Evaluating the modulation of the intracellular production of H2S in the functionality and metabolic activity of primary cultures of pancreatic islets

UNIVERSITY OF SEVILLE Master Thesis Evaluating the modulation of the intracellular production of H2S in the functionality and metabolic activity of primary cultures of pancreatic islets Master in Biomedical Research Andalusian Center of Molecular Biology and Regenerative Medicine 21 Feburary 2022 – 5 September 2022 Inmaculada Pino Pérez In Seville, 5 of September of 2022 Tutor: Alejandro Martín-Montalvo Sánchez Co-tutor: Iván Valle Rosado Date of the defense: 22 of September of 2022 Abstract Diabetes Mellitus is a metabolic disease characterized by hyperglycaemia that currently affects one in ten people worldwide. Uncontrolled Diabetes is associated with long-term damage and dysfunction and failure of different organs. Although there are therapies for the treatment of diabetes, they are not strongly effective. In order to develop an appropriate therapy for diabetes, we are studying the modulation of the intracellular production of the gasotransmitter hydrogen sulfide (H2S) in pancreatic islets using a H2S donor (compound α). Mice treated with two doses (5 and 10 μM) of compound α showed increased metabolic activity and oxygen consumption rate in islets exposed to high concentrations of glucose (22 mM) when compared with the untreated control, suggesting that mitochondria are one of the main targets of H2S effects. Importantly, compound α showed increased insulin secretion under high glucose (22 mM) conditions. Lentiviral-mediated overexpression of Cystathionine-beta-synthase and Cystathionine-gamma-lyase, main enzymes that synthesise endogenous H2S, in primary pancreatic islets also showed increased metabolic activity. Taken together, these data indicate that compound α, a H2S generator, increases metabolic activity and produces greater insulin secretion in murine primary pancreatic islets. INDEX I. Antecedents ................................................................................................... 1 II. Objectives ...................................................................................................... 7 III. Material and Methods .....................................................................................8 IV. Results and Discussion .................................................................................. 14 V. Conclusions ................................................................................................... 24 VI. Bibliography .................................................................................................. 25 1 I. Antecedents The incidence of Diabetes Mellitus (DM) is fasting increasing worldwide1. This endocrine and metabolic disease is defined by the American diabetes Association as a group of metabolic diseases characterised by hyperglycaemia resulting from insulin secretion deficiency or by its compromised biological function2,3. Over the last few years, the number of people with this multifactorial disease has increased rapidly. According to the International Diabetes Federation (IDF), in 2021 approximately 537 million adults (20-79 years) were living with DM and the total number of people living with diabetes is projected to rise to 643 million by 2030 and 783 million by 2045. DM is traditionally divided into type 1 diabetes (T1D) and type 2 diabetes (T2D), although other types of DM also exist (American diabetes association, 2021). In most cases, type 1 diabetic patients have a complete lack of insulin due to a failure of β-cell-specific self-tolerance by T-lymphocytes. The lack of self-tolerance sets in motion an autoimmune attack and the elimination of insulin-producing cells in the endocrine pancreas4. This type of DM commonly manifests during childhood5. On the other hand, T2D is a non-insulin- dependent DM6,7. Insulin-producing cells (β-cell) are one of the main types of endocrine cells of the endocrine pancreas8. The pancreatic endocrine cells are settled into clusters, called islets of Langerhans and there are five principal types: the β cells (insulin-producing cells), the α cells (glucagonproducing cells), the δ cells (somatostatin-producing cells), the ε cells (ghrelin-producing cells) and PP cells (pancreatic polypeptide-producing cells)9. These pancreatic cells clusters are highly vascularized, and this allows that the insulin is secreted into the circulation to target the insulin-sensitive organs, such as the liver or the skeletal muscle10. β and α cells play an important role in glucose sensing and the maintenance of glucose homeostasis. Specifically, pancreatic β-cells sense changes in plasma glucose levels and are responsible for the synthesis and secretion of insulin11. In type 2 diabetics, insulin-producing cells do not fabricate enough insulin, or they cannot secrete it effectively. These effects result in high blood glucose levels and can be due to influencing factors such as heritable genetic correlation (40% of first-degree relatives of patients with T2D develop DM) or environmental factors (sedentary lifestyles, unhealthy diet, obesity or ageing)12. Nowadays, this complex disease is diagnosed by measuring glucose in blood under fasting conditions or after challenging the body with a glucose load (glucose tolerance tests)13. Moreover, in the diagnosis of DM a determination of glycated haemoglobin 2 is normally used, which allows a determination of long-term hyperglycaemia. Nevertheless, despite being relatively easily diagnosed, in Europe over 1 in 3 adults (36%) living with DM are undiagnosed (IDF, 2021). DM that is not well controlled can have an important impact on mortality and morbidity, including being a health risk for obesity, stroke, renal dysfunction, heart and vascular diseases, blindness, and neuropathy14,15. In fact, DM was responsible for 6,7 million deaths in 2021 (IDF, 2021). Current therapies for the different types of DM are not, in general, highly effective. Therefore, it is necessary to develop novel strategies for managing DM. The lifestyle strategies frequently include ordinary physical activity, avoid smoking, avoid obesity and the use of healthy diet with low glycaemic index and high fibre content14. Symptoms of DM can be treated by pharmacological therapy with exogenous insulin and other molecules as glucagon-like peptide-1 (GLP-1) receptor agonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, thiazolidinediones and sulfonylurea derivates, but these therapies are, in the majority of the cases, not very successful16. For this reason, it is necessary to bet on new strategies for this complex disease. On this point, there is evidence that hydrogen sulfide (H2S) is produced endogenously in different organs by enzymatic and non-enzymatic production. The non-enzymatic production of H2S is responsible for approximately 10-50% of total H2S production. The enzymatic production of H2S is organ-specific. H2S endogenous production plays a regulatory role in physiological processes, such as adipogenesis and adipose tissue metabolism which are promoted by endogenous H2S17. Previous reports indicate that H2S could be endogenously produced in islet β-cells, liver and adipose tissue, suggesting that this molecule could play a significant role in the survival or functionality of metabolic tissues. Furthermore, it has been found that H2S might be involved in the regulation of insulin sensitivity and insulin secretion. In addition, further research has indicated that H2S levels are decreased in diabetic subjects when compared with healthy individuals, suggesting a link between H2S and diabetogenesis18. In mammal tissues, enzymatic production occurs primarily through the actions of four enzymes19: Cystathionine-beta-synthase (CBS), Cystathionine-gamma-lyase (CSE or CTH), 3- mercaptopyruvate sulfurtransferase (3-MST) and cysteine aminotransferase (CAT)20 (Figure 1). CBS and CTH catalyse the production from L-cysteine and/or homocysteine, while 3-MST can catalyse 3-mercaptopyruvate to produce H2S19. 3 Figure 1. H2S generation via enzymatic pathway. Modified from Zhu et al., 202020. The distribution of this enzymes is tissue specific18. CTH has its main role in the production of H2S in the peripheric system (pancreas, liver, adipose tissue, cardiovascular system, and respiratory system), whereas CBS is abundantly expressed in the central nervous system21. Furthermore, in pancreatic β-cells it has been discovered a generation of H2S through CBS (exocrine and endocrine pancreatic cells) and/or CTH (exocrine pancreatic cells)18. 3-MST has an important role in the production of H2S in those tissues with high cysteine concentration, such as the kidney, but its principal expression is in the gastrointestinal tract22. In addition to the enzymatic production, H2S is produced via a non-enzymatic endogenous generation that is less well understood. In this H2S production, the reducing power (NADPH) that is generate by different metabolic pathways, including glycolysis or phosphogluconate, is used to reduce oxidized glutathione (GSSG) to glutathione (GSH) which allows the generation of H2S by the reduction of polysulfides (Figure 2)23. Figure 2. H2S generation via non-enzymatic pathway. Modified from Kolluru et al., 201323. 4 Despite of been known for its toxicity over the years, H2S, a “toxic gas with strong odor of rotten eggs” has gone from a toxic gas to a signalling molecule24, which was first described as an endogenous neuromodulator by Abe and Kimura in 199625. H2S is a gasotransmitter that has the ability to diffuse freely through cells membranes26 and in physiological concentrations this molecule mediated important biological actions as vasorelaxation, changes in brain neurotransmission and the effect on K+ ATP channel27. Evidence suggests that physiological effects of H2S are linked to the post-translational modification S-Sulfhydration of target proteins in cysteines (persulfidation). In this reaction, H2S transfer a sulfhydryl group (-SH) to a cysteine residue of a protein and this contribute to the modification of functions, stability, and localization of cells28. This post-translational modification favoured by H2S could be an important signalling mechanism of metabolism. Alternatively, H2S effects might originate from the action of Sulfide Quinone Oxidoreductase (SQOR). SQOR is a mitochondrial inner membrane-anchored flavoenzyme which catalyses the first step in the mitochondrial H2S oxidation pathway29. This oxidation by SQOR is coupled to the reduction of the coenzyme Q30. In low concentrations (<20 μM) H2S stimulates mitochondrial respiration donating an electron to the electron transport chain (ETC) resulting from its oxidation by SQOR. Specifically, the electrons are captured by ubiquinone and are incorporated into the ETC at the level of Cytochrome C reductase (Complex III)31. This activity can stimulate oxidative phosphorylation and ATP production in the cell22. In the field of DM, different implications of H2S have been discovered. Several studies have demonstrated that DM is associated with lower circulating concentrations of H2S32,33. Several reports have showed in patients and experimental animals (rats and mice) that diabetic individuals have lower circulating H2S concentrations than age-matched non-diabetic individuals34. Focusing on pancreatic β-cells, Zhang et al., 2014, have found that the effect of increased H2S production could limit insulin secretion in Zucker diabetic fatty rats. The increase in H2S production is due to the enzymatic action of CTH (main producer of H2S in the peripheric system, which includes the endocrine pancreas) because the addition of an enzymatic inhibitor of CTH recovers the normal insulin secretion18. Pancreatic β-cells work as sensors of glucose circulation levels. When circulating glucose concentrations are high, β-cells secrete insulin, and this mechanism is mediated by the ATP- dependent inhibition of KATP channels (Figure 3). In β-cells glucose enters via GLUT2 11 10% FBS, Pen-strep, 2 mM glutamine, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol and 10 mM HEPES (RPMI complete media). The following day, healthy islets were separated and treated with three different conditions: DMSO 0,1% as a control, compound α 5 µM and compound α 10µM (in DMSO). Oxygen consumption rate and extracellular acidification of the media Mitochondrial function was assessed in islets using an Agilent SeaHorse XF24 according to the instructions of the manufacturer (Agilent Technologies). XF DMEM assay media was supplement with 3 mM glucose and 1 % FBS (MA Media) to run the whole islets and the Seahorse XF Sensor Cartridge was hydrated with Seahorse XF Calibration Buffer at 37 °C overnight. The day of the assay, 30 islets per well were placed in Agilent SeaHorse XF24 Islet Capture Microplates along with 500 µL of MA Media. Islets were captured with a capture screen in each well. Then, the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured. OCR and ECAR were determined in basal conditions and through consecutive injections of glucose (20 mM), oligomycin (5 µM), carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP; 1 µM) and rotenone (5 µM) with antimycin A (5 µM). The durations of the injections were basal conditions 3 cycles (1 cycle: mix, wait and measure), glucose 5 cycles, oligomycin 5 cycles, FCCP 5 cycles and rotenone with antimycin A 7 cycles. Thiazolyl blue tetrazolium bromide (MTT) assay The islets cell metabolic activity was determined using the Cell Proliferation kit according to the recommendations of the manufacturer (Roche, Spain). Firstly, 30 islets/well of each condition were cultured into a 24-well plate. MTT assay reagent (60 µL thiazolyl blue tetrazolium bromide) was added to the medium, and incubated at 37 °C, 5% CO2 for 2 hours before adding 600 µL MTT solubilization solution. The next day, the Optical density was determined at 575 nm with a reference wavelength of 690 nm using a Varioskan Flash spectrophotometer (Thermo Scientific, Spain). Insulin Secretion Groups of 10 islets of each condition (DMSO 0,1%, Compound α 5 µM and Compound α 10µM) were washed in Krebs-Ringer Bicarbonate HEPES buffer (KRBH-BSA) (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2 PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 12 mM HEPES, 0.1% BSA) and incubated for 1 h at 37ºC in KRBH-BSA solution containing 3mM glucose (KRBH-BSA-Glucose) and supplemented with their treatment condition. Then, ten size-matched islets per condition were incubated for 30 min at 37ºC with either 3 mmol/l glucose (low glucose), 22 mmol/l glucose (high glucose) or 20 mmol/l KCl. Secreted and total insulin were quantified using an Ultra Sensitive Mouse Insulin ELISA Kit according to the manufacturer´s instructions (Crystal Chem). RNA Extraction and RT-PCR Quiagen RNeasy Micro kits were used for the extraction of total RNA from the pancreatic islets according to the instructions of the manufacturer (Quiagen). After extraction, the quantification of the RNA was done using a DS-11 Spectrophotometer/ Fluorometer (DeNovix). To perform the real-time PCR, single-stranded cDNA was synthetized with the iScript cDNA Synthesis Kit according to the instructions of the manufacturer (BIO-RAD). The gene-specific primers (RPS29, GLUT2, INS1, INS2, GLUCOKINASE, CBS, CTH, SQOR, GFP) were obtained from Biomol and the RT-PCR was performed on a QuantStudio 7 Pro using the RT-PCR SYBR Green Kit according to the instructions of the manufacturer (BIO-RAD). Expression levels of the housekeeping RSP29 gene were used for normalization. Lentivirus Amplification and Purification Lentivirus production was done using 5 x 106 Hek293T cells that were seed in a 100mm petri dish for virus package for 24 hours. The following day, cells were transfected with 3 plasmids: 1) 2,5 µg of interest plasmid (CBS, CTH, CONTROL), 2) 1 µg of envelope plasmid (pMD2.G), 3) 1,5 µg of capsid plasmid (psPAX2). The transfection was performed following the protocol of LipoD293. LipoD293 is diluted with Dulbecco's Modified Eagle Medium D5796 (DMEM D5796) supplemented with FBS 10%, Pen/Strep 1X y Glutamine 10% and allows transfection by making pores in cells. After transfection, for 72 hours lentivirus were harvested and then purified using a 0,45 µm Whatman Puradisc filter and concentrated by ultracentrifugation in a Optima XPN-100 Ultracentrifuge (Beckman Coulter, United States) at 22000 rpm for 2 hours at 4ºC. Virus particles were resuspended in DMEM D5796, distributed in aliquots according to the conditions and stored at -80ºC. Hek293T cells were transduced with the viral particles obtained and the viral titer was estimated by flow cytometry analysis (FAC-SCalibur, BD Biosciences, Spain). This analysis allows to determine the Plaque Forming Units/mL (PFU/mL) based on GFP (Green Fluorescent Protein) emission. 13 Islets Infection After isolation by collagenase digestion, islets were picked and cultured in a petri dish with RPMI 1640 for 3 hours at 37ºC. After the incubation, islets were separated in the same number in three 15mL falcon tube, for separate each lentivirus condition: lentivirus control, lentivirus CBS and lentivirus CTH. Subsequently islets were incubated in 0,5X Trypsin- Ethylenediaminetetraacetic acid (diluted in Hanks Balanced Salt Solution (HBSS)) for 3 minutes in a cell culture incubator (37ºC, 5% CO2). After trypsinization, 1000 μL of RPMI complete media were added to each falcon. Islets were centrifuged at 50 x g for 2 minutes and placed in a Round-bottom polystyrene tube. Islets were infected with 20 Plaque Forming Units per cell (PFU/cell) in free serum RPMI, assuming that a single islet has 1000 cells. Finally, to achieve the optimal lentivirus transduction, islets were incubated at 37ºC overnight. The following day, media were changed (Complete RPMI media) and islets were incubated overnight until the experiment. Flow Cytometry The efficiency of the transduction was evaluated following the protocol previously described (Jimenez-Moreno et al., 2015) with a Flow Cytometry Analysis. Around 20 islets per condition (Lentivirus Control, Lentivirus CBS, and Lentivirus CTH) were picked and placed in a 5mL Round-bottom polystyrene tube in a final volume of 500 μL of Complete Islets Media. Then, islets were disaggregated using Trypsin- EDTA 1X for 5 minutes at 37ºC and centrifuged at 200 x g for 5 minutes. After trypsinization, the endocrine cells were resuspended in PBS and comparing with a non-infected cells the GFP positive cells were estimated using FAC-SCalibur, BD Biosciences, Spain. Statical Analysis Data were analysed by one-way or two-way ANOVA depending on the experiment. All results are reported as mean ± SEM and in all cases, P-values ≤ 0.05 were considered significant. Analyses were performed using GraphPad Prism 7.04. 14 IV. Results and Discussion Compound α treatment The H2S donor compound α increases metabolic activity and oxygen consumption in pancreatic islets. Primary islets treated with 5 μM and 10 μM of compound α have shown increased MTT activity when compared with the untreated control (Figure 4). MTT test measure indirectly the cellular metabolic activity (viability) by the reduction of the tetrazolium compound (yellow) to water insoluble formazan precipitates (blue-magenta) using NADH/NADPH41. Formazan crystals can be dissolved using an organic solvent and analyzed colorimetrically42. Figure 4. Determination of cell viability in pancreatic islets treated with compound α. Increased in MTT activity in pancreatic islets treated with 5 and 10 μM of compound α. n=4. Data shown as mean ± SEM. * p≤ 0.05 vs. Control (one way ANOVA test). The increase in cell metabolic activity by compound α might be an effect of its activity at the mitochondria. Mitochondria are considered one of the main targets of H2S effects, having a directly impact on the respiratory chain22,43. Therefore, we measured parameters of mitochondrial function. We examined mitochondrial performance using Seahorse XF technology in pancreatic islets in response to compound α (Figure 5). We found that oxygen consumption rate (OCR) was increased in islets exposed to high concentrations of glucose (22 mM) when treated with compound α at 10 μM. This technology is based on the inhibition or activation of the different complexes of the respiratory chain, which makes possible the evaluation of parameters such as maximum cellular respiration, basal respiration, or ATP-linked OCR. 15 Figure 5. Determination of cellular oxygen consumption. Oxygen consumption of pancreatic islets treated with 5 and 10 μM of compound α in basal conditions, exposed to high concentrations of glucose (22 mM), and to modulators of respiration (Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), Rotenone, and Antimycin). n=7 (Untreated), 6 (Compound α 5 μM), 7 (Compound α 10 μM). Data shown as mean ± SEM. * p≤ 0.05 vs. control (two-way ANOVA test). As previously mentioned, mitochondria are essential for the production of ATP in the cells44. Módis et al., 201645 showed the existence of a persulfidation mechanism at the level of the respiratory chain, specifically in Complex V (ATP synthase). In this report the authors determined that high levels of H2S produced an increased in the persulfidation of 2 cysteine residues of ATP5A1. As a consequence, persulfidated ATP5A1 exhibited increased ATP synthase activity when compared with ATP synthase variants lacking persulfidated cysteins (mutations in the target cysteines of persulfidation). The effects of the H2S donor compound α could be mediated through this mechanism, leading to a marked increase in mitochondrial energy production and cellular respiration on conditions of high glucose concentration in the cell culture media22. In addition, other mechanisms by which the H2S donor compound α may be driving an increase in oxygen consumption, is via increased electron flow in the ETC resulting from the oxidation of H2S by SQOR. These electrons are captured by ubiquinone and transferred to Complex III (Cytochrome C reductase) of the respiratory chain22. This process could also contribute to increase the oxygen consumption and, consequently, ATP production. The Seahorse XF technology also measures the extracellular acidification rate (ECAR). The increase of this biochemical process is indicative of an acidification of the medium produced by the increase of lactic acid and protons resulting from glycolysis. Pancreatic islets showed no 16 effects in pH on exposure of 10 μM compound α, while there was a significant increase in islets exposed to high concentration of glucose when treated with compound α at 5 µM (Figure 6). Figure 6. Extracellular acidification of the medium. Extracellular acidification of the medium of pancreatic islets treated with 5 and 10 μM compound α in basal conditions and when they are exposed to high concentrations of glucose (22 mM) and modulators of respiration (Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), Rotenone, and Antimycin). n=7 (Untreated), 6 (Compound α 5 μM), 7 (Compound α 10 μM). Data shown as mean ± SEM. * p≤ 0.05 vs. control (two-way ANOVA test). The H2S donor compound α increases Insulin-secretion in pancreatic islets. Figure 7. Glucose-promoted insulin secretion. Increased Insulin secretion of pancreatic islets treated with 10 μM compound α when they are exposed to high glucose (22 mmol/l)). n=7. Data shown as mean ± SEM. * p≤ 0.05 vs. control (two-way ANOVA test). Insulin-secretion tests have determined that under high glucose (22 mM), compound α (10 µM) increased insulin secretion in pancreatic islets (Figure 7). Therefore, the functionality of the pancreatic islets is altered by compound α. 17 Relative gene expression in pancreatic islets treated with 5 and 10 µM compound α. Using the RT-PCR SYBR Green Kit we examined the relative gene expression of the two main enzymes that synthesize H2S (CBS and CTH), as well as elements of glucose metabolism such as the transmembrane protein transporter that enables the passive movement of glucose across cell membranes (GLUT2), Insulin 1 (INS1), Insulin 2 (INS2) and the phosphorylating enzyme that controls the entry of glucose into the glycolytic pathway (GCK) (Figure 8). These results are preliminary data because there are not enough replicates available to apply statistics. Additional RT-PCR is required to obtain a larger number of replicates, but preliminarily, it seems that pancreatic islets treated with 5 and 10 μM compound α increase the relative expression of CTH, INS1 and INS2. The increased relative expression of INS1 and INS2 genes may be to the increased insulin secretion of pancreatic islets. Figure 8. Relative Gene Expression by RT-PCR. Relative gene expression in pancreatic islets treated with 5 and 10 μM compound α with repeat-positive reverse transcription polymerase chain reaction (RT-PCR). Expression levels of the housekeeping RSP29 gene were used for normalization. (A) Cystathionine beta synthase expression (CBS); (B) Cystathionine y-lyase expression (CTH); (C) Glucokinase expression (GCK); (D) Glycotransporter 2 expression (GLUT 2); (E) Insulin 1 expression (INS1); (F) Insulin 2 expression (INS2). Data shown as mean ± SEM. No statistical analysis for having a small number of replicates to have statistical power. 18 Lentivirus infection The production of lentiviruses was performed using the following plasmids: - Interest plasmid: CBS, CTH or CONTROL (Figure 9). - Envelope plasmid: pMD2.G (Figure 10). - Capsid plasmid: psPAX2 (Figure 11). Figure 9. CBS and CTH Lenviral Vector Human (pLenti-GIII-CMV-GFP-2A-Puro). The CBS, CTH and CONTROL (gene insert) expression is driven by a CMV promoter, with a GFP reporter. The gene inserts are flanked by and can be excised using NheI and BamHI as long as inserts do not contain any internal NheI or BamHI sites. On the other hand, the envelope plasmid (Figure 10) is a plasmid expressing the VSV-G envelope, whose cloning method is the restriction enzyme. Figure 10. Envelope Plasmid (pMD2.G). Finally, the plasmid that makes up the viral capsid (Figure 11) is a second-generation lentiviral packaging plasmid, which also features the restriction enzyme as a cloning method. 19 Figure 11. Capsid plasmid (psPAX2). Viral titer in Hek293T by flow cytometry analysis. Lentiviral vectors were produced according to the experimental method described in Material and Methods section. The viral titer was validated using Flow Cytometry Analysis in Hek293T cells. Using 10 μL of each lentivirus condition, 59,35% of Hek293T were GFP-positive (Figure 12B), 22,45% for lentivirus CBS (Figure 12C) and 35,22% for lentivirus CTH (Figure 12D). These indicated a concentration of viruses of 2,490 x E4 Titer Units (TU)/μL for lentivirus control, 6,512 x E3 TU/μL for lentivirus CBS and 1,008 x E4 TU/ μL for lentivirus CTH. A B 20 C D Figure 12. Viral Titer in Flow Cytometry Analysis. The red circle (%gated) shows the percentage of HEK293T cells that were GFP-positive. Each image was obtained with Flow Cytometry Analysis (FAC-SCalibur, BD Biosciences, Spain) by simply adding 10 μL of each lentivirus. (A) Media; (B) Lentivirus CONTROL; (C) lentivirus CBS; (D) lentivirus CTH. Evaluation of the efficiency of infection in pancreatic islets by flow cytometry analysis. Pancreatic islets were infected using the method described in Materials and Method section using a final concentration of 20 PFU/cell. The efficiency of the transduction in pancreatic islets was evaluated using Flow Cytometry Analysis. 68,92% of islets were GFP-positive when compared with non-infected cells for lentivirus control (Figure 13-B), 42,06% for lentivirus CBS (Figure 13-C) and 47,80% for lentivirus CTH (Figure 13-D). Taken together, these data indicate that the infection protocol was highly efficient. 27 26. Wang R. Physiological implications of hydrogen sulfide: A whiff exploration that blossomed. Physiol Rev. 2012; 92(2):791–896. 27. Zaorska E, Tomasova L, Koszelewski D, Ostaszewski R, Ufnal M. Hydrogen Sulfide in Pharmacotherapy, Beyond the Hydrogen Sulfide-Donors. Biomolecules. 2020; 10(2):323. 28. Gheibi S, et al. Regulation of carbohydrate metabolism by nitric oxide and hydrogen sulfide: Implications in diabetes. Biochem Pharmacol. 2020; 176:113819. 29. Landry AP, Ballou DP, Banerjee R. Hydrogen Sulfide Oxidation by Sulfide Quinone Oxidoreductase. Chembiochem. 2021; 22(6):949. 30. 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Pharmacol Res. 2016; 113:116-124. 29 UNIVERSITY OF SEVILLE Master Thesis Evaluating the modulation of the intracellular production of H2S in the functionality and metabolic activity of primary cultures of pancreatic islets Master in Biomedical Research Andalusian Center of Molecular Biology and Regenerative Medicine 21 Feburary 2022 – 5 September 2022 Inmaculada Pino Pérez In Seville, 5 of September of 2022 Tutor: Alejandro Martín-Montalvo Sánchez Co-tutor: Iván Valle Rosado Date of the defense: 22 of September of 2022