed with PBS with Ca2+ and Mg2+ and incubated with isopropanol 60% for 5 minutes. After this, the stock solution of Oil Red Staining OTM at 0,05% was diluted in water for a final concentration of 0,02% and then filtrated. This solution was incubated with the fixated Sertoli cells for 20 minutes. After this incubation, the cells were washed with a solution of isopropanol at 50% and then with PBS until the oil red residues disappear. Then, the coverslips were removed from the wells and placed in a clean 12-wells plate and dried at 37 ºC. Finally, 150 µL of isopropanol were added for the removing of the oil red accumulated in the lipid droplets of the cell and then transferred to a 96-wells plate for quantification through absorbance at 510 nm using BioTek Synergy HT (BioTek, Winooski, VT, U.S.A). Resulting values were divided by the mean of the control group and expressed in fold variation versus the control group. 46 3.12. Mitochondrial membrane potential Mitochondrial membrane potential reveals the functional status of the mitochondria and cell heath (184). Depolarization of the mitochondrial membrane means that the organelle is damaged and that is not able to satisfy cellular energy demands. Hyperpolarization of the mitochondrial membrane is a marker of cellular damage, being the increased presence of ROS the main culprit of the damage (185). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide or JC-1 is a dye which allows to measure mitochondrial membrane potential. Unhealthy cells emanated green florescence caused by JC-1 monomers. On the other hand, healthy cells emitted red florescence from the aggregates of JC-1. TM4 Sertoli cells were seeded in black 96-well plates and incubated with the different treatments’ medium. After the 24h treatment with dapagliflozin, exenatide, and the combined therapy, the treatment medium was removed and replaced with Sertoli cell medium with JC-1 at 2 µM diluted in FBS (1% of medium volume). Then, the cells were incubated at 37 ºC for 30 minutes in a dark environment. After incubation, the medium was removed and media without JC-1 was added to allow the measurement of the fluorescence of each sample at the excitation wavelength of 485 nm and 535 nm and an emission wavelength of 530 and 590 nm with BioTek Synergy HT (BioTek, Winooski, VT, U.S.A). Results were treated as the ration aggregates/monomers, divided by the mean of the control group and expressed in a fold variation versus the control group. 3.13. Statistical analysis Statistical significance among the experimental groups’ samples was assessed by ordinary one-way ANOVA plus uncorrected Fisher’s LSD, with a single pooled variance test. This test was performed in all the results except when were compared the results from the combined therapy of dapagliflozin plus exenatide with its control. In this situation was performed an unpaired T-test student. All experimental data are shown as mean ± SEM. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA). Outliers were identified using the Grubbs method with Alpha equal to 0,2 and the results were considered significant when p < 0,05. 47 Chapter IV RESULTS 48 4. RESULTS 4.1. GLP-1 receptor and SGLT2 are expressed in Sertoli cells GLP-1 receptor has already been identified in mouse testes (167) and in human Sertoli cells (186). Herein, we examined if GLP-1 receptor was expressed in TM4 Sertoli cell line derived from mouse testes. We evaluated the mRNA expression for this receptor, using specific primer set, and obtained a 253 bp amplicon (Figure 7A). Similarly, SGLT2 expression in human testes was also already described (122). In the TM4 Sertoli cell line we were able to detect the presence of SGLT2 mRNA, using a specific primer set, which resulted in a 104 bp amplicon (Figure 7B). 4.2. Exposure of TM4 Sertoli cells to exenatide 4.2.1. Exenatide decreased Sertoli cell membrane integrity and metabolic viability, without affecting cell proliferation Results from techniques such as SRB, LDH release and MTT assays are essential in studies that aim to evaluate the cytotoxic effect of a chemical or drug. In our case, cytotoxicity assays allowed us to determine if the different treatments would affect cell viability or metabolic viability in a way that would invalidate further studies. SRB assay data showed that exenatide at concentrations of 2,5 pM (1,05 ± 0,03 – fold variation to control), 25 pM (1,06 ± 0,05 – fold variation to control), or 250 pM (1,03 ± 0,04) did not have any significant effect on TM4 Sertoli cells proliferation, as compared with Sertoli cells on control conditions (Figure 8A). In the case of the LDH release assay, exenatide decreased membrane integrity in relation to control at every concentration tested. Exenatide showed to increase LDH leakage by TM4 Sertoli cells at the concentrations of 2,5 pM (1,44 ± 0,10 – fold variation to control), 25 pM (1,68 ± 0,12 – fold variation to control), and 250 pM (1,82 ± 0,12 – fold variation to control) (Figure 8B). Finally, data obtained from of the MTT assay showed that the cells incubated with the pharmacological concentration (25 pM) of exenatide did not have a A B Figure 7: Confirmation of the expression of GLP-1 receptor (A) and SGLT2 (B) in TM4 Sertoli cells by reverse transcriptase polymerase chain reaction. Mouse lung, testis, and kidney total RNA were used as positive controls. Legend: N - negative control; SC - TM4 Sertoli cells; T - Testis; L - Lung; K - Kidney. 49 significantly different metabolic viability (1,00 ± 0,03 – fold variation to control) in relation to control. However, the Sertoli cells exposed to the other exenatide concentrations showed a decrease in the metabolic viability. The cells exposed to exenatide 250 pM (0,87 ± 0,03 – fold variation to control) were the ones in which metabolic viability decreased the most. The cells exposed to the concentration of 2,5 pM (0,88 ± 0,02 – fold variation to control) also showed a significant decreased of their metabolic viability in relation to the ones of the control group (Figure 8C). C ell P ro life ra tio n (fo ld v aria tio n to C o n tro l) C T R 2,5 p M 25 p M 250 p M 0.0 0.5 1.0 1.5 A B C Figure 8: Evaluation of the cytotoxic profile of the different concentration of exenatide on TM4 Sertoli cells. The data shows results from the experimental groups of sub-pharmacological concentration (2,5 pM), pharmacological concentration (25 pM), and supra-pharmacological concentration (250 pM) of exenatide. To evaluate the treatments cytotoxicity, the effect of the different concentrations on cellular proliferation (A), membrane integrity (B), and metabolic viability (C) was studied through SRB assay, LDH release assay, and MTT assay, respectively. The data is organized in pooled data of independent experiments and the results are expressed as mean ± SEM (N=6 for each condition). Significantly different results (P < 0.05) are indicated as: a – relative to control (CTR). LD H R elea s e (fo ld v aria tio n to C o n tro l) C T R 2,5 p M 25 p M 250 p M 0.0 0.5 1.0 1.5 2.0 2.5 a aa M e ta b olic A ctivity (fo ld v aria tio n to C o n tro l) C T R 2,5 p M 25 p M 250 p M 0.0 0.5 1.0 1.5 aa LD H R elea s e (fo ld v aria tio n to C o n tro l) C T R 2,5 p M 25 p M 250 p M 0.0 0.5 1.0 1.5 2.0 2.5 a aa 50 4.2.2. Exenatide maintained glucose consumption by Sertoli cells while increasing the expression of GLUT2, when at pharmacological concentrations Sertoli cells nourish developing sperm cells through all the steps of spermatogenesis. With this in mind, glucose consumption is essential for the formation of new spermatozoa. Ideally, Sertoli cells should be able to maintain glucose consumption to sustain the delivery of metabolites such as lactate and acetate to developing germ cells. In our study, the data showed no statistical differences in the Sertoli cell glucose metabolism of cells treated or not with exenatide. Taking the glucose consumed by the control group as reference (12127 ± 4212 nmol/106 cells), the treatment with exenatide at 2,5 pM (6589 ± 1497 nmol/106 cells), at 25 pM (6416 ± 1154 nmol/106 cells), and at 250 pM (8313 ± 1734 nmol/106 cells) seemed to decrease glucose consumption although this tendency did not reach statistical significance (Figure 9A). In contrast, GLUT2 expression levels were increased in cells treated with exenatide concentration of 25 pM (3,18 ± 1,02 – fold variation to control). On Sertoli cells exposed to the concentrations of 2,5 pM and 250 pM, the enhanced expression was not significantly different (2,59 ± 0,49 and 2,63 ± 0,75 – fold variation to control, respectively) from that observed in the cells from the control group (Figure 9B). G LU T2 E xp re ssio n (fo ld v aria tio n to C o n tro l) C T R 2,5 pM 25 p M 250 pM 0 1 2 3 4 5 a A B GLUT2 (50 kDa) Figure 9: Effect of exenatide at 2,5 pM, 25 pM, and 250 pM on glucose consumption by TM4 Sertoli cells. The figure shows pooled data of independent experiments, indicating glucose consumption (A) and protein levels of glucose transporters 2 (GLUT2) and a representative immunoblot (B) performed with Sertoli cells treated with the different concentrations of exenatide and compared with the control group. The results are expressed as mean ± SEM (N=6 for each condition). Significantly different results (P < 0.05) are indicated as: a – relative to control (CTR). G luco se C onsu m ptio n (n m o l/106c e lls ) C T R 2,5 pM 25 p M 250 pM 0 5000 10000 15000 20000 G LU T2 E xp re ssio n (fo ld v aria tio n to C o n tro l) C T R 2,5 pM 25 pM 250 pM 0 1 2 3 4 5 a 51 4.2.3. Exenatide treatment maintained the secretion of monocarboxylates and alanine in TM4 Sertoli cells Lactate and acetate are two monocarboxylates essential for spermatogenesis. The secretion of these metabolites by TM4 Sertoli cells exposed to exenatide did not show any difference in relation to those of the control group. Lactate production by Sertoli cells exposed to 2,5 pM of exenatide (21682 ± 3934 nmol/106 cells), or 25 pM (24447 ± 4990 nmol/106 cells), or 250 pM (18532 ± 4493 nmol/106 cells) did not reach statistical difference, when compared that of cells from the control group (15370 ± 2397 nmol/106 cells) (Figure 10A). Concurrently, the results obtained for the expression levels of LDH in the TM4 Sertoli cells of the different experimental groups treated with exenatide did not vary significantly. Sertoli cells treated with 2,5 pM presented an LDH expression level of 1,50 ± 0,26 – fold variation to cells of the control group. Similarly, cells treated with 25 pM and 250 pM had LDH expression levels of 1,30 ± 0,34 and 1,54 ± 0,41 – fold variation to control group, respectively (Figure 10B). LDH activity also did not show any difference in the diverse treatments. When compared to the control group (90,1 ± 17,5 nmol/min/mg of protein), Sertoli cells treated with exenatide at 2,5 pM (112 ± 23 nmol/min/mg of protein), at 25 pM (99,4 ± 21,4 nmol/min/mg of protein), and at 250 pM (101 ± 18 nmol/min/mg of protein) maintained LDH activity (Figure 10C). 52 Acetate is also an important substrate for the development of the germ cells. Our results showed that exenatide did not alter acetate secretion by Sertoli cells in a statistically significant way (Figure 11A). However, treatment of TM4 cells with 2,5 pM (-26 ± 67 nmol/106 cells) and 25 pM (-32 ± 64 nmol/106 cells) reverted the production trend of acetate, observed in the cells of the control group (115 ± 28,6 nmol/106 cells) in which acetate is being produced. It is worth noticing that this fact could be due to the low concentration of acetate in the medium. The remaining suprapharmacological concentration led to an acetate production of 89,9 ± 60,5 nmol/ 106 cells, which is not statistically different from that observed in the cells from the control group. We also determined the MCT4 expression levels in TM4 cells of the different experimental groups. The cells exposed to the concentrations of 2,5 pM (2,78 ± 1,48 – fold variation to control), 25 pM (1,44 ± 0,41 – fold variation to control), and 250 pM (1,72 ± 0,61 – fold variation to control), although LDH (37 kDa) Lacta te P ro d uc tio n (n m o l/106c e lls ) C T R 2,5 p M 25 p M 250 p M 0 10000 20000 30000 40000 LD H E xp ress ion (fo ld v aria tio n to C o n tro l) C T R 2,5 p M 25 p M 250 p M 0.0 0.5 1.0 1.5 2.0 2.5 Figure 10: Effect of exenatide concentrations (2,5 pM, 25 pM, and 250 pM) on lactate production in TM4 Sertoli cells. The figure shows pooled data of independent experiments, indicating lactate production (A), the quantification of lactate dehydrogenase (LDH) expression levels and representative immunoblot (B), and intracellular LDH activity (C) by Sertoli cells under the different exenatide treatments and compared with the control group (CTR). The results are expressed as mean ± SEM (N=6 for each condition). A B C LD H A ctivity (n m o l/m in /m g ) C T R 2,5 p M 25 p M 250 p M 0 50 100 150 LD H A ctivity (n m o l/m in /m g ) C T R 2,5 p M 25 p M 250 p M 0 50 100 150 53 showing an increasing tendency, did not exhibit an alteration on the expression of MCT4 in relation to the control group (Figure 11B). Similarly, the expression of the MCT4 B1ATM2 isoform (Uniprot: B1ATM2) with lower molecular weight remained unchanged in the TM4 Sertoli cells after exposure to exenatide. In cells exposed to 2,5 pM of exenatide, MCT4 B1ATM2 isoform levels were 1,66 ± 0,42 – fold variation to control and in those exposed to 25 pM of exenatide the expression levels were 2,0 ± 0,5 – fold variation to control, whereas cells exposed to supra-pharmacological concentrations (250 pM) of exenatide had an expression level of 1,54 ± 0,27 – fold variation to control (Figure 11C) for this MCT4 isoform. Like lactate and acetate, alanine is also an important substrate for developing germ cells. In addition, the equilibrium between lactate and alanine is important for the redox state of the cell (187). In Sertoli cells cultured in control conditions, alanine production was 376 ± 85 nmol/106 cells. When TM4 Sertoli cells were treated with 2,5 pM of exenatide, the production of alanine was 383 ± 58 nmol/106 cells, and in those treated with 25 pM, the production was 471 ± 122 nmol/106 cells. Finally, in cells treated with 250 pM of exenatide, we observed an alanine production of 342 ± 60 nmol/106 cells, statistically similar to that observed in the other experimental groups (Figure 11D). 54 4.2.4. Sub-pharmacological concentration of exenatide decreases mitochondrial membrane potential in TM4 Sertoli cells Sertoli cells need to ensure not only the energetic requirements of germ cells during spermatogenesis but also its own metabolic and energetic needs. To do so, Sertoli cells utilize the TCA cycle and the pentose phosphate pathway (121). A proper mitochondrial function is imperative to produce energy through the TCA cycle. Impaired mitochondrial membrane potential is a marker of unhealthy mitochondria and dysregulated energy synthesis. JC-1 assay allows us to measure the depolarization of mitochondrial membrane. MCT4 (50 kDa) M C T4 E xp ressio n (fo ld v aria tio n to C o n tro l) C T R 2,5 p M 25 p M 250 p M 0.0 0.5 1.0 1.5 2.0 Figure 11: Effect of exenatide concentrations (2,5 pM, 25 pM, and 250 pM) on metabolite production and secretion in TM4 Sertoli cells. The figure shows pooled data of independent experiments, indicating acetate production (A), monocarboxylate transporter 4 (MCT4) expression levels and representative immunoblot (B), MCT4 B1ATM2 isoform expression levels and representative immunoblot (C), and alanine production (D) by Sertoli cells under the different exenatide treatments and compared with the control group (CTR). The results are expressed as mean ± SEM (N=6 for each condition). A cetate P ro d u ction (n m o l/106c e lls ) C T R 2,5 p M 25 p M 250 p M -200 -100 0 100 200 A lanin e P ro du c tio n (n m o l/106c e lls ) C T R 2,5 p M 25 p M 250 p M 0 200 400 600 800 M C T4 (B 1A TM 2 ) E xp ress io n (fo ld v aria tio n to C o n tro l) C T R 2,5 p M 25 p M 250 p M 0 1 2 3 MCT4 (30 kDa) A B C D A cetate P ro d u ction (n m o l/106c e lls ) C T R 2,5 p M 25 p M 250 p M -200 -100 0 100 200 A lanin e P ro du c tio n (n m o l/106c e lls ) C T R 2,5 p M 25 p M 250 p M 0 200 400 600 800 M C T4 (B 1A TM 2 ) E xp ress io n (fo ld v aria tio n to C o n tro l) C T R 2,5 p M 25 p M 250 p M 0 1 2 3 61 compared with that of cells from the control group (Figure 16A). Interestingly, LDH expression shows a tendency to increase when cells were treated with increasing concentrations of dapagliflozin, without ever reaching statistical significance. TM4 Sertoli cells treated with 50 nM of dapagliflozin had an LDH expression of 1,32 ± 0,40 – fold variation to control, whereas those treated with 500 nM of dapagliflozin had an LDH expression of 1,48 ± 0,37 – fold variation to control. Finally, cells treated with 5000 nM of dapagliflozin had an LDH expression of 2,10 ± 0,57 – fold variation to control (Figure 16B). Similarly, intracellular LDH activity did not show any significant change when cells were treated with increasing concentrations of dapagliflozin in relation to the control group. Intracellular LDH activity in Sertoli cells treated with control conditions (90,1 ± 17,5 nmol/min/mg of protein) did not had a significant variation in relation to the Sertoli cells treated with 50 nM (101 ± 15 nmol/min/mg of protein), with 500 nM (105 ± 17 nmol/min/mg of protein), or with 5000 nM (103 ± 16 nmol/min/mg of protein) of dapagliflozin (Figure 16C). 62 On the other hand, acetate production was not significantly different between the cells of the various experimental groups. Sertoli cells from the control group exhibited an acetate production of 115 ± 28 nmol/106 cells, whereas those of the groups treated with 50 nM, 500 nM, and 5000 nM exhibited an acetate production of 196 ± 34; 206 ± 41; and 202 ± 33 nmol/106 cells, respectively (Figure 17A). Moreover, MCT4 expression levels in cells treated or not treated with dapagliflozin was not altered. When incubated with 50 nM dapagliflozin, the TM4 Sertoli cells’ expression levels of MCT4 were 1,57 ± 0,28 – fold variation to control. Those treated with dapagliflozin at a concentration of 500 nM had MCT4 expression levels of 1,68 ± 0,37 – fold variation to control, while those treated with 5000 nM of dapagliflozin had MCT4 expression levels of 1,68 ± 0,40 – fold variation to control (Figure 17B). Contrastingly, the expression of the MCT4 Lacta te P ro d uc tio n (n m o l/106c e lls ) C T R 50 n M 500 n M 5000 n M 0 10000 20000 30000 40000 a LD H E xp ress ion (fo ld v aria tio n to C o n tro l) C T R 50 n M 500 n M 5000 n M 0 1 2 3 LDH (37 kDa) A B C Figure 16: Effect of dapagliflozin concentrations (50 nM, 500 nM, and 5000 nM) on lactate production in TM4 Sertoli cells. The figure shows pooled data of independent experiments, indicating lactate production (A); the expression levels of lactate dehydrogenase (LDH) and representative immunoblot (B); and intracellular LDH activity (C) by Sertoli cells under the different treatments of dapagliflozin and compared with the control group. The results are expressed as mean ± SEM (N=6 for each condition). Significantly different results (P < 0.05) are indicated as: a – relative to control (CTR). LD H A ctivity (n m o l/m in /m g ) C T R 50 n M 500 n M 5000 n M 0 50 100 150 LD H E xp ress io n (fo ld v ariatio n to C o n tro l) C T R 50 n M 500 n M 5000 n M 0 1 2 3 LD H A ctivity (n m o l/m in /m g ) C T R 50 n M 500 n M 5000 n M 0 50 100 150 63 B1ATM2 isoform was altered in the presence of dapagliflozin. Interestingly, there was a significant increase in the isoform expression when cells were treated with the pharmacological concentration (500 nM) of dapagliflozin, reaching a 2,18 ± 0,42 – fold variation to control. The treatment of TM4 Sertoli cells with 50 nM (1,94 ± 0,44 – fold variation to control) and 5000 nM (1,27 ± 0,36 – fold variation to control) of dapagliflozin did not alter significantly the expression levels of the MCT4 B1ATM2 isoform when compared with that of the cells from the control group (Figure 17C). Similarly to what happened with lactate, alanine production also peaked when Sertoli cells were treated with the pharmacological concentration (500 nM) of dapagliflozin. When incubated with the control medium, Sertoli cells alanine production was 376 ± 85 nmol/106 cells. However, when the cells were treated with the pharmacological concentration of dapagliflozin (500 nM), alanine production was significantly enhanced to 863 ± 174 nmol/106 cells. Contrastingly, treatment with the 50 nM of dapagliflozin (539 ± 102 nmol/106 cells) and 5000 nM of dapagliflozin (728 ± 178 nmol/106 cells) did not change significantly the production of alanine in relation to that of cells from the control group (Figure 17D). 64 M C T4 (B 1A TM 2 ) E xp ress io n (fo ld v aria tio n to C o n tro l) C T R 50 nM 500 n M 5000 nM 0 1 2 3a A lanin e P ro du c tio n (n m o l/106c e lls ) C T R 50 nM 500 n M 5000 nM 0 500 1000 1500 a M C T4 E xp ressio n (fo ld v aria tio n to C o n tro l) C T R 50 nM 500 n M 5000 nM 0.0 0.5 1.0 1.5 2.0 2.5 MCT4 (50 kDa) MCT4 (30 kDa) A B C D Figure 17: Effect of dapagliflozin concentrations (50 nM, 500 nM, and 5000 nM) on acetate and alanine production in TM4 Sertoli cells. The figure shows pooled data of independent experiments, indicating: acetate production (A); the monocarboxylate transporter 4 (MCT4) expression levels and representative immunoblot (B); the expression levels of MCT4 B1ATM2 isoform and representative immunoblot (C); and alanine production (D) by Sertoli cells under the different treatments of dapagliflozin and compared with the control group. The results are expressed as mean ± SEM (N=6 for each condition). Significantly different results (P < 0.05) are indicated as: a – relative to control (CTR). A cetate P ro d u ction (n m o l/106c e lls ) C T R 50 nM 500 n M 5000 nM 0 100 200 300 A lanin e P ro du c tio n (n m o l/106c e lls ) C T R 50 n M 500 nM 5000 n M 0 500 1000 1500 a 65 4.3.4. Supra-pharmacological concentration of dapagliflozin decreases the amount of lipid droplets on TM4 Sertoli cells Our data showed that dapagliflozin increased the secretion of lactate and alanine without increasing glucose consumption. This fact made us investigate which could be the source of the extra carbons used for the synthesis of the extra substrates noticed at pharmacological concentrations. First, we evaluated the glycogen reserves of the Sertoli cells because glycogen can be quickly mobilized and converted into glucose to undergo glycolysis. The quantification of glycogen on the cells from the different experimental groups showed no differences in glycogen content. The Sertoli cells from the control group had similar glycogen content (15,2 ± 1,4 nmol/mg protein) to those treated with concentration of 50 nM of dapagliflozin (13,9 ± 1,1 nmol/mg protein), to those treated with 500 nM of dapagliflozin (12,5 ± 0,6 nmol/mg protein), and to those treated with 5000 nM of dapagliflozin that had a glycogen content of 11,5 ± 1,6 nmol/mg protein (Figure 18A). We also focused our attention on the mobilization of cytoplasmic lipid droplets. Oil red o staining assay results showed that the cells treated with dapagliflozin at supra-pharmacological concentrations of 5000 nM (0,74 ± 0,03 – fold variation to control) had decreased content of lipid droplets when compared to those of the control group. Cells from the other experimental groups did not show any alteration in the lipid droplets content in relation to cells from the control group, with the cells exposed to 50 nM of dapagliflozin showed a lipid content of 0,92 ± 0,07 – fold variation to control, and those exposed to 500 nM of dapagliflozin showed a lipid content of 0,89 ± 0,08 – fold variation to control (Figure 18B). 66 4.3.5. Dapagliflozin maintains the mitochondrial membrane potential of Sertoli cells Dapagliflozin at a supra-pharmacological concentration decreases lipid droplets in TM4 Sertoli cells, which could indicate that β-oxidation could be favored. To evaluate this hypothesis, cells treated with the different concentrations of dapagliflozin had their mitochondrial membrane potential analyzed by JC-1 assay. The data obtained from the cells treated with dapagliflozin at 50 nM (1,13 ± 0,28 – fold variation to control), at 500 nM (1,15 ± 0,33 – fold variation to control), and at 5000 nM (1,12 ± 0,24 – fold variation to control) did not significantly differ from that obtained for the cells of the control group (Figure 19A). Similarly, lactate/alanine ratio was also maintained in treated TM4 Sertoli cells. Control group had a ratio of 31,8 ± 2,0, whereas Sertoli cells treated with 50 nM, 500 nM, and 5000 nM had a ratio of 34,3 ± 2,5; 31,6 ± 1,9; and 33,7 ± 1,3; respectively, never reaching statistical relevance (Figure 19B). Lip id A cc u m u la tio n (fo ld v aria tio n to C o n tro l) C T R 50 nM 500 n M 5000 nM 0.0 0.5 1.0 1.5 a A B Figure 18: Effect of dapagliflozin treatments (50 nM, 500 nM, and 5000 nM) on carbon storage by TM4 Sertoli cells. The data shows the concentration of glycogen reserves (A) and the quantification of lipid droplets (B) of Sertoli cells when incubated with the different concentrations of dapagliflozin. The data is organized in pooled data of independent experiments and the results are expressed as mean ± SEM (N=6 for each condition). Significantly different results (P < 0.05) are indicated as: a – relative to control (CTR). G lycogen C on tent nm o l/m g p rotein C T R 50 n M 500 n M 5000 n M 0 5 10 15 20 Lip id A cc u m u la tio n (fo ld v aria tio n to C o n tro l) C T R 50 nM 500 n M 5000 nM 0.0 0.5 1.0 1.5 a 67 4.4. Exposure of TM4 Sertoli cells to exenatide plus dapagliflozin 4.4.1. Combined treatment with exenatide plus dapagliflozin decreases Sertoli cells proliferation and metabolic viability without decreasing membrane integrity With this study, we tried to evaluate the impact that the treatment with the combination of pharmacological concentrations of both exenatide (25 pM) and dapagliflozin (500 nM) could have on Sertoli cell development and metabolism. First, we assessed the cytotoxicity of this combined treatment on Sertoli cell proliferation. SRB assay results showed that the combined treatment with exenatide plus dapagliflozin decreased cell proliferation (0,81 ± 0,03 – fold variation to control) in relation to the control group (Figure 20A). Moreover, a cytotoxic effect was also noted when evaluating the metabolic viability through MTT assay. Sertoli cells treated with the combination of exenatide plus dapagliflozin (0,92 ± 0,02 – fold variation to control group) presented decreased metabolic viability (Figure 20B). Contrastingly, LDH release did not significantly change in relation to the control group when the cells were treated with the combination of exenatide plus dapagliflozin (0,93 ± 0,04 – fold variation to control) (Figure 20C). L ac ta te /A la n in e R atio C T R 50 n M 500 n M 5000 n M 0 10 20 30 40 JC -1 R atio (fo ld v aria tio n to C o n tro l) C T R 50 n M 500 n M 5000 n M 0.0 0.5 1.0 1.5 A B Figure 19: Effect of dapagliflozin (50 nM, 500 nM, and 5000 nM) on the mitochondrial function of TM4 Sertoli cell line. The data shows the ratio of the JC-1 assay (A) and the ratio of lactate/alanine (B) of Sertoli cells incubated with the different concentrations of dapagliflozin and compared with the control group (CTR). The data is organized in pooled data of independent experiments and the results are expressed as mean ± SEM (N=6 for each condition). L ac ta te /A la n in e R atio C T R 50 n M 500 n M 5000 n M 0 10 20 30 40 68 4.4.2. Combined treatment with exenatide plus dapagliflozin maintains the glucose consumption and GLUT2 expression on TM4 Sertoli cells The exposure of TM4 Sertoli cells to the combination of the pharmacological concentration of exenatide and dapagliflozin did not alter glucose consumption. Sertoli cells from the control group exhibited a glucose consumption of 22390 ± 7834 nmol/106 cells, whereas, when cells were treated with the drug combination, the glucose consumption was 41303 ± 11800 nmol/106 cells (Figure 21A). GLUT2 expression levels of cells treated with exenatide plus dapagliflozin (1,04 ± 0,26 – fold variation to control) did not differ from that observed in cells from the control group (Figure 21B). C ell P ro life ra tio n (fo ld v aria tio n to C o n tro l) C T R E + D 0.0 0.5 1.0 1.5 a LD H R elea s e (fo ld v a ria tio n to c o n tro l) C T R E + D 0.0 0.5 1.0 1.5 A B C Figure 20: Evaluation of the cytotoxicity profile of the combined treatment (E+D) (25 pM exenatide plus 500 nM dapagliflozin) on TM4 Sertoli cells. To evaluate the cytotoxicity of the combined treatment on cellular proliferation (A), metabolic viability (B), and membrane integrity (C) were evaluated using the SRB assay, MTT assay, and LDH release assay, respectively. The data is organized in pooled data of independent experiments and the results are expressed as mean ± SEM (N=6 for each condition). Significantly different results (P < 0.05) are indicated as: a – relative to control (CTR). M etab olic V ia b ility (fo ld v a ria tio n to c o n trol) C T R E + D 0.0 0.5 1.0 1.5 a M e ta b olic A ctivity (fo ld v aria tio n to C o n tro l) C T R E + D 0.0 0.5 1.0 1.5 a LD H R elea s e (fo ld v aria tio n to C o n tro l) C T R E + D 0.0 0.5 1.0 1.5 69 4.4.3. Combined treatment with exenatide plus dapagliflozin maintained the production of monocarboxylates and alanine on TM4 Sertoli cells Cells exposed to control medium exhibited a lactate production of 31518 ± 10556 nmol/106 cells, while those treated with the combination of exenatide plus dapagliflozin had a lactate production of 48730 ± 12274 nmol/106 cells, which were not statistically different (Figure 22A). Similarly, LDH expression and activity levels were not altered by exposure to the combined treatment with exenatide plus dapagliflozin. Sertoli cells treated with the combination exenatide plus dapagliflozin showed an LDH expression level of 0,90 ± 0,19 – fold variation to control (Figure 22B). In addition, cells exposed to exenatide plus dapagliflozin had an intracellular LDH activity (106 ± 20 nmol/min/mg of protein) similar to those of the control group (96 ± 11 nmol/min/mg of protein) (Figure 22C). Moreover, neither MCT4 or MCT4 B1ATM2 isoform levels expression were altered in relation to the control group (0,75 ± 0,05 and 0,43 ± 0,05 – fold variation to control, respectively) by the combined treatment with exenatide plus dapagliflozin (Figure 22D). These transporters are also capable of export other monocarboxylates that are produced by the cells, namely acetate. The amount of acetate exported by Sertoli cells exposed to the combined treatment (108 ± 45 nmol/106 cells) was not different from that exported by cells from the control group (296 ± 116 nmol/106 cells) (Figure 22E). The production of alanine by Sertoli cells was also G luco se co n sum ption (n m o l/106c e lls ) C T R E + D 0 20000 40000 60000 G LU T2 E xpress io n (fo ld v aria tio n to C o n tro l) C T R E + D 0.0 0.5 1.0 1.5 A B Figure 21: Effect of the combined treatment (E+D) (25 pM exenatide plus 500 nM dapagliflozin) on glucose consumption by TM4 Sertoli cells. The figure shows pooled data of independent experiments, indicating glucose consumption (A) and protein levels of glucose transporters 2 (GLUT2) and the respective representative immunoblot (B) of Sertoli cells under the treatment of the combined treatment and compared with the control group (CTR). The results are expressed as mean ± SEM (N=6 for each condition). GLUT2 (50 kD) G LU T2 E xpress io n (fo ld v aria tio n to C o n tro l) C T R E +D 0.0 0.5 1.0 1.5 G luco se C ons um ptio n (n m o l/1 06c e lls ) C T R E + D 0 20000 40000 60000 70 statistically similar between the cells from the group exposed to the combined treatment with exenatide plus dapagliflozin (1428 ± 304 nmol/106 cells) to that from cells from the control group (637 ± 258 nmol/ 106 cells) (Figure 22F). LD H A ctiv ity (n m o l/m in /m g ) C T R E + D 0 50 100 150 Lacta te P ro d u c tio n (n m o l/106c e lls ) C T R E + D 0 20000 40000 60000 80000 LD H E xp ress ion (fo ld v aria tio n to C o n tro l) C T R E + D 0.0 0.5 1.0 1.5 M C T4 E xp ressio n (fo ld v aria tio n to C o n tro l) C T R E + D C T R E + D 0.0 0.5 1.0 1.5 A lanin e P ro du c tio n (n m o l/106c e lls ) C T R E + D 0 500 1000 1500 2000 LDH (37 kDa) MCT4 (50 kDa) MCT4 (37 kDa) MCT4 MCT4 isoform A B C D E Figure 22: Effect of the combined treatment (E+D) (25 pM exenatide plus 500 nM dapagliflozin) on monocarboxylates (lactate and acetate) and alanine production in TM4 Sertoli cells. The figure shows pooled data of independent experiments, indicating lactate production (A), quantification of lactate dehydrogenase (LDH) expression levels and respective representative immunoblot (B), intracellular LDH activity (C), the expression levels of monocarboxylate transporter 4 (MCT4) and MCT4 B1ATM2 isoform and respective representative immunoblots (D), acetate production (E) and alanine production (F) by Sertoli cells under the treatment of the combined treatment and compared with the control group (CTR). The results are expressed as mean ± SEM (N=6 for each condition). A cetate P ro d u ction (n m o l/106c e lls ) C T R E + D 0 100 200 300 400 500 F LD H E xp ress ion (fo ld v aria tio n to C o n tro l) C T R E + D 0.0 0.5 1.0 1.5 77 Sertoli cells in the study mentioned above. This fact is one more example of the adaptative abilities and metabolic plasticity of the Sertoli cells to different external conditions (186, 187). Without an alteration of the glucose consumption, as observed in our study, TM4 Sertoli cells did not have the necessity of mobilizing their glycogen and lipid reserves as we report. However, TM4 Sertoli cells were not insensitive to exenatide. Sub-pharmacological concentrations (2,5 pM) increased the lactate/alanine ratio. This ratio is often associated with the redox state of the cell (187). Interestingly, lactate/alanine ratio increased in the Sertoli cells exposed to this same concentration, which further suggests a decreased metabolic viability in these cells. In fact, when TM4 Sertoli cells were exposed to exenatide at concentrations of 2,5 pM, we also observed depolarization of the mitochondrial membrane. With this data, we hypothesized that exenatide at 2,5 pM enhanced NADH/NAD+ ratio, which decreased the metabolic viability demonstrated by the mild depolarization of the mitochondrial membrane on TM4 Sertoli cells. In the cells exposed to the other exenatide concentrations, lactate and alanine production and the ratio NADH/NAD+ was not altered, which did not correlate with any mitochondrial membrane depolarization. The study of the metabolic secretome of TM4 Sertoli cells exposed to dapagliflozin showed that glucose consumption did not suffer any significant change. One of the main pharmacological property of this drug is to block the SGLT2 and it has been shown that SGLT2 inhibitors decrease cellular glucose consumption in other cellular systems (198). However, our study revealed that cells incubated with 5000 nM of dapagliflozin had enhanced GLUT2 expression levels, one important glucose mobilizer on Sertoli cells (124). Enhanced expression of GLUT2 could compensate for the blockage of the SGLT2 and sustain the glucose consumption in these Sertoli cells. Moreover, even without increasing glucose consumption, exposure to pharmacological concentrations of dapagliflozin (500 nM) enhanced the lactate secretion on mice Sertoli cells. This fact could be positive for male fertility since lactate is the preferential energy substrate for developing germ cells (20, 187). MCTs are the membrane transporters responsible for the secretion of the lactate to the developing germ cells. The major MCT in Sertoli cells is MCT4 and its expression levels were not changed when TM4 Sertoli cells were treated with dapagliflozin. Still, we are able to describe for the first time the expression of on specific cataloged MCT4 isoform (Uniprot: B1ATM2), which has a very similar primary and secondary structure in relation to the original isoform. However, the molecular weight is approximately 30 kDa in contrasts with the one of the original MCT4 isoform which has a molecular weight of approximately 50 kDa. Moreover, the transcription of B1ATM2 isoform gene was only found in mice (199) up until now. The B1ATM2 78 MCT4 isoform had its expression levels increased after the treatment with pharmacological concentrations (500 nM) of dapagliflozin. This fact can explain the superior lactate levels observed in the extracellular medium. However, due to the lack of studies assessing the 3D structure and functional properties of this MCT4 protein isoform further studies are needed to support this suggestion. To further understand the mechanism of action of dapagliflozin, we investigated if other carbon sources were being used for the increased production of lactate observed in the cells exposed to the pharmacological concentrations (500 nM). It has been described that Sertoli cells are capable of utilizing glycogen for the production of lactate in conditions with lower glucose availability and maintain their energetic needs (119, 189). Our results showed no changes in TM4 Sertoli cells glycogen content exposed to dapagliflozin. We must highlight that our study was performed with a culture medium supplemented with high glucose levels, mimicking the ones found in hyperglycemic individuals (3). In these conditions, it has been described that rodent Sertoli cells increased their glycogen reserves (200). Hence, we can hypothesize that both drug treatments and the hyperglycemic medium had opposite effects on glycogen reserves which could explain the nonalteration of the glycogen levels on TM4 Sertoli cells. After this, we evaluated the quantity of lipid droplets in the TM4 Sertoli cells after the treatment with dapagliflozin. It is widely described that Sertoli cells can metabolize lipids and mobilize its reserves when needed (119, 201). However, this was not the case for TM4 Sertoli cells treated with pharmacological concentrations of dapagliflozin (500 nM). Thus, our hypothesis for the enhanced lactate and alanine production on TM4 Sertoli cells exposed to 500 nM of dapagliflozin is that this treatment enhances the glycolytic efficiency of the cells. This could be proved through alanine production which is often considered a marker of glycolytic activity (202). Alanine synthesis is fundamental for the maintenance of the lactate/alanine ratio, which is not altered in the Sertoli cells exposed to the pharmacological concentrations of dapagliflozin (500 nM). Although our results showed that LDH expression and activity was maintained in these conditions. It is known that this enzyme is able to catalyze not only the forward reaction but also the reversible reaction. Thus, LDH can be prioritizing lactate synthesis when cells are exposed to this concentration of dapagliflozin. Is also worth noticing that dapagliflozin has the ability to diminish lipid droplets on TM4 Sertoli cells at supra-pharmacological concentrations. This evidences the anti-obesogenic ability of the dapagliflozin. In fact, it has already been described that people undergoing dapagliflozin treatment have enhanced levels of ketone bodies in circulation (171). In addition, diabetic patients that added dapagliflozin (5 mg/day) to its 79 natural treatment had their liver fat accumulation decreased during the 6-month treatment (203). Our study points out that dapagliflozin at supra-pharmacological concentrations (5000 nM) is also able to decrease intracellular fat deposits in Sertoli cells. Combined treatment of exenatide plus dapagliflozin did not alter the glycolytic flux of TM4 Sertoli cells. Moreover, enhanced lactate and alanine production and increased MCT4 B1ATM2 isoform expression levels observed in cells treated with pharmacological concentration of dapagliflozin were not evident when treated with the same dapagliflozin concentration but also in the presence of exenatide. Taking these results in consideration, combined therapy of exenatide and dapagliflozin does not seem to have an additive effect at a cellular level, at least in the glucose metabolism of TM4 mice Sertoli cells. The presence of exenatide in the medium seemed to promote the metabolic homeostasis of TM4 Sertoli cells. In fact, the drugs seem to have more of a synergic effect to each other at a cellular level rather than the additive effect seen in human clinical trials. Still, we must be aware that our study was developed using TM4 Sertoli cell line, which is an immortalized cell line. TM4 Sertoli cells maintain the hormone responsiveness yet, it is not the same magnitude of healthy human Sertoli cells. Moreover, TM4 Sertoli cells are under constant division which contrasts with post-pubertal humans’ Sertoli cells that stop their division, which can also alter the cell metabolism. However, we believe that besides these limitations our work is indicative of what happens in Sertoli cells when treated with the different therapies here tested. 80 Chapter VI CONCLUSION 81 6. CONCLUSION Metabolic diseases, such as DM and obesity, are rising among males in reproductive age. Along with this increase, subfertility and infertility incidence is also rising. To counteract this tendency, prevention with a healthy lifestyle should be the priority. However, in specific cases, pharmacological treatments allow to control glycemia levels and provoke weight loss in a safe manner. Often, two target pharmacological agents used in those treatments are the GLP-1 receptor agonist exenatide and the SGLT2 inhibitor dapagliflozin. Both can be used in combined treatment and have shown to efficiently control metabolic diseases comorbidities with relative safety. Although, as they modulate glucose metabolism, they can impact Sertoli cells glucose metabolism and influence spermatogenesis. To evaluate that, we incubated TM4 Sertoli cells with subpharmacological, pharmacological, and supra-pharmacological concentration of both exenatide and dapagliflozin. Moreover, the combination of both pharmacological concentrations was also tested and its effect on Sertoli cell viability and glucose metabolism were evaluated. In sum, with this work, we were able to notice that pharmacological concentrations of exenatide do not modulate Sertoli cell division or metabolic viability. Likewise, Sertoli cell glucose metabolism was maintained after exposure to 25 pM of exenatide. Dapagliflozin pharmacological concentration caused a mildly detrimental effect on Sertoli cell proliferation and metabolic viability. However, the same concentration was capable of increasing lactate production on Sertoli cells due to enhanced glycolytic efficiency, which can be a good indicator for spermatogenesis. Moreover, dapagliflozin at 5000 nM showed to have anti-obesogenic properties at a cellular level. Interestingly, the combined therapy, while it still had an impact on cell division and metabolic viability, it restored the glycolytic flux, indicating that both drugs have a synergic effect on maintaining metabolic homeostasis on treated Sertoli cells. Further studies will be needed to confirm the hypothesis presented in this work and to further unveil the mechanism of action of each drug on Sertoli cells metabolism and male fertility. As future perspectives would be interesting to evaluate the effect of the therapies at shorter time points with human Sertoli cells or animal models and evaluate if the alterations observed in the metabolism of these cells can jeopardize spermatogenesis or steroidogenesis. These studies would increase our knowledge about the side effects of the pharmacological agents used in this study. 82 Chapter VII REFERENCES 83 7. REFERENCES 1. Cho NH, Shaw JE, Karuranga S, Huang Y, da Rocha Fernandes JD, Ohlrogge AW, et al. 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