The influence of anthocyanin treatment on the mesenteric adipose tissue expression of enampt in animal models of obesity

THE INFLUENCE OF ANTHOCYANIN TREATMENT ON THE MESENTERIC ADIPOSE TISSUE EXPRESSION OF eNAMPT IN ANIMAL MODELS OF OBESITY MARIA MARGARIDA NOBRE PROJECTO A dissertation submitted in partial fulfillment of the requirements for the Degree of Masters in Human Nutrition and Metabolism Dissertação para obtenção do grau de Mestre em Nutrição Humana e Metabolismo at Faculdade de Ciências Médicas | NOVA Medical School of Universidade NOVA de Lisboa September, 2022 ii THE INFLUENCE OF ANTHOCYANIN TREATMENT ON THE MESENTERIC ADIPOSE TISSUE EXPRESSION OF eNAMPT IN ANIMAL MODELS OF OBESITY Maria Margarida Nobre Projecto Supervisors: Marta P Silvestre, Invited Assistant Professor at Faculdade de Ciências Médicas | NOVA Medical School of Universidade NOVA de Lisboa Ana Faria, Assistant Professor at Faculdade de Ciências Médicas | NOVA Medical School of Universidade NOVA de Lisboa A dissertation submitted in partial fulfillment of the requirements for the Degree of Masters in Human Nutrition and Metabolism September, 2022 iii This study was supported by National Funds through FCT – Fundação para a Ciência e a Tecnologia (FCT) within the project PTDC/BAA-AGR/7419/2020. iv ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisors, Professora Doutora Marta Silvestre, and Professora Doutora Ana Faria, for their professionalism, guidance, and availability. Sharing their knowledge and vast experience was a great asset to me and the elaboration of this thesis. I also want to thank and acknowledge the entire faculty members of this Master for the quality of teaching and the encouragement of scientific investigation, right from the beginning. In particular, to Professora Doutora Diana Teixeira and Professor Doutor João Araújo for the essential support provided in this work. Just as important is my gratitude to Pedro, Shámila, and especially Maria João for the fundamental support, collaboration, and company during the laboratory experience. Finally, a big thank you to my family for their support and words of encouragement throughout my academic journey, which allowed me to get here today. v ABBREVIATIONS AT Adipose Tissue ANKO Adipose Tissue-Specific Nampt Knockout AUC Area Under the Curve BAT Brown Adipose Tissue BE Standard Diet + Blackberry Extract BMI Body Mass Index C Standard Diet Cy-3-G Cyanidin-3-O-β-Glucoside eNAMPT Extracellular Nicotinamide Phosphoribosyltransferase FRD Fructose Rich Diet GSIS Glucose-Stimulated Insulin Secretion GTT Glucose Tolerance Test HFD High-Fat Diet HFDBE High-Fat Diet + Blackberry Extract HRP Horse-Radish Peroxidase iNAMPT Intracellular Nicotinamide Phosphoribosyltransferase IL-6 Interleukin 6 IR Insulin Resistance IS Insulin Sensitivity ITT Insulin Tolerance Test mAT Mesenteric Adipose Tissue NAD Nicotinamide Adenine Dinucleotide NAMPT Nicotinamide Phosphoribosyltransferase NAMPT +/− NAMPT-deficient Heterozygous NF-KB Nuclear Factor Kappa B NMN Nicotinamide Mononucleotide NMNAT Nicotinic Acid Mononucleotide Adenylyltransferase NMS|FCM NOVA Medical School | Faculdade de Ciências Médicas PBS Phosphate-buffered saline PVDF Polyvinylidene difluoride vi RIPA Radio-Immunoprecipitation scAT Subcutaneous Adipose Tissue SD Standard Deviation SEM Standard Error of The Mean SDS-PAGE Sodium dodecyl-sulfate polyacrylamide gel electrophoresis STAT3 Signal Transducer and Activator of Transcription 3 T2D Type 2 Diabetes TPBS PBS containing 0,1% (vol/vol) Tween-20 TLR4 Toll-like Receptor 4 WAT White Adipose Tissue WB Western Blot vii ABSTRACT Introduction: Nicotinamide Phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for NAD biosynthesis. Its extracellular form (eNAMPT), mainly secreted by visceral fat, has been shown to have bimodal, concentrationand structure-functional-dependent effects in important metabolic pathways and has been connected to a wide variety of diseases. Data suggests that as serum eNAMPT concentration rises to pathophysiological levels, as in obesity and type 2 diabetes (T2D), eNAMPT adopts a monomeric form capable of proinflammatory NAD-independent effects. Strategies to block the actions of the eNAMPT monomer could represent promising therapeutic approaches for obesity-related metabolic disorders. Consumption of anthocyanin rich foods appears to prevent or treat obesity-related consequences, such as T2D, inflammation and oxidative stress, but the mechanism behind this is unknown. Moreover, anthocyanins have been shown to inhibit the secretion of eNAMPT in animal models. With this study, we aim to understand if treating a rat model of obesity with anthocyanins could abrogate the impact of a high fat diet in the expression of monomeric eNAMPT. Methods: Mesenteric adipose tissue (mAT) was obtained from four groups of male Wistar rats, treated with different diets: (C) standard diet; (BE) standard diet + blackberry extract; (HFD) highfat diet; (HFDBE) high-fat diet + blackberry extract. eNAMPT monomer’s protein expression was measured by Western Blotting, after protein extraction and quantification from mAT, to access the differences between the animals fed a standard diet and those of increased metabolic risk – HFD, with and without treatment with anthocyanins. Results: The mAT from HFD rats displayed a higher expression of eNAMPT monomer, compared to C rats (138,6% ± 11,4% HFBE vs 100% C, p=0,01). The anthocyanin’s treatment influence on mAT eNAMPT monomer expression was also assessed. mAT eNAMPT monomer expression was significantly decreased in the HFDBE group compared to HFD (-54,1 ± 15,3 % [-89.4, -18.8], p<0,01). Conclusions: Anthocyanin consumption might be an interesting dietary approach to abrogate the impact of a high fat diet on the rise of monomeric eNAMPT in mesenteric adipose tissue. Ultimately, our results suggest that long-term anthocyanin treatment/supplementation might be effective for sustaining lower levels of monomeric eNAMPT in the context of diet-induced obesity, potentially preventing or delaying the consequent metabolic impairments such as the alarming epidemic of T2D. Keywords: Adipose tissue, Anthocyanins, eNAMPT, Nicotinamide Phosphoribosyltransferase, Obesity, Type 2 Diabetes viii RESUMO Introdução: A Nicotinamida Fosforibosiltranferase (NAMPT) é a enzima limitante no processo de biossíntese de NAD. A sua forma extracelular (eNAMPT), secretada principalmente pelo tecido adiposo visceral, demonstrou ter efeitos bimodais, dependentes da concentração e da estrutura funcional, em importantes vias metabólicas e tem sido associada a uma ampla variedade de doenças. Dados sugerem que à medida que a concentração sérica de eNAMPT aumenta para níveis fisiopatológicos, como na obesidade e diabetes tipo 2 (T2D), a eNAMPT adota uma forma monomérica capaz de efeitos pró-inflamatórios independentes do NAD. Possíveis estratégias que bloqueiem as ações do eNAMPT monomérico podem representar abordagens terapêuticas promissoras para os distúrbios metabólicos relacionados com a obesidade. O consumo de alimentos ricos em antocianinas parece prevenir ou tratar consequências relacionadas com a obesidade, como T2D, inflamação e stress oxidativo, mas o mecanismo subjacente é ainda desconhecido. Para além disso, as antocianinas demonstraram inibir a secreção de eNAMPT em modelos animais. Com este estudo, pretendemos compreender se o tratamento de um modelo animal de obesidade com antocianinas poderia anular o impacto de uma dieta rica em gordura na expressão de eNAMPT monomérico. Métodos: O tecido adiposo mesentérico (mAT) foi obtido a partir de quatro grupos de ratos Wistar machos, tratados com diferentes dietas: (C) dieta standard; (BE) dieta standard + extrato de amora; (HFD) dieta rica em gordura; (HFDBE) dieta rica em gordura + extrato de amora. A expressão proteica do eNAMPT monomérico foi medida por Western Blotting, após a extração e quantificação das proteínas do mAT, para avaliar as diferenças entre os animais alimentados com dieta standard e os que apresentam risco metabólico aumentado – HFD, com e sem tratamento com antocianinas. Resultados: No mAT de ratos HFD, a expressão do monómero eNAMPT foi 38,6% mais elevada quando comparado com C (138,6% ± 11,4% HFBE vs 100% C, p=0,01). A influência do tratamento com antocianinas na expressão do eNAMPT monomérico no mAT também foi avaliada em ratos alimentados com HFD e C. O eNAMPT monomérico diminuiu significativamente em ratos alimentados com HFDBE em comparação com HFD (-54,1 ± 15,3 % [-89.4, -18.8], p<0,01). Conclusão: O consumo de antocianinas parece ser uma abordagem dietética interessante para anular o impacto de uma dieta rica em gordura no aumento de eNAMPT monomérico no tecido adiposo mesentérico. Em última análise, os nossos resultados sugerem que o tratamento/suplementação com antocianinas, a longo prazo, pode ser eficaz para sustentar níveis mais baixos de eNAMPT monomérico no contexto de uma obesidade induzida pela dieta, contribuindo para combater outras alterações metabólicas, como a alarmante epidemia de T2D. Palavras-chave: Antocianinas, Diabetes tipo 2, eNAMPT, Nicotinamida Fosforibosiltranferase, Obesidade, Tecido Adiposo ix 3 When surveying the tissue distribution of iNAMPT in mice, Revollo et al. demonstrated that iNAMPT protein expression levels were the highest in BAT, liver, and kidney. Heart, WAT, lung, spleen, testis, and skeletal muscle showed intermediate to very low levels of iNAMPT, while in the brain and pancreas it was undetectable. These findings suggest that iNAMPT-mediated NAD biosynthesis might significantly differ amidst different tissues (15). The extracellular form of NAMPT (eNAMPT), also known as pre-B cell colony enhancing factor or visfatin, exhibits robust, even higher NAD biosynthetic activity than iNAMPT (15,17). The exact mechanism of eNAMPT’s secretion is still under investigation, but it seems to be secreted mainly by adipocytes, through a non-classical pathway (15,18), being lately one of the adipokines with greater research interest. The physiological role of eNAMPT, other than the enzymatic one, has been a matter of much debate. It has been shown that eNAMPT leads to different biological reactions by diverse types of cells, possibly through the activation of intracellular signalling pathways (19). Results from different studies have been controversial and the mechanisms by which eNAMPT exerts proinflammatory properties or is protective against cellular stress or toxicity is still subject of study. Indeed, the relationship between NAMPT and cellular homeostasis is highly complex (19). For instance, it has been shown that eNAMPT binds to Toll-like receptor 4 (TLR4) and activates the nuclear factor kappa B (NF-KB) signalling pathway (20,21). This causes the expression of various markers of inflammation and oxidative stress, as well as cell survival and death pathways regulation (22,23). In fact, there are several reports of eNAMPT having proinflammatory and immunomodulating properties. After murine eNAMPT administration in mice, circulating levels of interleukin 6 (IL-6), a cytokine that is strongly implicated in inflammatory and immunological processes, arose (3). Some authors observed that recombinant eNAMPT dose-dependently induced mRNA expression levels of cytokines such as IL-1, IL-6, IL-10, and TNF-α and provoked the production of these cytokines in human lymphocytes and monocytes, in which IL-6 up-regulation and production was the most pronounced (3,21). For the first time in 2008, investigators found that eNAMPT could promote cell survival in macrophages, by inhibiting endoplasmic reticulum stress-induced apoptosis. This mechanism occurs by activating an IL-6/Signal Transducer and Activator of Transcription 3 (STAT3 ) signalling pathway (24). Macrophages have an important and preponderant role in the pathophysiology of comorbidities associated with obesity, such as atherosclerosis (25), inflammation (26), and cancer (27). Therefore, something that changes the dynamics of survival-apoptosis of these immune cells will have an impact on the emergence of diseases related to obesity. Prolonged macrophage survival was suggested to prop up the inflammatory response, known as a crucial factor underlying obesity-related metabolic diseases (24). Interestingly these findings were not dependent on the enzymatic activity of eNAMPT, corroborated by the fact that STAT3 phosphorylation was not induced by NMN – NAMPT’s enzymatic product - and because the 4 inhibitor of NAMPT’s enzymatic activity - FK866 - also did not prevent eNAMPT's ability to stimulate phosphorylation of STAT3 (24). As Y. Li et al. also report, it seems questionable that cells release an enzyme that is capable of activating certain metabolic pathways in a non-enzymatic way. Later, a possible justification for this, related to structural and functional changes of the eNAMPT protein, will be addressed. 1.3 NAMPT In The Context of Metabolic Disease – Obesity and Diabetes eNAMPT has recently been shown to have an important connection to a wide variety of diseases. There is in fact evidence showing enhanced mRNA expression of NAMPT and higher circulating levels of eNAMPT in obesity, T2D, cancer, and other inflammatory conditions (18,28,29), including inflammatory bowel diseases ((3,30)) rheumatoid arthritis (31), atherosclerosis, myocardial infarction, and severe generalized psoriasis (3,13,30). The biological action of NAMPT in obesity and obesity-related disorders, such as T2D, has been interestingly studied since Fukuhara et al. demonstrated that eNAMPT exerted a glucoselowering effect in a diabetic mouse model. However, the article ended up retracted due to the controversy regarding the insulin receptor-binding activity of eNAMPT that could not be reproduced (32). Despite the significant interest, there is a lot of controversy over the association between circulating levels of eNAMPT and obesity-related parameters, and the effects of eNAMPT in the context of T2D, particularly regarding the role of this protein in beta cell function. The results from a meta-analysis of 13 observational studies involving 644 subjects showed a significant increase in eNAMPT concentration in participants with overweight/obesity compared to normal body mass index (BMI) controls (28)). Moreover, Fukuhara et al. demonstrated that eNAMPT was more abundantly secreted by vAT in both humans and mice, than in scAT (13,32). As mentioned, the article was retracted due to the lack of reproducibility of the main result – eNAMPT insulin-mimetic effect – however other authors corroborated the significant association between eNAMPT levels and visceral fat (19,33,34). To investigate the function of eNAMPT specifically secreted and regulated by AT, Yoon et al. created and analysed adipose tissue-specific Nampt knockout (ANKO) mice, which showed reductions in plasma eNAMPT levels and NAD levels, resulting also in severe impairments in IS and glucose-stimulated insulin secretion (GSIS) (35). In line with this knowledge, AT may be fundamental to regulate beta cell function through secretion of eNAMPT and consequent extracellular biosynthesis of NMN, as fully differentiated adipocytes are a natural producer of eNAMPT (13,15,35). Revollo et al. showed that NAMPT-deficient heterozygous (NAMPT +/−) mice had a defect in NAD biosynthesis and in GSIS in pancreatic islets. Administration of NMN could completely ameliorate these defects, strongly indicating that they were due to a lack of NAMPTmediated NAD biosynthesis (15). Adipocyte dysfunction, with ultimate dysregulation of adipokines, particularly eNAMPT, can lead to alterations in IS and the development of T2D, with simultaneously worsening of inflammation and obesity (20,36,37). Consistent evidence shows 5 that T2D patients present higher levels of eNAMPT compared to healthy controls (36–38)). In a 2011 meta-analysis, this evidence was reinforced as it was shown that eNAMPT was significantly increased in subjects diagnosed with T2D (28). This relationship between NAMPT activity and T2D appears to be related to pancreatic tissue being more susceptible to changes in NAMPTmediated NAD biosynthesis, compared to other tissues. Because of the almost total absence of iNAMPT, islets rely on extracellular sources of NAD intermediates, such as NMN, to maintain their NAD levels adequate for their functions (14,15). Moreover, available evidence suggests that the elevated eNAMPT concentration in T2D may be a physiologic protective response to a hyperglycemic environment (28). Although, at supraphysiological levels, the effect apparently turns deleterious (20,39). As discussed earlier, according to some data, the inhibitor of NAMPT’s activity - FK866 - has been shown to be ineffective, indicating that at least part of NAMPT’s effects is independent of its enzymatic activity ((18,24)). Dimerization of eNAMPT is essential for its enzymatic function, and at physiological levels, in healthy individuals, eNAMPT is found mainly in the dimeric form (~100kDa). Yet, Li et al. showed that NAMPT mutants, which were unable to dimerize, maintained a cytokinelike activity (24). For example, as discussed previously, the anti-apoptotic effect of NAMPT does not require dimerization, suggesting that it is the monomeric form (~50kDa) that maintains this pro-inflammatory effect (13). A study deepened the characterization of this structure-functional difference of eNAMPT in the deterioration of pancreatic beta cell function and its relationship with T2D, in order to understand the contradictory effects of eNAMPT on beta cell function (protective or deleterious), previously described in the literature (40). The study concluded that concentration played an important role in these changes in structure and function, and that dimeric eNAMPT, found at low physiological levels promotes NAD biosynthesis, while at a supraphysiological state eNAMPT adopted a monomeric form capable of pro-inflammatory mechanisms, independent of NAD, deleterious for beta cell function (40). 1.4 Anthocyanins - Health Promotion and Potential Effect on eNAMPT in Context of Metabolic Disease Despite obesity being the main risk factor for T2D (37,41), diet is an unquestionable modifiable factor that may influence the risk of both obesity and diabetes (42). A high fat diet in the context of overnutrition leads to increased lipid deposition in both adipose and non-adipose tissues (43,44). In vivo studies have shown that animals fed high-fat diets develop obesity and diabetes, within weeks (45–47)). Furthermore, a high fat diet has been shown to induce alterations in blood lipid profile and in circulating proinflammatory cytokines (48,49). In contrast to the typical “western diet”, high in sugar and fat, a fruit and vegetables rich diet has been associated with a lower risk of obesity and related co-morbidities (50,51). In particular, observational studies show that a higher intake of fruits and vegetables has been inversely associated with the risk of T2D 6 (52–55). However, the number of studies is limited and findings regarding these associations have not been consistent (56–58). In recent years, berries have been a field of research that has grown considerable interest, due to their potential, as a natural product, for health promotion and disease prevention (59–61). Berries have been associated with cognitive function and anti-inflammatory and antioxidant responses (62,63), as well as with the reduction of adipocyte death and lower adipocyte size in mAT (46,61). In addition, a growing body of evidence, including prospective cohort studies, suggests that higher intakes of berries are inversely associated with T2D risk (52–54). Considering this, in other findings, including those from clinical trials, berries have shown an improvement in glucose response and in IS (52–54). These beneficial outcomes of fruit, in particular berries, seem to be connected to their composition in terms of dietary fiber and polyphenols (64,65). Dietary phytochemicals (such as polyphenols) may result in synergistic activity that can offer advantages over treatments with single chemicals that may comprehend adverse side effects (59,60). Anthocyanins are a group of polyphenolic natural pigments that belong to a broad class of secondary metabolites called flavonoids, abundant in various brightly colored fruit and vegetables (59–61). Despite the limited number of clinical studies, several in vitro and in vivo studies confirmed that the consumption of anthocyanins rich food prevents or treats obesityrelated consequences like T2D, inflammation and oxidative stress. (66,67). The studies that have been conducted to evaluate the biological functions of dietary anthocyanins on inflammation and obesity have shown that their anti-obese properties rely on their ability to control food consumption and energy metabolism and improve inflammatory response. However, these favorable effects were dependent on several physiological, immunological, neurological, and metabolic interconnected events (66,67). Dietary anthocyanins also appear to ameliorate IR (42,46,61,64,65,68). Experimental studies have demonstrated that the benefits in T2D associated with anthocyanins may be related to improvements in IS (60), through diverse mechanisms ranging from inhibiting carbohydrate digestion in the gut, pancreatic beta cell protection, and insulin secretion to enhancing IS in vital organs (59). Cyanidin-3-O-β-glucoside (Cy-3-G), an abundant anthocyanin, was able to regulate the expression of adipokines in BAT, which were disrupted by a high fat diet supplemented with cholesterol (8). Furthermore, supplementation with purified anthocyanins decreased eNAMPT in patients with prediabetes or newly diagnosed diabetes and showed improvements in glucolipid metabolic parameters (69). Taken together, these studies suggest a beneficial effect of anthocyanins as preventive strategy for obesity–related metabolic abnormalities, potentially through a pathway involving NAMPT. However, the mechanism by which anthocyanins play a role in obesity through eNAMPT has not been elucidated nor was the relationship between anthocyanin treatment and the specific concentration of eNAMPT in its dimeric/monomeric form. 7 2. AIMS This study seeks to help closing the gap in current literature, by investigating the differences in eNAMPT’s expression, in its monomeric form, in the mesenteric adipose tissue of animal models in the context of diet-induced obesity and increased metabolic risk (fed a high fat diet). Moreover, the study aims to understand how the effect of such obesogenic diet in the expression of monomeric eNAMPT is impacted by an anthocyanin treatment. Ultimately, the study aims to give us an insight into early mechanisms of disease prevention, potentially contributing to the therapeutic strategies for metabolic disorders. 8 3. METHODOLOGY 3.1 Experimental Design This study protocol obtained ethical approval by the Ethics Committee of NOVA Medical School | Faculdade de Ciências Médicas (NMS|FCM), NOVA University of Lisbon (nº113/2021/CEFCM). An overview of the experimental protocol is represented in Figure 2. Figure 2 - Overview of experimental protocol and timeline. mAT - mesenteric Adipose Tissue. 3.2 Animals and Sample Collection The mAT samples used in this study were previously collected during a research project in 2012 (62). Animal handling and housing protocols followed European Union guidelines (86/ 609/EEC) and the Portuguese Act (129/92) for the use of experimental animals. The study obtained ethical approval from the Ethical Committee of the Faculty of Medicine, University of Porto and the São João Hospital Center (46). Twenty-four male Wistar rats, acquired from Harlan Laboratories (Santiga, Spain), were divided into four groups (n = 6 rats per group), after two weeks of acclimatization, as follows: (C) standard diet; (BE) standard diet + blackberry extract; (HFD) highfat diet; (HFDBE) high-fat diet + blackberry extract. Animals were fed ad libitum with “standard” (Teklad 2014, Harlan Laboratories, Santiga, Spain) or “high-fat” diets (D1245 Research Diets, New Brunswick, USA), according to their groups, for 17 weeks. Blackberry anthocyanin rich extract (25 9 mg/kg body weight/day) was dissolved daily in sterile water and embedded in food pellets that animals had daily access to. Animals were maintained at 23–25 °C with a 12/12 h light–dark cycle and housed two per cage. Food ingestion was measured twice a week. Fasting blood glucose, and glucose and insulin tolerance tests were also assessed then, and the detailed methodology is described in the paper from Meireles et al. 2016 (46). This data is presented in this thesis to contextualize further results obtained in this project and for interpretation purposes. After sacrifice, mAT was collected, immediately frozen in liquid nitrogen, and stored at −80 °C until use. In the present study, protein extraction and quantification were performed from these mAT samples. 3.3 Protein Extraction Approximately 300µg of each mAT sample were grounded in liquid nitrogen with a mortar and pestle and transferred to new Eppendorf tubes (1,5mL). Reconstructed RadioImmunoprecipitation (RIPA) Lysis Buffer with proteases inhibitors (10μL PMSF solution, 10μL sodium orthovanadate solution, and 10-20μL protease inhibitor cocktail solution per mL of 1X RIPA Lysis buffer) (Cat#sc-24948A, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the tubes, following the supplier recommendations, and homogenized using an electric homogenizer. RIPA Buffer enables efficient tissue and cell lysis, and protein solubilization while avoiding protein degradation. The samples were refrigerated at -20ºC, for at least 2h and then centrifuged at 10000 x g, for 15 min at 4ºC. The tubes were gently removed from the centrifuge and placed on ice. The supernatant was aspirated from each sample (± 600 µL) and placed in fresh identified Eppendorf tubes kept on ice. The lipid upper phase and the pellet were discarded. 3.4 Protein Quantification For protein quantification, an EZQ® Protein Quantitation Kit (R33200, Thermo Fisher Scientific, USA) was used. After preparing the microplate cassette with the assay paper, 1 well was left with nothing to ensure non-contamination of the assay paper (negative control 1), and 1µL of reconstructed RIPA Lysis Buffer was spotted onto another well (negative control 2). The samples were spotted onto assay paper in triplicate using 1µL of each protein extraction sample per well (protein extract was not diluted to avoid loss of protein). The paper was dried and fixed with methanol for 5 minutes with gentle agitation to remove contaminating substances. The EZQ® protein quantitation reagent was added into a staining tray and the protein-spotted assay paper was added into the stain solution and agitated gently on an orbital shaker for 30 minutes. After staining, the assay paper was washed twice for 1–2 minutes with a rinse buffer solution (10% methanol, 7% acetic acid) and after completely dried was replaced into the microplate. The fluorescence was measured in a SpectraMax i3x plate reader (Molecular Devices; Sunnyvale, CA) using excitation/emission settings of 450/618nm. The final protein concentration was determined using a standard curve of ovalbumin (Appendix 1), and the samples were frozen at -20ºC for later use. The samples had a very low protein concentration, insufficient to provide enough protein to 10 perform the Western blots (WB). In order to concentrate the samples, they were lyophilized overnight and resuspended in a smaller volume of ultrapure water (Milli-Q® systems). Protein quantification was then repeated according to the method described above. 3.4 eNampt Measurements - Protein Gel Electrophoresis and Western Blotting In order to separate proteins, a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDSPAGE) of 4-15% precast polyacrylamide gels (#4568034, Bio-Rad Laboratories Inc, USA) was used. SDS is an anionic detergent, used to denature tertiary and secondary protein structures, thus allowing a protein separation by size/molecular weight depending on their electrophoretic mobility in a polyacrylamide gel. After normalizing to 15μg of protein, adding 10μL of loading buffer (6X, 400/L saccharose, 2,5g/L bromophenol blue) and ensuring an equivalent volume of 40μL to load each lane, samples were heated at 100ºC for 10 minutes and centrifuged at 4,000 rpm for 20 seconds. No reducing agent was added to the protein samples prior to SDS-PAGE to allow detection of both monomeric and dimeric eNAMPT in biological samples. Before loading the samples, 3μL of a prestained protein ladder (R26619, PageRuler™Plus Thermo Fisher Scientific, USA) was loaded to one of the wells of each gel to serve as a size reference to which samples could be compared later. 40μL of the respective samples were loaded to the remaining gel wells, and electrophoresis was run at 100V for 90 minutes with migration/running buffer (3g/L TRIZMA® base, 14g/L glycine, 10mL/L SDS 10%) in mini-PROTEAN® Tetra system gel tanks (BioRad Laboratories Inc, USA). The evolution of electrophoresis was monitored by observing the migration of the bromophenol blue dye that was added to the loading buffer. Due to its low molecular weight and negative charge, it migrates along with proteins towards the positive anode. After the proteins have been separated by gel electrophoresis, the WB technique requires the transfer of the proteins to a membrane, in this case a polyvinylidene difluoride (PVDF) membrane, for subsequent densitometric quantification. PVDF membranes are highly hydrophobic, so prior to their utilization they were pre-wetted with ethanol for 1 minute, water for 10 minutes, and at least 10 minutes with transfer buffer (1.5g/L TRIZMA® base, 7.5g/L glycine). The transfer devices, with the black part of the cassettes facing bottom, were prepared in the following way for each cassette: 1 scratch sponge, 2 Whatman® blotting papers, SDS-PAGE gel, PVDF membrane, 2 Whatman® blotting papers, 1 scratch sponge, and everything was embedded in transfer buffer. The transfer cassettes were then inserted into the Mini Trans-Blot Cell® buffer tank (Bio-Rad Laboratories Inc, USA), which was filled with transfer buffer, and blotting was performed at 50 V for 95 minutes, so the negatively charged proteins could transfer onto the membrane, by migrating towards the positively charged electrode of the blotting chamber. The efficiency of transfer was controlled by the presence of the protein ladder on the membrane at the end. Following the transfer, membranes were washed twice with phosphate-buffered saline (PBS) containing 0,1% (vol/vol) Tween-20 (TPBS). Membranes were then blocked with 5% (wt/vol) dried 11 skimmed milk (Molico®) dissolved in TPBS (2,5 g per 50mL) for 60 minutes at room temperature under agitation, in order to prevent non-specific binding during the following antibody incubations. After quickly rinsing the blocked membranes three times with TPBS, a mouse primary antibody against NAMPT (Cat#sc-393444, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:500 dilution in TPBS containing 5% BSA) was incubated with the membranes overnight at 4ºC on a rocking platform shaker. Membranes were again washed three times with TPBS, with gently agitation on an orbital shaker for 10 minutes each time, to eliminate unbound antibody residues and posteriorly incubated with secondary antibody anti-mouse conjugated to horseradish peroxidase (HRP) (Cat#sc-542741, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:3000 dilution in TPBS containing 5% BSA) for 60 minutes at room temperature with gentle agitation. Following one quick wash with TPBS, membranes were again washed three times with TPBS, with gently agitation on an orbital shaker for 10 minutes each time. The proteins were then visualized in a ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories Inc, USA) after the membranes were washed for 1 minute with a chemiluminescent WB detection reagent (RPN2235, Amersham™ ECL Select™, cytiva), which allows the HRP to produce a luminescent signal. The signal obtained is proportional to the antibody-bounded protein, which appears as bands, therefore is used as the measurement for protein quantity. Densitometry of WB bands was calculated using Fiji software version V1.52 E. 3.5 Statistical Analysis The normality of the data was assessed by a Shapiro-Wilk test. Comparison between C vs HFD, C vs BE and HFD vs HFDBE groups was performed using unpaired t-tests. One way-ANOVA was undertaken to analyse the variance across the groups. Tukey’s post hoc test was then implemented to verify potential differences between the treatment groups. Data are expressed as mean ± standard error of the mean (SEM). Results were considered statistically significant for p≤0.05. All statistical analysis were performed in GraphPad Prism 8 software (GraphPad Software, San Diego, USA). 12 4. RESULTS 4.1 Data previously collected, analysed and described in Meireles et al. 2016 The animal experiments originally took place in 2012. All animals started the diet treatment at similar body weights (Table 1). The high fat diets - HFD and HFDBE - resulted in overweight rats, with increased body fat percentage. The effect of blackberry anthocyanin extract supplementation on body weight gain was not significant (62). Table 1 –Body weight of rats from different groups before (initial body weight) and after the 17 weeks of diet and blackberry anthocyanin extract supplementation (final body weight). Values are expressed as mean ± Standard Deviation (SD). Different superscript letters represent statistically significant differences (p<0,05): C – standard diet; BE – standard diet + blackberry extract; HFD – high-fat diet; HFDBE – high-fat diet + blackberry extract. Reproduced from Meireles et al., 2016 to contextualize further results obtained in the project here presented. Fasting blood glucose was assessed in these animals, showing significant differences between groups (p<0,05). The HFDBE had higher fasting glucose levels compared to C at the 15th week of treatment. (*p<0,05) (figure 3). Figure 3 – Fasting blood glucose levels of rats from the four groups (n = 6). Data are expressed as means ± SEM. Statistical significance was tested by one-way ANOVA, followed by a Tuckey’s post hoc analysis for comparison between groups (p<0,05). The HFDBE had higher fasting glucose levels compared to C (*p<0,05). Reproduced from Meireles et al., 2016 to contextualize further results obtained in the project here presented. C BE HFD HFDBE Initial body weight (g) 237,8±7,7 246,5±8.6 237,2±7.2 244,0±11,1 Final body weight (g) 429,8±27,8a 432,7±28,4a 550,7±67,6b 581,0±63,1b 19 improved all profiles associated with the condition, after receiving an antibody against the monomeric eNAMPT, such as decreased blood glucose, improved glucose tolerance and IR, improved pancreatic islet function, and reduced inflammation (73). On the other hand, upon administration of eNAMPT-monomer to the non-diabetic mouse model, they developed a diabetic phenotype, characterized by an increase in blood glucose, impaired glucose tolerance and insulin secretion and by the presence of a generalized inflammation, without changes in NAD levels (73). These results suggest that these pro-inflammatory and diabetic effects are independent of eNAMPT’s NAD biosynthetic activity, providing evidence that this monomeric form not only does not present enzymatic capacity, but is also responsible for the previously mentioned adverse effects. In agreement with the aforementioned study, other analysis conducted in individuals with obesity, diabetic and non-diabetic, for detection of eNAMPT’s dimeric and monomeric levels, showed that serum eNAMPT concentrations increased with progression of T2D. eNAMPT monomer was found to be significantly higher in T2D (40). These increasing concentrations did not correlate with serum NMN levels. This study also showed that eNAMPT concentration was a critical factor in these changes in structure and function. At low and physiological concentrations (1 ng/ml) eNAMPT remained in its dimeric form, optimizing beta cell function through its NADbiosynthetic capacity. However, at supraphysiological levels (5 ng/ml), characteristic of obesity and T2D, eNAMPT adopted a monomeric structure and deterioration of beta cell function was observed, resulting from pro-inflammatory mechanisms independent of NAD (40). Although is not yet clear how and why these structural differences occur during the rising concentrations, studies present different hypotheses, being one of them the possible necessity of a ligand-induced dimerization, that begins to be deficient at higher eNAMPT concentrations, leading to the breakup of the dimer into constitutive monomers (73). The other possibility described involves an obesity-mediated dysfunctional AT that is characterized by poorly differentiated adipocytes and immune cells, incapable of secreting eNAMPT dimer, as this form is said to be secreted by fully differentiated adipocytes (40). Either way, finding approaches that can stabilize eNAMPT in dimer form, by controlling its rising serum concentration, or that can block the actions of the monomer form, by neutralizing eNAMPT monomer appears to be a promising future for disease prevention and potentially contributing to the therapeutic strategies for metabolic disorders such as obesity and T2D. The main purpose of this study was also to analyse the impact of an anthocyanin treatment in eNAMPT’s expression on mAT of these animal model of increased metabolic risk. Diet is a key factor in terms of managing the risk of obesity and obesity-related disfunctions. Previous studies have shown that a dietary intervention with anthocyanins is effective in reducing the risk of obesity and preventing or treating obesity-related co-morbidities (66,67). The results in subjects 20 with obesity suggested that daily intake of dietary anthocyanins might aid in maintaining or reducing the body weight and attenuating some risk factors of metabolic syndrome (75,76). The anthocyanin extract from blackberry given to the animals in this study was able to attenuate the raise of mAT eNAMPT monomer in the context of an HFD. mAT eNAMPT monomer expression was significantly decreased in group HFDBE compared to HFD (-54,1 ± 15,3 % [-89.4, -18.8], p<0,01). In support of these results, a previous randomized placebo-controlled trial also aimed to examine the effects of purified anthocyanins supplementation on eNAMPT and another adipokine in Chinese adults with prediabetes and newly diagnosed, untreated T2D (69). Supplementation with purified anthocyanins for 12 weeks decreased serum eNAMPT in patients with prediabetes or newly diagnosed diabetes and showed improvements in glucolipid metabolic parameters (69). A significant difference in serum eNAMPT (-3,5 ng/mL [ -6,69, -0,31], p=0,03) was observed between the anthocyanins and placebo groups, however it is not mentioned if it is the monomeric or dimeric form that has been evaluated. Serum eNAMPT decreased by 9,7% (-3,57 ng/mL [-5,98, - 1,15], p=0.004) after anthocyanins treatment compared to the 0,2% decrease (-0,07 ng/mL [-2,07, 1,94], p=0,947) after placebo treatment (69). The authors had already demonstrated that anthocyanins inhibited the secretion of eNAMPT in a previous animal study (8), and an important conclusion from that experiment was that Cy-3-G, as the most abundant monomer of anthocyanins, could regulate the secretion of monomeric eNAMPT from brown adipocytes. After being incubated with palmitate, the release of monomeric eNAMPT from those cells was increased and could be inhibited by Cy-3-G (8). The main anthocyanin in blackberry is Cy-3-G (about 85%). As such, the anthocyanin extract from blackberry used for treating the animals on the project that led to our study is rich in Cy-3-G. As assessed by our working group in the original project from Meireles et al 2016, diet was a significant factor that influenced the glucose sensibility of the animals (46). The HFD showed a tendency to promote a higher fasting glucose in animals, an observation that was not attenuated by the anthocyanin treatment. The HFDBE had higher fasting glucose levels compared to C (p<0,05). As these animals gained weight along the weeks of treatment, especially the HFD groups, the risk of greater insulin resistance and glucose intolerance was higher, as these conditions are highly associated with obesity. As expected, animals from the HFD displayed a lower response to insulin as well as an impaired glucose sensibility (46). As discussed earlier, reductions in plasma biosynthesis of NMN, result in severe impairments in IS and GSIS (35). With this study we demonstrated that an obesogenic condition, imposed by a HFD, led to an increase in the monomeric eNAMPT in these animal’s mAT. This setting may have led to a greater secretion of the less biosynthetic monomeric eNAMPT into the circulation and consequently, to a decrease in serum NMN, being this the possible cause for the derangements in the target tissues most susceptible to these alterations, such as beta cells (Figure 9). However, the dimeric form of eNAMPT should have been analysed for a better comprehensive analysis of the results, including the ratio monomer/dimer instead of the absolute quantification of the monomeric eNAMPT 21 alone. If the ratio had increased, as would be expected at the outset, this would lead to a decrease in the production of NMN, as seen in other studies (40,73,74). Figure 9 - Schematic proposal of eNAMPT monomer's higher expression in obese adipose tissue and the consequent NMN deficiency effects on beta cell function. An obesogenic condition, imposed by a HFD, led to an increase in the monomeric eNAMPT in these animal’s mAT. This setting may have led to a greater secretion of the less biosynthetic monomeric eNAMPT into the circulation and consequently, to a decrease in serum NMN, being this the possible cause for the derangements in the target tissues most susceptible to these alterations, such as beta cells. Nonetheless, to a certain extent supplementation with blackberry anthocyanin extract was able to attenuate the impairment of the glycemic response caused by the HFD, as seen in the AUC of the HFDBE group, after 8 weeks. A possible hypothesis is that as the anthocyanin’s treatment was able to concurrently attenuate the increase of the mAT monomeric form of eNAMPT, as demonstrated in this study, the dimeric form may have been secreted in higher or normal levels from the AT in this group of animals, maintaining serum NMN levels adequate and as so protecting the beta cell from the deleterious effects previously discussed, particularly attenuating the impairment of the glucose sensibility, and optimizing the insulin response. After the 15th week of treatment HFBE group also had a better response to insulin than HFD rats (p<0,05). Together these data demonstrate that mAT levels of eNAMPT monomer were selectively elevated in HFD-fed mice, but the anthocyanin’s treatment was able to significantly minimize/abrogate this abnormal increase. However, this relation requires further investigation. It is necessary to carry out studies with a larger n and preferably a human study design to prove the hypothesis raised and deepen the technique that allows the quantification of the dimeric form, that can provide valuable 22 information. Being able to analyse both forms of eNAMPT would bring a more in-depth investigation of the molecular mechanisms resulting in the repercussions of the alterations in this adipokine. Further investigation will also be required to identify detailed mechanisms for the efficacy of the anthocyanins. Additional interventional studies, preferably long-term trials are needed to clarify and confirm our findings and test the potential of blackberry supplementation as a therapeutic intervention in humans. 23 6. CONCLUSIONS Despite NAMPT’s essential role in metabolism through its catalytic function in the rate-limiting step in NAD biosynthesis, the protein has become the center of attention due to the subsequent association of its monomeric-non-biosynthetic form with metabolic and inflammatory diseases. This study demonstrates that an anthocyanin treatment abrogates the elevation of monomeric eNAMPT protein in mAT from animals inflicted with a HFD - that negatively impacted their metabolism. Here we hypothesize that the anthocyanin treatment, by reducing the mAT monomeric eNAMPT levels, was able to attenuate some of the resulted impairments, such as glucose intolerance, and optimize the response to insulin. 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