Regulation of amino acid catabolism in rats fed diets with different protein content Silvia Agnelli Aquesta tesi doctoral està subjecta a la llicència Reconeixement- NoComercial 3.0. Espanya de Creative Commons. Esta tesis doctoral está sujeta a la licencia Reconocimiento - NoComercial 3.0. España de Creative Commons. This doctoral thesis is licensed under the Creative Commons Attribution-NonCommercial 3.0. Spain License. UNIVERSITAT DE BARCELONA Regulation of amino acid catabolism in rats fed diets with different protein content Silvia Agnelli Barcelona, 2016 Alla mia Famiglia : per la pazienza e l'amore infinito i ii “Dubium sapientiae initium” René Descartes Meditationes de prima philosophia iii iv Abbreviations ATF:Activating transcription factor 4 AP-1:Activator protein-1 ARG:Arginase ASL:Argininosuccinate lyase ASS:Argininosuccinate synthase BAT:Brown adipose tissue BCAA:Branched-chain amino acids BiP:Immunoglobulin binding protein BMI:Body mass index CAFD:Cafeteria diet CPS:Carbamoyl-phosphate synthase DAG:Diacylglicerol eIF2α:Eukaryotic translation initiation factor 2 eNOS:Vascular endothelial constitutive NOS ER:Endoplasmic reticulum GADD34:Growth arrest and DNA damage- inducible protein GLUT-4:Glucose transporter 4 GOT:Glutamic oxaloacetic transaminase GPT:Glutamate-pyruvate transaminase HDL:High density lipoprotein HFD:High fat diet HPD:High protein diet IKK:Inhibitor of κ kinase IL:Interleukin IMCL:Intramyocellular lipid iNOS:Inducibile NOS IR:Insulin resistance IRE1:Inositol requiring 1 IRS:Insulin receptor substrate JNK:c-jun N-terminal kinase LPS:Lipopolisaccharide MS:Metabolic sindrome NAFLD:Non-alcoholic fatty liver disease v NF-κB:Nuclear factor -light-chain-enhancer of activated cells nNOS:Neuronal or brain constitutive NOS NO·:Nitric oxide NOS:Nitric oxide synthase NRF2:Nuclear factor erythroid 2 OTC:Ornithine transcarbamylase PERK:PKR-like ER kinase PKC:Protein kinase C PP1:Protein phosphatase 1 SCFA:Short chain fatty acids SDH:Serine dehydratase SFA:Saturated fatty acid T2DM:Type 2 diabetes mellitus TAG:Triacylglycerol TLR:Tool-like receptor TNFα:Tumor necrosis factor TRAF2:Tumor necrosis factor receptor-associated factor 2 UC:Urea cycle UPR:Unfolded protein response VLDL:Very low-density lipoprotein WAT:White adipose tissue WHO:World Health Organization XBP1:X box-binding protein 1 vi Abstract Current lifestyle with high-energy diets and characterized by sedentary is triggering an alarming growth in obesity. Obesity along with metabolic syndrome-related co-morbidities (i.e. insulin resistance, atherosclerosis, sleep apnoea, depression, asthma, hypertension and the alteration of blood lipid transport) are the most apparent consequence of the excess energy. Under conditions of excess dietary energy, the body cannot easily dispose of the excess amino-N against the evolutively-adapted schemes that prevent its wastage; thus ammonia and glutamine formation and urea excretion are decreased. High lipid and energy availability limit the utilisation of glucose, and high glucose spares the production of ammonium from amino acids, decreasing the synthesis of glutamine and its utilisation by the intestine and kidney. In contrast, high protein diets enhance protein synthesis and growth, and the synthesis of non-protein- N-containing compounds. But these outlets are not enough; consequently, less-conventional mechanisms are activated, such as increased synthesis of NO∙followed by higher nitrite (and nitrate) excretion and changes in the microbiota. In this study we studied how the initial phase of development of metabolic syndrome can affects the function of liver as main site of amino-N metabolism, and to determine whether doubling the protein content in the diet induced significant changes in enzyme of amino acids metabolism along intestine and on liver. The common result obtained by these studies is that, both in case of hyperlipidic or hyperproteic diets, elimination of excess N is necessary but cannot be easily carried out through the metabolic pathways/tissues we evaluated, although possible alternative pathways have been taken into consideration. vii viii Contents 1. INTRODUCTION ............................................................................................................. 1 1.1 The metabolic syndrome ................................................................................................. 3 1.1.1 The insulin resistance ................................................................................................. 5 1.1.2 Obesity and high fat diets ......................................................................................... 17 1.2 Amino-N disposal in metabolic syndrome ................................................................... 20 1.3 High protein diets and their differences with high fat diets ...................................... 22 1.4 Outline of nitrogen metabolism .................................................................................... 24 2. OBJECTIVES ................................................................................................................. 39 3. METHODS...................................................................................................................... 43 3.1 Development of methods for the determination of ornithine carbamoyl transferase and argininosuccinate synthase activities in rat tissues ............................................. 44 4. RESULTS ........................................................................................................................ 63 4.1 Sabater D, Agnelli S, Arriarán S, et al. Altered nitrogen balance and decreased urea excretion in male rats fed cafeteria diet are related to arginine availability. Biomed Res. Int. 2014, 959420 (2014) .......................................................................... 64 ix 4.2 Agnelli S, Arriarán S, Oliva L, et al. Modulation of rat liver urea cycle and related ammonium metabolism by sex and cafeteria diet. RSC Adv 6, 11278–11288 (2016) ......................................................................................................................................... 74 4.3 Sabater D, Agnelli S, Arriarán S, et al. Cafeteria diet induce changes in blood flow that are more related with heat dissipation than energy accretion. PeerJ 4, e2302 (2016) ............................................................................................................................... 86 4.4 Agnelli S, Arriarán S, Fernández-López JA, et al. The activity of amino acid metabolism enzymes along the small intestine of male rats is slightly affected by high-protein diet. Under evaluation (European Journal of Nutrition) ..................... 98 4.5 Agnelli S, Arriarán S, Fernández-López JA, et al. The effects of high-protein diet on amino acid metabolism of lean and obese Zucker rats. Under evaluation (Food&Function) ......................................................................................................... 118 5. GENERAL DISCUSSION ........................................................................................... 137 6. SUMMARY ................................................................................................................... 145 7. CONCLUSIONS ........................................................................................................... 149 8. BIBLIOGRAPHY ......................................................................................................... 153 x List of tables and figures Table 1. Names given to the clustering of metabolic syndrome disorders. .................................. 4 Figure 1. Insulin Action. .................................................................................................................... 6 Figure 2. Lipid accumulation in adipose and non-adipose tissues ................................................ 8 Figure 3. Molecular mechanisms of skeletal muscle insulin resistance ........................................ 9 Figure 4. Hallmarks of metabolic inflammation in adipocyte ..................................................... 12 Figure 5. Diet-induced dysbiosis affects disease susceptibility .................................................... 16 Figure 6. Schematic representation of amino acid catabolism in mammals .............................. 25 Figure 7. The transamination reaction catalyzed by alanine aminotransferase. ....................... 26 Figure 8. Glutamate dehydrogenase reaction................................................................................ 27 Figure 9. Glutamine synthetase reaction. ...................................................................................... 28 Figure 10. Schematic representation of mechanism of action of L- glutaminase. ..................... 29 Figure 11. The enzymatic reaction of serine dehydratase. ........................................................... 29 Figure 12. The enzymatic reaction of AMP deaminase. ............................................................... 29 Figure 13. The enzymatic reaction catalyzes by glycine cleavage system. .................................. 30 Figure 14. The enzymatic reaction catalyzed by monoamine oxidase. ....................................... 31 xi Figure 15. Mechanism of chemical reaction catalyzed by L-amino acid oxidases. .................... 32 Figure 16. Schematic representation of urea cycle. ...................................................................... 34 xii Introduction 1. INTRODUCTION 1 Introduction 2