Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Sayrany, Melody
Master of Science - MSc
University of British Columbia
Age-related diseases encompass confounding biological insults, some of which are related to dysregulated amino acid and lipid metabolism. Glutamine is one of the most abundant and vital amino acid for cellular processes that is depleted during conditions of stress and aging. Besides glutamine, polyunsaturated fatty acid (PUFA) metabolism is also altered in aging. However, their inter-relationship in the context of the liver is unknown. My project was to investigate the effects of glutamine supplementation and lipid metabolism in middle-aged mouse livers. To this end, we selected a mouse deficient in fatty acid elongase 5 (Elovl5), with impaired PUFA bioconversion and another mouse deficient in endonuclease G (EndoG), which stays protected in aging due to a lack of endonuclease activity and attenuation of mitochondrial DNA breakdown. My first objective was to identify said relationship in middle-aged livers of Elovl5 and EndoG heterozygous mice by feeding glutamine or alanine, while keeping C57/Bl6 mice (wild type, WT) as controls (Chapter 2). We show that glutamine supplementation does not affect gene expressions of glutamine-related metabolic (glutaminase 2, glutamine synthase, and glutamine dehydrogenase), or PUFA bioconversion (fatty acid desaturase 1/2 and Elovl5), enzymes in WT, and they were decreased in EndoG+/- mice livers. In contrast, glutamine supplemented Elovl5+/- mice livers showed upregulated gene expressions for both. For my second objective in Chapter 3, we determined the impact of glutamine or alanine on inflammation and mitochondrial biogenesis markers in the same livers. Glutamine-fed WT mice demonstrated no change while EndoG+/- showed a decrease in gene expressions of pro-inflammatory or mitochondrial biogenesis markers. Elovl5+/- mice on the other hand, demonstrated increased gene expression both inflammation and mitochondrial biogenesis markers. Given the fact that Elovl5+/- livers had increased levels of lipogenic as well as inflammatory and mitochondrial biogenesis genes with glutamine, such results indicate that glutamine supplementation is probably harmful for the liver at an advanced age with existing metabolic defects but might be neutral or beneficial in healthier phenotypes. Overall, these results contribute to understanding the relationships between glutamine, lipid metabolism, and aging in middle-aged mouse livers.
i THE ROLE OF GLUTAMINE HOMEOSTASIS IN THE MIDDLE-AGED LIVER by Melody Sayrany B.Sc., University of California San Diego, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) May 2022 © Melody Sayrany, 2022 i The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: The Role of Glutamine Homeostasis in the Middle-Aged Liver submitted by Melody Sayrany in partial fulfillment of the requirements for the degree of Master of Science. Dr. Sanjoy Ghosh, Faculty of Science Supervisor Dr. Rajavel Elango, Faculty of Medicine Supervisory Committee Member Dr. Christopher West, Faculty of Education Supervisory Committee Member Dr. Angela Devlin, Faculty of Medicine University Examiner ii Abstract Age-related diseases encompass confounding biological insults, some of which are related to dysregulated amino acid and lipid metabolism. Glutamine is one of the most abundant and vital amino acid for cellular processes that is depleted during conditions of stress and aging. Besides glutamine, polyunsaturated fatty acid (PUFA) metabolism is also altered in aging. However, their inter-relationship in the context of the liver is unknown. My project was to investigate the effects of glutamine supplementation and lipid metabolism in middle-aged mouse livers. To this end, we selected a mouse deficient in fatty acid elongase 5 (Elovl5), with impaired PUFA bioconversion and another mouse deficient in endonuclease G (EndoG), which stays protected in aging due to a lack of endonuclease activity and attenuation of mitochondrial DNA breakdown. My first objective was to identify said relationship in middle-aged livers of Elovl5 and EndoG heterozygous mice by feeding glutamine or alanine, while keeping C57/Bl6 mice (wild type, WT) as controls (Chapter 2). We show that glutamine supplementation does not affect gene expressions of glutamine-related metabolic (glutaminase 2, glutamine synthase, and glutamine dehydrogenase), or PUFA bioconversion (fatty acid desaturase 1/2 and Elovl5), enzymes in WT, and they were decreased in EndoG+/- mice livers. In contrast, glutamine supplemented Elovl5+/- mice livers showed upregulated gene expressions for both. For my second objective in Chapter 3, we determined the impact of glutamine or alanine on inflammation and mitochondrial biogenesis markers in the same livers. Glutamine-fed WT mice demonstrated no change while EndoG+/- showed a decrease in gene expressions of pro-inflammatory or mitochondrial biogenesis markers. Elovl5+/- mice on the other hand, demonstrated increased gene expression both inflammation and mitochondrial biogenesis markers. Given the fact that Elovl5+/- livers had increased levels of lipogenic as well as inflammatory and mitochondrial biogenesis genes with glutamine, such results indicate that glutamine supplementation is probably harmful for the liver at an advanced age with existing metabolic defects but might be neutral or beneficial in healthier phenotypes. Overall, these results contribute to understanding the relationships between glutamine, lipid metabolism, and aging in middle-aged mouse livers. iii Lay Summary Aging is a natural, long, steady process that happens from a lifetime of biological stressors. Low amino acid levels and improper fat metabolism play a role. Studies have shown that the amino acid glutamine grows scarce during the aging process, which makes it a great therapeutic target for fighting against ARDs. Other research looking into omega-3 and -6 fats has found a key metabolic enzyme, elongase 5 (Elovl5), that can cause fatty liver disease if defective. Lastly, proper mitochondrial (cell organelle) functioning is needed to carry-out energy production and fat metabolism. Endonuclease G (EndoG) can cause cell death. If it is removed, it can help mitochondria and overall cell function during aging. I proposed that when glutamine is supplemented in the diet for middle-aged individuals, it can lessen the severity of damage caused by defective elongase 5 or impaired mitochondria. My study looked at middle-aged livers in normal, Elovl5 (poor fat metabolism), and EndoG (better cell health) mice. We found that, when given glutamine, no gene level changes are noticed in metabolic enzymes related to either fat or glutamine itself in normal mice. However, in the healthier EndoG mice, the enzymes were decreased, and in the unhealthy Elovl5 mice, the enzymes were increased. Next, we tried to understand inflammation and cell health status in glutamine-fed middle-aged livers. Like before, glutamine did not change anything in normal mice but either reduced such indicators in EndoG mice or increased them in Elovl5 mice. Therefore, we show that the benefits of glutamine supplementation depend on health status. If the cell is metabolically unhealthy from the beginning, glutamine feeding might not help them and cause more harm. Overall, these results contribute to understanding the relationship between amino acid and fat metabolism and their role in the aging process. iv Preface I am largely responsible for the generation of all experimental data; however, Sandy Davidson-Hunt assisted with animal feeding and euthanasia. Hansika Dadlani, M.Sc., helped with IPGTT administration. Sandy Davidson-Hunt also kindly collaborated on gas chromatography experiments. All animal experiments completed in this thesis were reviewed and approved by the UBC Animal Care Committee (certificate #A13-0305). v Table of Contents Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... iii Preface ........................................................................................................................................... iv Table of Contents .......................................................................................................................... v List of Tables ................................................................................................................................ vi List of Figures .............................................................................................................................. vii List of Abbreviations ................................................................................................................. viii Acknowledgements ....................................................................................................................... x Dedications.................................................................................................................................... xi Chapter 1: Introduction ............................................................................................................... 1 1.1 Overall Problem .............................................................................................................. 1 1.2 Aging............................................................................................................................... 1 1.3 Glutamine ........................................................................................................................ 9 1.4 Rationale ....................................................................................................................... 17 1.5 Hypothesis and Objectives ............................................................................................ 17 Chapter 2: Impact of Glutamine on Lipid Metabolism in Middle-Aged Mouse Livers ...... 18 2.1 Rationale ....................................................................................................................... 18 2.2 Aim and Novelty of Research ....................................................................................... 18 2.3 Elovl5 Transgenic Mouse Model and Lipid Metabolism ............................................. 19 2.4 EndoG Transgenic Mouse Model and Lipid Metabolism ............................................. 19 2.5 Background ................................................................................................................... 20 2.6 Materials and Methods .................................................................................................. 23 2.7 Results ........................................................................................................................... 32 2.8 Discussion ..................................................................................................................... 40 Chapter 3: Impact of Glutamine on Mitochondrial and Immune Markers in Middle-Aged Mouse Livers ............................................................................................................................... 44 3.1 Rationale ....................................................................................................................... 44 3.2 PUFAs, Mitochondria, and Inflammation .................................................................... 44 3.3 EndoG Transgenic Model, Mitochondria, and Inflammation ....................................... 44 3.4 Background ................................................................................................................... 45 3.5 Materials and Methods .................................................................................................. 46 3.6 Results ........................................................................................................................... 47 3.7 Discussion ..................................................................................................................... 53 Chapter 4: Conclusion ................................................................................................................ 56 4.1 Limitations of Thesis Work .......................................................................................... 56 4.2 Conclusion .................................................................................................................... 56 4.3 Significance................................................................................................................... 57 4.4 Future Directions .......................................................................................................... 58 Bibliography ................................................................................................................................ 59 vi List of Tables Table 2.1. Detailed Composition of Basal Diet ............................................................................ 24 Table 2.2. Macronutrient Composition of Experimental Diets ..................................................... 25 Table 2.3. Primer Sequences and Efficiencies .............................................................................. 27 Table 2.4. RIPA Buffer Recipe ..................................................................................................... 29 Table 2.5. SDB Buffer Recipe ...................................................................................................... 29 Table 2.6. Ponceau Stain Recipe................................................................................................... 30 Table 2.7. Primary Antibody Solution .......................................................................................... 30 Table 2.8. Secondary Antibody Solution ...................................................................................... 30 Table 2.9. Stripping Buffer Recipe ............................................................................................... 31 Table 2.10. Primary Antibodies used ............................................................................................ 31 Table 2.11. Secondary Antibodies used ........................................................................................ 31 Table 2.12. Relative proportions of certain FAs within WT and KO liver tissues, obtained by gas chromatography .................................................................................................................... 37 vii List of Figures Figure 1. Lipid metabolism during aging ....................................................................................... 4 Figure 2. Protein metabolism in the elderly .................................................................................... 5 Figure 3. The aging liver ................................................................................................................. 8 Figure 4. Whole-body glutamine use ............................................................................................ 11 Figure 5. Glutamine and glutamate metabolism in the cell .......................................................... 15 Figure 6. Polyunsaturated fatty acid (n-3 and n-6) bioconversion pathways ............................... 21 Figure 7. Relative proportions of fatty acids present in both glutamine and alanine diets ........... 32 Figure 8. Change in body weight across glutamine and alanine diets for WT, Elovl5+/-, and EndoG+/-............................................................................................................................... 33 Figure 9. Blood glucose levels of WT and KO mice for glutamine and alanine diets over time . 34 Figure 10. Elovl5+/- glutamine-fed mice express significantly greater PUFA bioconversion enzymes in liver tissue .......................................................................................................... 36 Figure 11. Glutamine increases SREBP1 gene levels in WT, Elovl5+/-, and EndoG+/- mice .... 38 Figure 12. Glutamine does not alter pACC/ACC protein levels in WT, Elovl5+/-, or EndoG+/- mice ....................................................................................................................................... 39 Figure 13. Triglyceride differences across diets in WT, and Elovl5 and EndoG KO livers ........ 40 Figure 14. Glutamine increases metabolic genes in Elovl5+/- mice while decreasing expression in EndoG+/- .......................................................................................................................... 48 Figure 15. Elovl5+/- and EndoG+/- glutamine-fed mice express increased GLS2 protein levels compared to WT ................................................................................................................... 49 Figure 16. EndoG+/- mice express decreased GDH protein levels. ............................................. 50 Figure 17. Elovl5+/- and EndoG+/- mouse livers express decreased ASCT2 protein levels ....... 50 Figure 19. Elovl5+/- mice express increased mitochondrial biogenesis and copy number genes 52 viii List of Abbreviations ACC Acetyl-CoA Carboxylase ALA Alpha Linolenic Acid ANOVA Analysis of Variance ARA Arachidonic Acid ARDs Age-Related Diseases ASCT2 Alanine Serine Cysteine Transporter 2 AVG Average AUC Area Under the Curve BSA Bovine Serum Albumin cDNA Complimentary deoxyribonucleic acid DGLA Dihomo-Gamma-Linolenic Acid DHA Docosahexaenoic Acid ELOVL2 Fatty Acid Elongase 2 ELOVL5 Fatty Acid Elongase 5 ELOVL6 Fatty Acid Elongase 6 ENDOG Endonuclease G EPA Eicosapentaenoic Acid FA Fatty Acid FAS Fatty Acid Synthase FADS1 Fatty Acid Desaturase 1 FADS2 Fatty Acid Desaturase 2 FAO Fatty Acid Oxidation GC Gas Chromatography GDH Glutamate Dehydrogenase GLA Gamma-Linolenic Acid GLN Glutamine GLS Glutaminase GS Glutamine Synthetase GSH Glutathione H2O Water ix IL1B Interleukin-1 Beta IL6 Interleukin-6 IPGTT Intraperitoneal Glucose Tolerance Test KO Knockout LA Linoleic Acid MCP1 Monocyte Chemoattractant Protein-1 mtDNA Mitochondrial DNA NAFLD Non-alcoholic Fatty Liver Disease ND5 NADH: ubiquinone oxidoreductase core subunit 5 n-3 Omega-3 n-6 Omega-6 OA Oleic Acid PA Palmitic Acid PCM Protein-Calorie Malnutrition PGC1α Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-α pACC Phospho-ACC PUFAs Polyunsaturated Fatty Acids qPCR Quantitative Polymerase Chain Reaction SA Stearic Acid SASP Senescence-Associated Secretory Phenotype SD Standard Deviation SRE Sterol Regulatory Element SREBP1 Sterol Regulatory Element Binding Protein-1 RIPA Radio-Immunoprecipitation Assay ROS Reactive Oxygen Species TCA Tricarboxylic Acid Cycle TG Triglyceride TNFα Tumor Necrosis Factor-α WT Wild type x Acknowledgements I would like to express my sincerest gratitude to my supervisor, Dr. Sanjoy Ghosh. I am forever appreciative for the opportunity you have given me to continue my education and to be a part of your incredible research. As a student in your lab, I have learned not only about science, but also a lot about myself. I am grateful for your unwavering support, encouraging pep talks, and endless wisdom. Thank you for always seeing the best in me and believing in my abilities, even when I didn’t. I am a better scientist and person because of your guidance and dedication to teaching. Thank you to my colleague Sandy, for positively working through a master’s degree with me. Your intelligence, helpfulness, and joyful presence in the lab, during the best and worst days, have helped me complete my research and graduate studies. I am also thankful to my loving, caring, and patient partner, Bobby, without whom I would not have made it this far. Thank you for being my greatest source of motivation and for being so steadfast and selflessly supportive during the past couple of years. Lastly, thank you to my dear friend, Gaurav, for being the best chef and always making sure I was fed. Thank you for your encouragement and the unconditional friendship that has been a great light during my graduate program. xi Dedications For my mom and dad. 1 Chapter 1: Introduction 1.1 Overall Problem Aging is a risk factor for many pathologies that limit lifespan, and it results from the dysregulated continuation of otherwise normal developmental processes and cellular programs.1 By 2030 approximately 16% of the world population will be aged 60 or older, and by 2050 it is expected to double.1,2 Within Canada alone, all provinces will more than double their number of individuals aged 75 and older.3 The overall senior population is estimated to grow by 68% within the next 20 years.3 The rapidly aging population poses both an individual and societal threat. An increase in aged individuals can correspondingly result in a greater number of age-related diseases (ARDs) and cause personal health concerns as well as overload healthcare systems. It is critical that societies respond to how we will provide the care needed for such patients. Until recently, medicinal approaches to ARDs have targeted one illness at a time; except to combat unhealthy aging, they must be examined holistically, prospectively, and longitudinally. 1.2 Aging Aging is a time-dependent process responsible for decreasing overall survival by reducing the physiological ability to self-repair, regulate, and adapt to the environment.4 It involves many complex mechanisms occurring at multiple levels within an organism that ultimately end in an inevitable and irreversible death. To-date, many theories have been presented regarding the natural biological mechanisms for aging, such as the free radical and immunological theories of aging.4 Some scientists believe a different perspective, where according to the ancient Roman phrase “senectus ipsa est morbus”, old age is a disease itself.1,5 There is currently no consensus within the scientific community on the basis for aging, however there is a common goal to promote successful aging and extend lifespan. To examine this, experts have identified seven mechanistic pillars shared by aging and ARDs: adaptation to stress, stem cell exhaustion, macromolecular damage, epigenetic modifications, loss of proteostasis, metabolism derangement, and inflammation.1 2 1.2.1 Middle versus Old Age Middle and old age life stages can be interpreted according to 1) definitions of aging versus senescence and 2) metabolic biomarker analysis. Aging is inferred to be the progressive deterioration of bodily functions, lending itself to multiple-organ system dysfunction over time. Senescence beings during development and continues throughout one’s lifespan. It is a forceful mechanism against cellular damage; it is responsible for inducing a stable cell cycle arrest and initiating phenotypic and metabolic alterations in damaged cells.6 Accumulated physiological insults during aging cause the deleterious effects of senescent cells to outweigh its protective role.6 Biomarkers of aging are used to predict health and life span and categorize stages of life.7 Metabolic biomarkers gauge ratios of molecular and phenotypic senescent cell types and age-related cellular abnormalities. Middle aged, 37-48 years old, precedes the onset of old age.8 Middle-aged individuals begin to exhibit changes in metabolic biomarkers: inflammation (IL-6 and TNFα), mitochondrial dysfunction (oxidative stress and ROS), lipid dysregulation (triglycerides), loss of proteostasis (frailty indexes), and cellular senescence.8,9 Older aged individuals, 56 onwards, experience high levels of metabolic abnormalities and chronic ailments.8 Elderly patients are often categorized according to irreversible changes noted in metabolic biomarkers and geriatric syndromes.2,10 1.2.2 Human versus Mouse Aging Mice are an extensively used human-alternative model for studying aging. Although mice have markedly shorter lifespans than humans, they exhibit accelerated rates of maturation.8 From birth to 1 month old, mice mature 150 times faster than humans.8 Between 1-6 months mice mature 45 times faster and starting at 6 months they are maturing 25 times faster.8 The average human lifespan is estimated to be around 77.8 years, while a mouse lifespan is between 2-7 years depending on the strain.8 Mature adults are categorized as having passed the developmental phase but not yet having reached senescence.6,8 A mouse is considered a mature adult between 3-6 months of age, which corresponds to 20-30 years old in humans.8 Middle age begins to occur when senescent changes become detectable in various biomarkers of aging. Middle-aged mice are between 10-14 months, while middle-aged humans are 37-48 years.8 In old age, senescent changes become profoundly notable and observable for most biomarkers of aging. Mice reach old age at 18 months until the upper limit of 24 months; certain strains can be affected by strain- 3 specific diseases, which impact the rates of biomarker changes.8 Humans reach old age from 56 years and onwards.8 1.2.3 Aging and Macronutrient Metabolism—an Overview i Carbohydrate Metabolism Aging results in an overall reduction of carbohydrate metabolism.11 Glucose intolerance particularly increases with age, exacerbating a plethora of age-related conditions related to oxidative, DNA, and other damage.11 Glucose disposal is jointly affected by both insulin- and non-insulin mediated pathways. Several studies have found non-insulin mediated peripheral glucose uptake, i.e., in muscle tissues, to be diminished under basal conditions in aging individuals.12,13 Such effects lead to the development of ARDs, notably diabetes. Currently, one-third of individuals 65 and older are diagnosed with diabetes, and it is projected to increase in prevalence two-fold for 65-74 years of age and four-fold for greater than 75.14 Age-associated comorbidities, including sarcopenia and inactivity, lead to impairment in glucose tolerance, which further aggravates frailty and geriatric syndromes.11 As insulin sensitivity and secretion turns dysfunctional during aging, insulin-mediated glucose uptake becomes impaired.12 The onset of which is provoked by 1) oxidative stress as result of mitochondrial dysfunction, 2) systemic inflammation, and 3) sarcopenia coupled with ectopic fat distribution.11 ii Lipid Metabolism Lipids are known for the outstanding roles they play in the storage and mobilization of energy and in the aging process. Aged individuals experience a decreased utilization of fat coupled with its increased accumulation in organs.15 In general, increase in fatty acid (FA) uptake and de novo lipogenesis amalgamate to promote the development of conditions such as fatty liver.15 At the same time, autophagic processes begin to decline along with mitochondrial integrity and quantity (Figure 2).15 The ultimate result is a reduction in fatty acid oxidation (FAO). FAO predominantly occurs in the mitochondria and is important for converting fatty acids to acetyl-CoA to be used as an energy precursor in metabolically active organs.15 Research has shown an upregulation of de novo lipogenesis enzymes, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), and transcriptional regulator, sterol regulatory element-binding protein1 (SREBP1) during aging.15,16 Researchers have observed quantitative differences in lipocyte activity in old versus young 4 adipose tissue: increases in lipid peroxidation coupled with decreases in lipid synthesis, antioxidants, and glucose metabolism6. Lipid peroxidation is a consequence of free radical reactions breaking down membrane polyunsaturated fatty acids. Such damage potently regulates lifespan, because lipid composition determines the resistance of cell membranes to oxidative damage.15,17 Ectopic fat distribution in older individuals in organs such as the liver, advances disease progression.18 Aging shifts the deposition of body fats from subcutaneous locations to dangerously visceral ones, and the capacity of respiring tissues to oxidize fats, weakens.15 The age-related reduction capacity of fat cells to properly store lipids in conjunction with negative effects of visceral fat accumulation lead to lipotoxic effects and metabolic complications.19,20 An ultimate product of such dysregulated lipid metabolism is low-grade chronic inflammation attributing to deteriorating liver conditions. With aging, adipose tissue itself has been implicated as having a pro-inflammatory phenotype along with increased cellular senescence, mitochondrial dysfunction, and metabolic declines.21 This promotes systemic pathophysiological changes that render age-related diseases. It is important to develop interventions to increase the oxidative or quantitative capacity of metabolically active tissue, which may occur as a result of glutamine supplementation.19,22–24 Figure 1. Lipid metabolism during aging. Image from Chung, 2021. 5 iii Protein Metabolism The aging process has a significant impact on protein metabolism and the gradual dysregulation of protein homeostasis (Figure 1).25 Proteostasis involves the tight and dynamic regulation of protein synthesis, trafficking, and degradation. The rates of protein renewal and repair concurrently decline with age.26 As such, aging results in an overall net loss of protein, which correlates to the progressive decline in skeletal mass and function known as sarcopenia.26 This musculoskeletal disorder affects 5% of adults aged 65 and about 50% of people aged 80 and older.26 It causes a decline in whole-body protein use and an increase in frailty and mortality.26 Current literature cites suboptimal nutrition and decreases in physical activity in the elderly as contributing factors to such issues.27,28 The outcomes of such dysregulated protein metabolism during aging lead to many of the age-related diseases that we see today. Among several strategies to mitigate this, elderly individuals must ingest increased amounts of protein compared to younger individuals to overcome anabolic resistance seen during aging.29 Figure 2. Protein metabolism in the elderly. Image from Chung, 2021. 1.2.4 Molecular Events in Aging i Cellular Senescence The accumulation of senescent cells is thought to contribute to the aged phenotype.7 Cellular senescence occurs as a results of various endogenous and exogenous cell mechanisms that 6 induce apoptosis.9 Senescent cell markers include DNA-damage responses, telomere shortening, oxidative stress, mitochondrial dysfunction, and senescence-associated secretory phenotype (SASP).7,6 Several immune proteins are associated with SASP: interleukin-6 (IL6), monocyte chemoattractant protein-1 (MCP1), and tumor necrosis factor-α (TNFα).7,21 Structural and functional changes in an aging cell produce aforementioned pro-inflammatory cytokines and SASP proteases, which contribute to ARDs.9 ii DNA and Chromosomes Macromolecular DNA damage demonstrates a causal relationship to aging.30 Genomic stability is crucial for cellular and organismal health, however it is largely prone to damage from endogenous and exogenous factors.7,9,30 Although all organisms maintain highly conserved DNA repair mechanisms, they cannot manage the accumulated insults over time.7,9 Increased DNA mutations eventually dysregulate gene expression and protein products.9 Additionally, telomere attrition during aging conduces cellular senescence. Telomeres are end-chain DNA sequences marked to protect from chromosomal damage. As they replicate throughout an organisms lifespan, they become increasingly shorter and therefore less protective.9 Finally, markers of epigenetic alterations, which include DNA methylation and histone modification, are tightly linked to ARDs. Recent evidence has correlated increased DNA methylation to premature mortality.9 iii Mitochondrial Health Mitochondria are key players in whole-body metabolism. They are subcellular organelles responsible for the production of energy via ATP synthesis.31 The role of mitochondria during the aging process is complicated and profound. The mitochondrial theory of aging postulates that the accumulation of reactive oxygen species (ROS) in a mitochondrion over time causes damage and renders it dysfunctional, however this has yet to be proven.31 Unarguably though, are the changes exhibited within aging mitochondria. Mitochondrial dysfunction—decreased oxidative capacity and increased oxidative damage—encompasses several molecular changes during aging.31 First, mitochondrial DNA (mtDNA) accumulates mutations 15-fold greater than nuclear DNA; when thresholds are reached, oxidative phosphorylation can become compromised as a result of faulty DNA replication and repair.31 Second, as ROS are produced and accumulated by 7 the mitochondrial electron transport chain, antioxidant defense mechanisms may become impaired and lead to cellular damage.31 Third, physical changes begin to take place, which affect structure and function. Advanced age shows declines in mitochondrial density in skeletal muscle and overall biogenesis.31 1.2.5 The Aging Liver The liver is a vital organ for the mediation of amino acid, lipid, and carbohydrate metabolism. Such processes are tightly regulated, however under various pathological conditions, such as seen in age-related non-alcoholic fatty liver disease (NAFLD), the liver’s ability to effectively carry-out its functions begins to deteriorate (Figure 3). The aging liver is similarly plagued with maladaptations and an inability to remedy failing structural and functional processes. Researchers have observed a 20-40% reduction in volume, a 35-50% decline in blood flow, and elevated hepatocyte numbers.35-38 Of note, there is a diminished number in overall liver mitochondrial count but an increase in individual mitochondrion volume. Biochemically, age-associated changes in the liver can be highlighted by virtue of faulty mitochondria, autophagy, and fatty acid oxidation.32,33 In an aging liver, increased inflammatory changes, decreased hepatic free-radical scavenging systems, and defective autophagy lead to detrimental effects on regenerative responses. 35-38 Additionally, aging livers exhibit increased fibrosis with a reduced percentage of fat on the liver. The aged liver experiences decreased ability to metabolize drugs, thus leading to drug-induced liver injury. As elderly patients may have multiple risk factors including hyper- lipidemia and glycemia, quality of life impairments, and an inefficiency to metabolize drugs, the simple use of glutamine supplementation to target multiple stressors may prove beneficial as a treatment.24,29,34 8 Figure 3. The aging liver. Image adapted from Lozada-Delgado et al., 2020. 1.2.6 Liver Metabolism—an Overview i Aging Liver and Carbohydrates The aging process disrupts proper liver functioning and thus, its carbohydrate metabolism. Post food absorption, blood glucose levels are regulated via hepatic glucose production and uptake by peripheral tissues.35 Hepatocytes take up plasma glucose via GLUT2 transporters. Research has demonstrated decreased gene expression of glucose transporters in senescent hepatocytes.36 Glycolysis and gluconeogenesis are regulated by means of enzymes. Glucose is subsequently converted to glucose-6-phosphate, for glycogen synthesis, by glucokinase (GCK). As mentioned, the aging process results in changes in DNA methylation patterns. Epigenetic modification of GCK has been linked to its decreased hepatic expression, thus promoting susceptibility to insulin resistance and diabetes.37 Opposingly, in a fasted state hepatic glucose production is found to be rapidly stunted in approximately 90% of aged individuals, specifically with hyperinsulinemia.35 Such effects can be attributed to slowed suppression of endogenous insulin release in the 9 elderly.35 The consequences of dysregulated carbohydrate metabolism, such as hyperglycemia are linked to the progression of fatty liver ARD.11,38 ii Aging Liver, Proteins, and Lipids As previously identified, derangement in protein metabolism increases during the aging process. Specifically within the liver, rates of protein synthesis and degradation decline.39 Disordered amino acid metabolism is often encountered in liver disease patients suffering from protein-calorie malnutrition (PCM).40 Elderly individuals frequently manifest with PCM, as they have suboptimal protein intake.28,27 In the senile liver, denser cell body compartments, such as lysosomes and lipofuscins- responsible for the degradation of damaged and denatured proteins, have been also found to decline.41 The aged liver experiences increased protein misfolding and trafficking along with a decrease in regeneration and repair capacities, overall contributing to ARDs.39–41 Dysregulated lipid metabolism can be observed in an aging liver. The onset of ARDs, such as NAFLD, can be attributed to a combination of 1) impaired lipid delivery and uptake, 2) increased de novo lipogenesis, and 3) changes in FAO. Free fatty acids (FFAs) are derived from the diet or adipose tissue.42 FFAs are taken up and used by the liver for energy, membrane synthesis, or triglyceride (TG) storage. In non-pathological conditions, adipose FFAs comprise 75% of liver lipid deposition.42 However, during aging and NAFLD FFAs only contribute 60% to liver TGs while 15% is accumulated from the diet.42 FAs can be synthesized from non-lipid precursors via de novo lipogenesis. In healthy livers, de novo lipogenesis accounts for approximately 5-10% of TG accumulation, whereas it is increased to greater than 25% in NAFLD patients.42 Newly synthesized FFAs are stored as TGs in the liver or undergo FAO to generate energy. Hepatocellular mitochondrial FAO is regulated by a multitude of enzymes, including CPT-1 and ACC, which are reduced in the mitochondrial membrane during aging and result in impaired FAO.42 1.3 Glutamine Glutamine is the most abundant and versatile amino acid in the body. It is a 5-carbon amino acid with the following elemental composition: 41.09% carbon, 32.84% oxygen, 19.17% nitrogen and 10 6.90% hydrogen.19 It plays vital metabolic roles spanning multiple organ systems (Figure 4). Glutamine is responsible for regulating glucose and fatty acid metabolism, nitrogen balance, and maintaining physiological homeostasis for multiple organs and cell types. It serves as a fuel source for actively dividing enterocytes, fibroblasts, and lymphocytes, and for the physiological health of hepatocytes and neurons.19-20 Glutamine transports nitrogen from sites of synthesis- liver, skeletal muscle, and lungs, and acts as an ammonia shuttle when moving to sites of utilization- kidney, intestine, neuron and immune cells.43-44 Glutamine’s role in nitrogen metabolism is vital for regulating acid-base balance and renal function. It is also a major precursor for the synthesis of nucleotides needed for cell proliferation. Additionally, glutamine plays a role in antioxidant defense systems. It is converted to glutamate, a direct precursor for the antioxidant glutathione, which is abundant in cells. Glutamine is predominantly synthesized by skeletal muscle and most substantially utilized by the intestines. It is present in high concentrations in the plasma at 0.5-1.0mM and in the skeletal muscles at 5-20mM.45 Glutamine is deemed conditionally essential, for its endogenous synthesis is insufficient to meet bodily demands during conditions of critical illness or catabolic stress.19-44 For instance, plasma glutamine levels become depleted during aging.29 Lower plasma glutamine concentrations relate to reduced amino acid stores in muscles. The sarcopenic loss in muscle mass that characterizes the catabolic state amid aging causes a depression in glutamine synthesis in skeletal muscle.29 The depletion of this vital amino acid can lead to organ dysfunction, ARDs, and increased morbidity and mortality. 11 Figure 4. Whole-body glutamine use. Image from Cruzat et al., 2018. 1.3.1 Glutamine Metabolism—an Overview i Aging, Proteins, and Glutamine Muscle protein catabolism is a direct producer of glutamine, as well as other branched-chain amino acids.19 The carbon skeletons of such amino acids are utilized for de novo glutamine synthesis. In the post-absorptive state, more than 50% of glutamine synthesis in skeletal muscles occurs through the uptake of glutamate from the bloodstream.19 Glutamine and alanine make up approximately 48% and 32% of the amino acids released by skeletal muscles.19 Glutamine-alanine exchange rates in tissues occur in excess of their supply, thus there is a constant need for de novo glutamine and alanine synthesis in skeletal muscles. Glutamine synthesis in skeletal muscles occurs at a rate of 50mmol/h, which is greater than all other amino acids.19 Therefore, glutamine and alanine production must occur from the interconversion of other amino acids. As overall protein levels decrease during aging, alanine glutamine synthesis and utilization decline 12 as well.29 To combat this, amino acid regulation can be influenced by supplementation, nutrition, and exercise.26,27,46 ii Aging, Lipids, and Glutamine Glutamine is a lipogenic precursor. As an indirect fuel source for the TCA, it stimulates production of citrate, which can further promote lipid synthesis by metabolizing acetyl-CoA via acetyl-CoA carboxylase (ACC) or fatty acid synthase (FAS) enzymatic action (Figure 3). Glutamine also has regulatory effects on sterol regulatory element-binding proteins (SREBPs). SREBPs constitute a family of transcription factors that control cholesterol and fatty acid metabolism22. SREBP membrane proteins are synthesized on the endoplasmic reticulum and their N-terminal halves work as transcription factors in the nucleus.22 Three SREBP isoforms are involved in lipid homeostasis. SREBP1c is the predominant isoform in human and mouse tissues linked to FA synthesis. It is highly expressed in the liver, skeletal muscle, and white adipose. SREBP2 is specific to cholesterol synthesis. SREBP1a is greatly expressed in most cell lines and tissues, and it is implicated in the control of both lipid pathways.22 Glutamine is reported to stimulate SREBP processing and gene expression of their targets in human hepatoma cells.22 Overexpression of SREBP1a causes increases in ACC, FAS, and stearoyl-CoA desaturase-1 (SCD-1) target genes, which increases accumulation of cholesterol and TG.22 The aging process exhibits impaired lipid metabolism via changes in the aforementioned enzymes.15 Correlations between depleted glutamine stores, SREBP, and ARDs have been widely researched; glutamine supplementation may aid to promote SREBP functioning and its downstream products.22,29,47 1.3.2 Aging, Inflammation, and Glutamine Our immune system is an intricately conserved defense system utilized to maximize survival and as such, involves inflammatory responses. Inflammaging describes the upregulated inflammatory response seen with advanced aging and is the result of chronic low-grade inflammation over time.21 Researchers believe the root cause of inflammaging to be a cumulation of lifetime exposure to antigenic, immune-stimulating substances. Research has demonstrated that aged individuals exhibit increased levels of the proinflammatory cytokines: IL1, IL6, and TNF.21 Consequentially, the negative effects of such a prolonged pro-inflammatory state include greater susceptibility to ARDs (i.e., NAFLD) and gradual systemic failure. Susceptibility to 13 inflammaging can determine rate of aging. Genetic, environmental, and autophagic factors can all play a role in the upregulation of proinflammatory factors and the downregulation of a balanced anti-inflammatory response. Nutritional factors regulate the prevention, onset, and development of immune diseases.48 Immune cells have different nutrient requirements depending on their roles. Glucose and glutamine are the primary fuel supplies for lymphocytes, and both are metabolized through the tricarboxylic acid cycle (TCA). Immune cells use glutamine at higher rates during catabolic conditions: critical illness, sepsis, and aging.19 In such instances, glutamine becomes preferentially utilized at concentrations greater than or equal to glucose. Glutamine is a vital energy substrate for immune cell proliferation, tissue repair, and intracellular pathways related to pathogen recognition. Activation of B and T lymphocytes to induce an adaptive immune response requires high energy and increased nutrient metabolism. T cell activation causes a large increase in glutamine transport and enzymes needed for entry into the TCA.49,50 T cells depend on glutamine-specific stimulation of their receptors (CD3 and CD28). Glutamine depletion impairs T cell activation and proliferation, and neither glutamine precursors nor other amino acids can function as substitutes.50 High extracellular glutamine is needed for B cell growth via ligation of B-cell antigen receptors.51 B lymphocyte activation increases expression and affinity for glutamine transporter ASCT2.51 Macrophage and neutrophil innate immune cells depend on glutamine for pro-inflammatory cytokine synthesis and ROS driven antimicrobial activity.49 Glutamine stimulates the expression of lymphocyte cell surface markers (CD), as well as the production of immunomodulating cytokines: TNFα, IL-6, IL-10, and IFNγ. Research studies have found allergy patients to demonstrate decreased ASCT2 in B cells, IL-10 expression, and anti-inflammatory effects.48 Glutaminolysis—conversion of glutamine into glutamate, aspartate, and alanine—is able to maintain B cell IL-10 expression, thus presentencing as a potential therapeutic target for allergies.19,48 Aging induces immune system dysregulation that affects glutamine protein synthesis. Glutamine deficiency can trigger a low-level inflammatory process with decreased transcription of inflammatory regulators and cytokine protection.19 14 1.3.3 Glutamate, a Metabolite of Glutamine Glutamine is broken down via glutaminase into glutamate and ammonia. Glutamate is a non-essential amino acid that can be produced in adequate quantities in vivo. It is most metabolized by the brain, intestines, pancreas, and liver. Glutamate is widely utilized, and it has important roles in protein structure and function.52 It is incorporated into proteins during synthesis or post-synthesis as polyglutamyl tails.53 It is needed for hepatic nitrogen metabolism, production of glutathione antioxidant, and it is a major excitatory neurotransmitter in the central nervous system. As a result of muscle wasting and other age-related organ deficits, aging results in a decline in glutamate levels. Reduced glutamate in the motor cortex is related to brain shrinkage during aging.54 Experiments in naked mole rats and C. elegans have shown correlations between decreased lifespan and decreased plasma glutamate and transporter levels.53 1.3.4 Glutamine and Glutamate metabolism Glutamine is transported through cells via SLC class plasma membrane transporters, namely alanine, serine, cysteine transporter 2 (ASCT2). Once inside, intracellular glutamine metabolism is regulated via two principle enzymes: glutamine synthetase (GS) and glutaminase (GLS).55 GS synthesizes glutamine from glutamate and ammonia, while GLS performs the reverse reaction and hydrolyzes glutamine to glutamate (Figure 3).19,55 GS is mainly found in the cytosol and utilizes cytoplasmic nucleotides and proteins.19 Active GLS is primarily found in the mitochondria and catalyzes glutamine conversion for entry into the TCA.19,55 Glutaminase has two isoforms. GLS1, “kidney-type”, is most concentrated in the kidneys and mitochondria. GLS2, “liver-type”, is predominantly expressed in the liver. Highly proliferative replicating cells are avid glutamine consumers, therefore they contain greater amounts of GLS than GS.29 Organs such as, skeletal muscle and lungs, which synthesize and release glutamine into the bloodstream, contain greater amounts of GS.29 The liver alternates from glutamine production to utilization depending on nutritional conditions, therefore it contains both enzymes. Glutamate serves as an anaplerotic precursor and energy metabolite for the TCA. Glutamate is irreversibly catalyzed to α-ketoglutarate and ammonia via glutamate dehydrogenase (GDH) in the mitochondria.56 Glutamate is also responsible for the biosynthesis of glutathione antioxidant through further biochemical reactions.56 15 Figure 5. Glutamine and glutamate metabolism in the cell. Adapted from Choi & Park, 2018. 1.3.5 Glutamine Supplementation in Humans Acute stress, aging, and other pathological conditions can result in a drop in 30% plasma and 50% intracellular glutamine concentrations and overwhelm the body’s capacity for de novo glutamine synthesis.29 Since glutamine plays fundamental roles in the maintenance of various cellular processes, medical professionals have turned to glutamine administration for recovery. Over the past decades, many clinical trials have illustrated the benefits of glutamine supplementation in patients experiencing cancer, surgical stress, and critical illnesses. Glutamine can be administered orally, enterally, or parenterally.57 Scientists have found that administration of high doses of parenteral glutamine were beneficial to critically ill patients, albeit enteral administration is preferred. Comprehensive reviews determine that glutamine supplementation is associated with a reduction in mortality, low complication rates post-surgery, and shorter hospitals stays during recovery.24-58 The optimal dose of glutamine to be given to patients has yet to be determined, though many studies suggest that supplementation up to 0.5g per kg per day is safe.24 16 In terms of safety, Galera et al. found no significant negative effects of oral glutamine use in middle-aged and elderly individuals.59 Glutamine supplementation can greatly enhance the body’s antioxidant capabilities by regulating glutathione (GSH) content. A study by Song et al. demonstrated anti-aging effects following regulation of glutamine-glutamate metabolism in D-galactose induced aging rats. The chemical components of Scutellaria baicalensis Georgi flowers, rich in flavonoids, were analyzed in aging rats to determine the basis for its antioxidant and anti-inflammatory protective properties. Their findings indicated significant increases in glutamine content, glutamate-cysteine ligase, and GSH in the liver.60 Glutamine-supplemented nutrition has demonstrated an ability to protect the liver and improve survival during hepatic injury through the preservation of GSH stores.61 Badole et al., have discovered a cardioprotective role of L-glutamine in ischemic heart disease; for example, in a streptozotocin-nicotinamide induced diabetic rat model, glutamine was found to exhibit beneficial modulation of antioxidant systems.62 Ballard et al., showed that supplying L-glutamine during lipid infusion, which is often required for patients receiving nutritional support, reduced lipid peroxidation and change in blood glucose regulation, which would otherwise normally occur.63 Critically ill patients, particularly if elder, suffer from major loss in skeletal mass issuing a depletion of the glutamine pool.29,64 As a result, glutamine-supplemented feeding is required to increase survivability. Furthermore, glutamine supplementation has shown to preserve gastrointestinal tract integrity- protective of gut barrier functioning, acid-base balance, and promotion of the immune system23,55,65–72 Although there is a plethora of glutamine benefits, research has determined inconclusive and detrimental results of its use as well. Glutamine-induced hepatotoxicity is a widely noted point of concern.73 Case studies have reported liver failure in body-builders due to elevated plasma glutamine levels post powder consumption.73 Some studies promote the idea that the benefits of glutamine provision may be solely due to a correction in its deficiency.74 Unnecessary overconsumption may pose several risks. First, glutamine and other amino acids share cellular transporters; glutamine competition may hinder the transport of other amino acids to various tissues. In a study of healthy volunteers, infusion of a glutamine-tyrosine amino acid solution resulted in impaired methionine and phenylalanine uptake in limb tissues, which poses metabolic complications.75 Second, glutamine supplementation can result in alterations in its metabolism. 17 Namely, an accumulation of glutamine metabolites due to glutamine breakdown, and precursors due to the lack of endogenous glutamine synthesis.74 Acute liver failure and cirrhosis patients present increased release of blood and splanchnic glutamine-derived ammonia post oral glutamine intake, which may cause toxicity.76 The liver demonstrates specific alterations in the regulation of acid-base balance in cirrhotic patients by decreasing ureagenesis from peripheral hepatocytes coupled with enhanced glutamine release from perivenous hepatocytes, following glutamine administration.74 Lastly, glutamine can alter immune functioning; it is important in the proliferation and differentiation of immune cells.74,19 Several studies have demonstrated a dose-dependent effect of glutamine addition and increased spleen lymphocytes and interleukin-2 cytokines in culture medium.74 The adverse health effects of such immune changes are largely unknown. More concerningly is glutamine’s relationship to tumor growth. Cancer cells are avid consumers of glutamine. Hepatoma cells exhibit increased GLS, ASCT2, and tumor growth via intracellular glutamine signalling.77 Given that there are both beneficial and detrimental roles of glutamine, it is imperative we understand the conditions under which glutamine induces specific beneficial or detrimental responses. This conundrum precipitated the study as described below. 1.4 Rationale As the aging population increases exponentially, it is imperative to understand the relationship between aging and metabolism. While there are many studies on the underlying mechanisms of aging: dysfunctional mitochondrial and dysregulated protein and lipid metabolism, little is known about the interactions between them, especially with respect to glutamine. Further, results of glutamine supplementation are contradictory and do not provide an integrated understanding of glutamine and age-related metabolic derangement. There would be great public benefit in illuminating glutamine’s mechanisms of action in an aging liver model to better understand its effects as a therapeutic target in such cases. 1.5 Hypothesis and Objectives In concert with the theories of aging and dysregulated protein and lipid metabolism, I hypothesized that: Glutamine supplementation can mitigate the effects of dysregulated lipid metabolism and improve liver health in middle-old age populations. 18 To test this hypothesis, I proposed two objectives: First objective: Investigate the impact of glutamine on lipid metabolism in middle- aged livers Second objective: Investigate the impact of glutamine on inflammation and mitochondria in middle-aged livers Chapter 2 of this thesis uses in vivo techniques to focus on the first objective. Chapter 3 of this thesis uses similar in vivo techniques to concentrate on the second objective. Chapter 2: Impact of Glutamine on Lipid Metabolism in Middle-Aged Mouse Livers 2.1 Rationale Impaired elongase activity and depleted glutamine stores have both been implicated in aging.29,78 Little is known regarding fatty acid elongase 5 (Elovl5) activity during aging and its correlation to glutamine metabolism. Dysfunctional mitochondria can ultimately promote the progression of diseases and contribute to aging.31 Evidence of relationships between endonuclease G (EndoG) to glutamine metabolism during aging are scarce and similarly unknown. Thus, deficiencies in elongase activity can be used to illuminate glutamine mechanisms of action for a dysregulated, aged lipid profile. Glutamine may also be able to promote more efficient fatty acid metabolism in a healthier mitochondrial model. Management and rectification of energy metabolism is a possible target for age-related disease therapies.31 Its mechanisms of action and therapeutic effects require further investigation. 2.2 Aim and Novelty of Research In collaboration with current research implicating glutamine, Elovl5, and EndoG in fat metabolism, we aim to provide novel insight into the connectedness of their metabolic processes, specifically during aging. First, we hypothesize that glutamine supplementation can aid in PUFA 19 metabolism in an Elovl5 deficient model. Second, we hypothesize that glutamine supplementation can further improve lipid metabolism in the middle-aged liver. 2.3 Elovl5 Transgenic Mouse Model and Lipid Metabolism Functional elongase activity is required for proper PUFA metabolism. Studies have demonstrated that knockout Elovl5 mice display reduced PUFA synthesis corresponding with the development of fatty liver.79 An Elovl5+/- (fatty acid elongase 5 KO) mouse model was chosen to study the effects of glutamine supplementation on dysregulated lipid metabolism in middle-old age livers. Elongase exists in seven different isoforms.38 Elovl1, Elovl3, and Elovl6 elongate saturated and monounsaturated fatty acids.38 Of note, Elovl2 and Elovl5 are involved in the PUFA bioconversion pathways. Most studies related to elongase activity have been focused on adipose and liver tissues. Elovl5 has shown to break down hepatic triglycerides in obese C57/Bl6 mice.38 Additionally, obese patients have experienced improvements in fatty acid metabolism via increases in adipose elovl5 gene levels.80 These findings are indicative of the important role that Elovl5 plays in fatty acid catabolism. Ghosh et al. identified that Elovl5+/- mice decreased expression of antioxidant-associated genes in conjunction with causing fat accumulation.81,82 2.4 EndoG Transgenic Mouse Model and Lipid Metabolism The liver utilizes glucose and fatty acids as the main metabolic fuel sources for energy.83 Glucose is converted to fatty acids (de novo lipogenesis) which then undergo β-oxidation in the mitochondria.83 Alternatively, hepatocytes can obtain fatty acids for energy from the bloodstream post adipose tissue release or from food absorption.83 β-oxidation of long-chain fatty acids is the final step of energy production from FAs.84 Deficiencies in the mitochondria can disrupt homeostasis and impair such energy metabolic processes.85 Restricted β-oxidation activity can inhibit fatty acid transportation and utilization.86 Endonuclease G has been implicated in energy homeostasis of cells. Heat production occurs in brown adipocytes and relies on mitochondrial uncoupling protein-1 to initiate lipolysis.87 EndoG has been shown to be highly increased in brown adipose tissue, and EndoG KO mice have demonstrated increased expression of fatty acid metabolism genes such as CPT1b.88 Mice lacking EndoG have also exhibited decreased adiposity, with an overall reduction of white adipocyte size.88 Further, Pardo et al. showed that EndoG KO mice have improved glucose homeostasis.88 Such results indicate a 20 healthier overall metabolic profile. As healthy mitochondria are a requirement for proper energy production and fatty acid catabolism, an EndoG+/- (endonuclease G KO) mitochondrial mouse model was selected to determine the effects of glutamine supplementation in middle-old age mice. 2.5 Background 2.5.1 Polyunsaturated Fatty Acid Metabolism Fats are essential for maintaining a well-balanced diet, and they are needed for proper physical and intellectual development.89,90 Fatty acids are either synthesized de novo or ingested.91 Polyunsaturated fatty acids (PUFAs) are not produced endogenously and must therefore be taken through the diet. PUFAs include two series of fatty acids: omega-6 (n-6) and omega-3 (n-3).90 Once inside the body, n-6 and n-3 PUFAs are modified on the endoplasmic reticulum using key enzymes: desaturase and elongase.91 As mentioned, fat metabolism depends on cellular metabolic capacity. The localization and use of PUFA enzymes can be found in the mitochondria. Impediments in desaturase or elongase activities is disruptive of downstream generation of n-6 and n-3 PUFA products.92,79 We selected two specific genotypes, Elovl5+/- and EndoG+/-, which have impaired versus healthy mitochondrial function. 21 Figure 6. Polyunsaturated fatty acid (n-3 and n-6) bioconversion pathways. Adapted from Igarashi et al., 2007. 2.5.2 Desaturation Fatty acid desaturation removes two hydrogen atoms from fatty acids to form carbon-carbon double bonds. Desaturation is performed by FADS1 and FADS2 membrane-bound enzymes. FADS2 bioconversion takes place from 18:2n-6 to 18:3n-6 and 18:3n-3 to 18:4n-3.93 FADS1 desaturation occurs between 20:3n-6 to 20:4n-6 and 20:4n-3 to 20:5n-3 (Figure 4).93 FADS1 and FADS2 are located in clusters (FADS cluster) on human chromosome 11 with a head-to-head orientation; mouse homologues are on chromosome 19.91,94 FADS1 and FADS2 are widely expressed in tissues, and they are highest in the liver. FADS cluster polymorphisms have been associated with varying metabolic phenotypes during aging: alterations in fatty acid membrane compositions, changes in enzymatic activity and lipid accumulation in the liver.95 The single 22 nucleotide polymorphism (SNP), rs174537, has been associated with age-related changes in serum lipid long-chain PUFAs, changes in FADS1, and oxidative stress in middle-aged men.94,95 rs174537GG SNP increases ARA and FADS1 activity. Higher ARA concentrations can influence production of proinflammatory eicosanoids and elevate low-level systemic inflammation during aging. rs174537GG similarly increases low-density lipoprotein cholesterol and products of oxidative stress: oxidized low-density lipoproteins, in middle-aged men.94 FADS1 and FADS2 SNPs, rs147547 and re147576, influence levels of total cholesterol and triglycerides. Both of which are commonly measured metabolic biomarkers in aged or diseased patients.96 FADS crucially regulate biological functions, and desaturase activity can be affected by age-related inflammatory changes. Inhibition of FADS1 has been linked with systemic inflammatory responses. FADS2 deficient mice models result in brain dysfunctions, infertility, and intestinal diseases.97 2.5.3 Elongation Elongation of fatty acids requires the addition of two carbons into the carbon chain. The two enzymes required for PUFA metabolism, Elovl2 and Elovl5, are located on human chromosome 6. Elovl5 enzyme is responsible for bioconverting C18 to C20 and C20 to C22. Elovl2 bioconverts C20 to C22 and C22 to C24 (Figure 4).93 It does so via the addition of acetyl-CoA generated from the mitochondria.98 Due to metabolic-specific requirements, expression of elongases is time and tissue dependent. The liver has high gene expression for Elovl2 and Elovl5, while the kidney and lungs only present with high amounts of Elovl5.91 Cardiomyocytes express Elovl5, however Elovl2 expression remains undetectable. Research on elongase polymorphisms is currently lacking, however it has been shown that changes in elongase activity profoundly impact lipid metabolism during aging. Epigenetic alterations in Elovl2 are strongly correlated with age prediction. Impaired Elovl2 activity accelerates the aging phenotype by increasing endoplasmic reticulum stress, mitochondrial dysfunction, and disrupting lipid synthesis.82 Increased hepatic expression of Elovl5 correlates with decreased liver triglyceride and blood plasma levels through increased triglyceride catabolism.99 Lipogenesis functions in contrast to triglyceride catabolism. It is regulated by sterol regulatory element-binding protein 1 (SREBP1) nucleus transcription factor. Sterol regulatory elements (SREs) are SREBP1 transcription binding sites. Recently, two conserved SREs were identified as direct targets for 23 Elovl5. SRE human targets were located in exon 1 and -10kb upstream of the transcription start site in the genome.100 SREBP1 activates and increases Elovl5 expression, thus regulating de novo lipogenesis.100 Other research has demonstrated that increased Elovl5 gene expression silences SREBP1 via feedback-regulated PUFA products: ARA, DHA, and EPA. Importantly, a deletion or defect of Elovl5 results in deficiencies in C18 PUFA elongation. Elovl5 deficient mice have been shown to upregulate SREBP1 expression, ultimately leading to hepatic steatosis.79 2.5.4 Endonuclease G Endonuclease G (endoG), encoded by mtDNA, is a protein responsible for caspase-independent cell apoptosis.101 EndoG is mitochondria specific—during conditions of oxidative stress, it translocates from the intermembrane space of the mitochondria to the nucleus to cleave chromatin DNA into fragments.101 Nucleosomal DNA fragmentation is a hallmark of programmed cell death. Aging is accompanied by deterioration of tissues and loss of specialized cell types partly due to cell death. Many studies have documented the effects of age-dependent apoptosis. Regulation of apoptosis, or programmed cell death, is vital in tissue aging.102,103 Mice with genetic defects in stress-related apoptotic pathways demonstrate significantly extended lifespans.104 On the other hand, disorders caused by deficiencies in apoptosis can lead to tissue impairments that lead to aging.105 EndoG-related cell death similarly affects the liver. Bajt et al. recognized that endoG is responsible for the DNA fragmentation that occurs during drug (acetaminophen)-induced toxicity.106 2.6 Materials and Methods 2.6.1 Animal Model and Diet The common house mouse, Mus Musculus, is a widely used species for biomedical research. They have long been the preferred animal model for studying human disease, because they bear large genetic, physiological, and anatomical similarities to humans107 Mus Musculus are economically advantageous to use, as they are easy to breed and house. Mouse models are excellent for researching aging and age-related diseases.8 Research has shown that female homozygous knockout Elovl5 (Elovl5-/-) mice have impaired reproductive abilities, while males maintain normal fertility.79 It is also known that females accumulate higher levels of long-chain 24 PUFAs and more fat for the purposes of reproduction when compared to males.108 Therefore, aged Elovl5+/- (KO, mixed sex, n=7) and Elovl5+/+ (WT, C57/Bl6, female, n=8) mice were used to evaluate the effect of glutamine supplementation on dysregulated PUFA metabolism. Detailed research investigations have shown no physical abnormalities in EndoG+/- or EndoG-/- mice.109,110 EndoG+/- have demonstrated increased resistance against cell death.110 Aged EndoG+/- (female, n=9) and EndoG+/+ (WT, C57/Bl6, female, n=8) were used to investigate the effect of glutamine supplementation in a healthier mitochondrial model. Transgenic mice were purchased (KOMP, University of California, Davis, CA, USA) and bred in the animal care facility at the University of British Columbia, Okanagan. All animal experiments were approved by UBC’s Animal Care Committee under protocol A15-0240. Mice were weaned from their mothers at 3 weeks of age and experimental diets began at 7-19 months old. Mice were randomly assigned to either a) standard chow with alanine diet, or b) standard chow with glutamine diet. Experimental groups contained random distributions of male and female mice aged between 7-19 months old. A standard powdered chow diet (Bio Serv, AIN-76A) was mixed with 40g/kg diet of either alanine or glutamine supplement (diet compositions in table 2.1). Food was prepared biweekly and stored at -20°C. Mice received daily 5g food portions in order to reduce oxidation and prevent spoilage; food and water were provided ad libitum. Final diet compositions contained 3.79 kcal/g energy. The % energy by weight and by energy are shown below in Table 2.2. Table 2.1. Detailed Composition of Basal Diet Ingredients Composition % W/W Sucrose 50 Casein 20 Corn Starch 15 Corn Oil 5 Cellulose 5 Mineral Mix 3.5 Vitamin Mix 1 DL-Methionine 0.3 Choline Bitartrate 0.2 25 Table 2.2. Macronutrient Composition of Experimental Diets Mouse weights were recorded every other day. After 6 weeks of experimental feeding, the animals were ethically euthanized by administration of the inhalant anesthetic, isoflurane, followed by an overdose of carbon dioxide.111 Tissues of interest: aorta, heart, adipose, skeletal muscle, and liver, were collected and immediately flash frozen in liquid nitrogen. Half of the samples were collected in RNase-free RNAlater (Thermo Fisher Scientific, AM7020, Waltham, MA, USA) for qPCR and half in PBS for remaining experiments (section 2.5 and 3.7). Blood was collected in tubes containing pre-chilled protease inhibitor solutions (Thermo Fisher Scientific, Pierce 88666, Waltham, MA, USA). Serum was obtained from blood via centrifugation at 1000 rpm for 10 minutes. Tissues were placed in -80°C for future use. 2.6.2 Intraperitoneal Glucose Tolerance Test Mice intraperitoneal glucose tolerance tests (IPGTT) were conducted at the end of the sixth week to evaluate fasting glucose levels. Prior to testing, body weights were measured, and mice were moved to clean cages. Animals underwent a 6-hour fast where they only had access to water. Post fast, mice were intraperitoneally injected with a 1g/kg body weight glucose solution—prepared by diluting glucose in saline via sterile filtration through 0.2-micron filters (VWR, 28145-501, AB, Canada). Blood was collected from tail-veins, and glucose levels were recorded using glucose test strips and a glucometer (OneTouch® test strips and UltraMini® meter). Blood withdrawals and glucose measurements were repeated for 15-, 30-, 60-, and 120-minute timepoints post glucose injection. Food was returned to the mice after the procedure. Macronutrients % W/W % Energy Normal Chow Diet Protein 18.1 19.0 Carbohydrate 65.2 68.9 Fat 5.1 12.1 Energy density 3.79 kcal/g diet Amino Acid Diet + 40g/kg amino acid 26 2.6.3 Fatty Acid Extraction and Gas Chromatography Total and individual fatty acid analysis was conducted via gas chromatography (GC) (GC, TRACETM 1300, Bio-Rad) with a flame ionization detector (FID).112 First, FAs were extracted from liver tissue samples using a combined extraction and methylation protocol.113 Fatty acid methyl esters were created from glycerolipids and sterol esters by way of methylation.114 The inorganic compound, boron trifluoride, was used as the acid catalyst for esterification.114 Liver tissues were cut into 30mg pieces and mixed into a 2.4ml 1:1 hexane:boron trifluoride-methanol solution. Samples were heated on a hot plate at 90-100°C for 1 hour. Next, 1ml of water (H2O) was added to each sample and centrifuged for 1 minute. The supernatant, containing methylated and extracted FAs, was removed and placed into a glass vial for GC analysis. Fatty acid methyl esters were analyzed on a Trace 1300 Gas Chromatograph with an FID employing a Supelco Famewax column (30m x 0.32 mmID x 0.5 microns). GC peaks were identified using a 37-peak standard (Sigma Aldrich, St. Louis, MO, USA). The area under the curve for each identified peak was determined and the % area, corresponding to % FA, was calculated for each FA in that sample. Analysis was completed using Chromeoleon7™ Chromatography Data System Software (ThermoFisher Scientific, Waltham, MA, USA). 2.6.4 Triglyceride Assay Triglyceride (TG) concentrations were measured using a Triglyceride Colorimetric Assay Kit (Cayman Chemicals, 10010303, Ann Arbor, MI, USA). First, 350mg of liver tissues were weighed out and homogenized in 2ml of NP40 substitute assay reagent. Next, samples were centrifuged at 10,000g for 10 minutes at 4°C. Supernatants were stored at -80°C or used for TG analysis. Triglycerides were analyzed as a result of enzymatic reactions. TGs were hydrolyzed to glycerol and free fatty acid molecules. Fluorometric absorbance values of released glycerol was measured at 530-550nm using a plate reader. 2.6.5 Total RNA Extraction and Quantitative PCR Total RNA was extracted from frozen liver tissues using the Fibrous Tissue RNeasy Mini Kit (Qiagen, Hilden, Germany) employing the manufacturer’s protocol instructions. Complementary DNA (cDNA) was synthesized using a OneScript Hot cDNA synthesis kit (ABM Inc, Richmond, BC, Canada). All desired mRNA primers were synthesized by Integrated DNA Technologies 27 (IDT; Coralville, Iowa) (table 2.3). Quantitative polymerase chain reactions (qPCR) were performed using a Bio-Rad CFX Manager 2.0 machine. The reagent used for gene amplification was Blastaq Green 2X qPCR MasterMix (ABM Inc, Richmond, BC, Canada). Samples were run in duplicates, and the final volume of each reaction was 10ul. Polymerase chain reactions (PCR) were performed under the following cycling conditions: 95C for 30s, 50 cycles of 95C for 5s, 58C for 5s, 95C for 10s, and a melt curve analysis was performed for 5s at 65C-95C in 0.5C increments. CFX Manager Software version 3.0.1224.1015 was used to quantify gene expression using 18s rRNA as a reference gene for all reactions except ND5, which used beta-actin. Primer efficiencies were collected using LinREG software and are shown in Table 2.3 below. Data was log transformed to normalize. Relative gene expression was quantified via the ΔΔCt method 148. Equations are as follows: ΔCq = Cq (target) – Cq (reference) ΔCq expression = 2-ΔCq ΔCq expressions were normalized to experimental control to obtain ΔΔCq. Table 2.3. Primer Sequences and Efficiencies Gene Primer Sequence (5’ → 3’) Primer Efficiency GS F: CAAGTGTGTGGAAGAGTTACC R: AAAGGTACTAGAGCCATCAAAG 1.90 GLS2 F: CAGCTGAAGGGCACATTG R: TGAAGCAGTTTGACCACCTC 1.93 GDH F: CATTTGTTGTTCAGGGATTTG R: TCTCCAACACCAACACATTTAG 1.91 ELOVL2 F: GAAGGTGATGTCCGGGTAGC R: CATGGACGCGTGGTGATAGA 1.96 ELOVL5 F: CGAGGCATCCTGCAGTTGTA R: ACACACCTGTCACCAACTCA 1.92 FADS1 F: ACGCGCTACTTTACTTGGGA R: CACAAAAGGATCCGTGGCAT 1.91 FADS2 F: CACTATGCCACGTCACAAC R: TTCTTCAGTGAACTCACAATGTC 1.95 FAS F: GGCTGCAGTGAATGAATTTG R: TTCGTACCTCCTTGGCAAAC 1.93 28 SREBP1 F: CTGGAGACATCGCAAACAAGC R: ATGGTAGACAACAGCCGCATC 1.92 IL-6 F: TTCTCTCCGCAAGAGACTTC R: GGTCTGTTGTGGGTGGTATC 1.97 IL-1B F: ACACGGGTTCCATGGTGAAG R: TCAGCACCTCTCAAGCAGAGC 1.96 TNFα F: CAACAAGGAGGAGAAGTTCC R: GATCTGAGTGTGAGGGTCTG 1.90 MCP-1 F: CTGGAGAGCCAGATGTCAGAATTA R: GTACTCTCTGGATGGGTAGGTGG 1.94 ND5 F: ACAGCTATTTGTGCCCTCACCCAA R: GCATGGCTTTGAAGAATGCGTGGG 1.96 18s-rRNA F: CGG CTACCACATCCAAGGAA R: GCTGGAATTACCGCGGCT 1.97 Beta-actin F: GCCAACACAGTGCTGTCTG R: TACTCCTGCTTGCTGATCCA 1.92 2.6.6 Protein Extraction Liver proteins were extracted by cutting and immersing 20-30mg of frozen tissue in a 500ul solution containing radio-immunoprecipitation assay buffer (RIPA buffer) (recipe in table 2.4), phosphatase inhibitor (Alfa Aesar, J61022, Tewksbury, MA, USA), and a protease inhibitor cocktail (Thermo Fisher Scientific Pierce, 88666, Waltham, MA, USA). Homogenization beads were added to each tube and samples underwent three homogenizations, in a Retsch Mixer Mill M400 homogenizer, at 30hz for 2 minutes each, with one-minute rest intervals in between (Retsch, Germany). Homogenated samples were centrifuged at 20,000 rpm for 15 minutes at 4°C. The supernatant of each sample was removed and added into a new tube. Isolated supernatants, consisting of extracted proteins, were stored at -20°C for further use. Total protein concentrations were quantified using a Bradford protein assay (BioRad, 500-0006, California, USA). The Bradford protein assay is a fast and sensitive colorimetric detection method that employs bovine serum albumin (BSA) as a protein standard (Kruger, 2009; Schaffner & Weissmann, 1973). First, protein extracts were diluted with Bradford reagent and incubated at room temperature for 5 minutes. Proteins were quantified at 595nm by a spectrophotometer (Offenburg, Germany). Dilution calculations were made to equalize protein concentrations across 29 the samples. Samples were diluted in deionized H2O and 50ul of sample digestion buffer (SDB) (recipe in table 2.5) to be used for western blotting. Table 2.4. RIPA Buffer Recipe Ingredients Amount 0.1% SDS 0.2 g 0.5% Sodium Deoxycholate 1.0 g 1% Triton-X 2.0 mL 150mM Sodium Chloride 17.5 g Table 2.5. SDB Buffer Recipe Ingredients Amount Mercaptoethanol 2.0 mL 0.1% Bromophenol blue 4.0 mL 0.5m Tris 4.8 mL 10% SDS 8.0 mL 100% Glycerol 10.0 mL ddH2O 11.2 mL 2.6.7 Western Blot Western blotting is a common laboratory technique used to identify and quantify extracted proteins from cells or tissues. The procedure employs three essential steps: electrophoresis, electrotransfer, and blocking followed by antibody incubation.115 Samples were prepared via protein extraction, and protein concentrations were equalized by diluting with distilled water and loading buffer (recipe in table 2.4). Gel electrophoresis constituted of running 25μl of denatured diluted proteins on SDS-polyacrylamide gel. Electrotransfer, or blotting, involved transferring the separated protein mixture to nitrocellulose membranes. Post transfer, membranes were stained with Ponceau (recipe in table 2.6) to visualize bands.116 Membranes were blocked with a solution containing 5% skimmed milk in washing buffer, TBS-T, for 10 minutes at room 30 temperature with constant shaking. Blocking is a crucial strategy to prevent excessive background noise in images. Membranes were washed with TBS-T and incubated, either overnight at 4°C or for 2 hours at room temperature, with 5ml of primary antibody solution (recipe in table 2.7) and constant revolutions at 2 rpm/min. Following, membranes underwent 3 5-minute TBS-T washes. Membranes were incubated with 5ml of secondary antibody solution (recipe in table 2.8) for 1 hour with constant revolutions at 2 rpm/min. Membranes were coated with chemiluminescent ECL western blotting substrate (Thermo Fisher Scientific Pierce, 32109, Waltham, MA, USA) and visualized using Image Studio Density version 3.1. Band intensities were quantified via ImageJ software and expressed as a ratio to ponceau stains. Multiple antibodies were probed on the same blot using stripping buffer (pH 2.2, recipe in table 2.9).117 Primary and secondary antibodies used are listed in tables 2.10 and 2.11. Table 2.6. Ponceau Stain Recipe Ingredients Amount Ponceau S 1 g Acetic Acid (glacial) 50 mL ddH2O 950 mL Table 2.7. Primary Antibody SolutionIngredients Amount Bovine Serum Albumin 0.25 g TBST-T 5 mL Table 2.8. Secondary Antibody Solution Ingredients Amount Casein 0.25 g TBST-T 5 mL 31 Table 2.9. Stripping Buffer Recipe Ingredients Amount Glycine 15 g Sodium Dodecyl Sulfate 1.0 g Tween 20 10.0 mL ddH2O 990 mL Table 2.10. Primary Antibodies used Table 2.11. Secondary Antibodies used 2.6.8 Statistical Analysis Data is graphically represented as bar graphs and includes standard error of the mean. In Chapters 2 and 3, an unpaired T-test or a two-way ANOVA was used. In the case of non-normal data: Mann-Whitney post hoc comparisons were used following T-tests, or non-parametric Tukey’s post hoc analyses were utilized following two-way ANOVAs. Statistical significance was set at p<0.05. GraphPad Prism software 8.0 was used for all statistical analyses and graphical representation (GraphPad Software Inc., San Diego, CA, USA). All groups display their respective n values, with a minimum n=5. For GC results displayed in Table 2.12, standard deviation was calculated using Microsoft Excel. 1°Antibody Isotype Dilution Catalogue # Source GLS2 Rabbit 1:1000 NBP3-04094 Novus Biologicals GDH Rabbit 1:1000 NBP1-54962 Novus Biologicals ASCT2 Rabbit 1:1000 20350-1-AP Proteintech FAS Rabbit 1:1000 3180T Cell Signaling Technology ACC (Ser523) Rabbit 1:1000 36767 Cell Signaling Technology ACC-P (Ser79) Rabbit 1:1000 11818T Cell Signaling Technology 2°Antibody Dilution Catalogue # Source Anti-Rabbit 1:2000 7074S Cell Signaling Technology 32 2.7 Results 2.7.1 Mice Fed Specific Diets Did Not Demonstrate Differences in Weight Gain Gas chromatography was conducted on rodent diets in order to determine the relative proportions of fatty acids present in the diet. Mouse diets were a mixture of powdered standard chow and powdered amino acid (glutamine or alanine). Both diets contained the same proportions of fatty acids (Figure 5). Gas chromatographic analysis confirmed no significant differences between diets containing amino acids of interest. Figure 7. Relative proportions of fatty acids present in both glutamine and alanine diets. Gas chromatography results showing relative percentages of various fatty acid types within the amino acid experimental diets. To ensure food consumption, mice were weighed every other day and body weight measurements were recorded for the entirety of the diet experiment. Post six weeks of feeding, overall changes in weight were determined by subtracting the final body weight from the initial body weight. Wild-type (WT), Elovl5+/-, and EndoG+/- mice fed glutamine or alanine diets did not experience any significant differences in body weights (Figure 8). 33 Figure 8. Change in body weight across glutamine and alanine diets for WT, Elovl5+/-, and EndoG+/-. Change in body weight was determined by subtracting the final weight (grams) from the initial. Males represented via open-circle points; females represented via closed. Data was analyzed using unpaired T-test and two-way ANOVA followed by Mann-Whitney and Tukey’s post hoc comparisons (WT n=8, Elovl5+/- n=6, EndoG+/- n=9). 2.7.2 Mice Fed Specific Diets Displayed Genotypic Differences in Glucose Metabolism Intraperitoneal glucose tolerance tests (IPGTT) are frequently performed to assess glucose metabolism.118 Post six weeks of feeding, IPGTT results reveal that WT mice fed glutamine and alanine diets experienced significant differences in glucose metabolism compared to Elovl5+/- amino acid fed mice (Figure 9A). Both WT alanine and glutamine fed mice demonstrate decreased glucose clearance compared to Elovl5+/- alanine and glutamine groups. These results were further supported through area under the curve (AUC) analysis, which demonstrated various genotypic differences between diets (Figure 9B). WT mice from the glutamine diet group have significantly increased blood glucose levels compared to alanine. Group differences demonstrate decreased baselines in Elovl5+/- for both alanine and glutamine as compared to WT. The EndoG+/- glutamine group displayed higher blood glucose levels compared to Elovl5+/- glutamine. 34 35 Figure 9. Blood glucose levels of WT and KO mice for glutamine and alanine diets over time. A) IPGTT fasting blood glucose concentrations and subsequent blood glucose measurements at 15-, 30-, 60-, and 120-minutes post glucose injection for WT, Elovl5+/-, and EndoG+/- (WT alanine vs Elovl5+/- alanine *p=0.0026; **p=0.0011; WT glutamine vs Elovl5+/- glutamine +=0.0209; ++=0.0077). B) Area Under the Curve for IPGTT values for genotypes (*p=0.0057; WT glutamine vs Elovl5+/- glutamine +=0.0142; WT alanine vs Elovl5+/- alanine ++=0.0021; Elovl5+/- alanine vs EndoG+/- alanine +++=0.0010). Data was analyzed within genotypes using an unpaired T-test and between genotypes via two-way ANOVA followed by Mann-Whitney and Tukey’s post hoc comparisons (WT n=9, Elovl5+/- n=7, EndoG+/- n=8). 2.7.3 Glutamine drives Elovl5-mediated PUFA bioconversion To investigate glutamine’s effects on lipid metabolism, qPCR was performed for PUFA bioconversion genes in all WT, Elovl5+/-, and EndoG+/- mice. WT displayed no significant differences in PUFA bioconversion genes. However, in Elovl5+/- mice, glutamine significantly increased gene levels for FADS1, FADS2, and Elovl5, while Elovl2 gene levels were significantly decreased (Figure 10). In EndoG+/- mice, FADS1, FADS2, and Elovl5 were significantly downregulated. Opposingly, Elovl2 was upregulated in these samples (Figure 10). Significant diet differences between genotypes observed for all four enzymes (Figure 10). 36 Figure 10. Elovl5+/- glutamine-fed mice express significantly greater PUFA bioconversion enzymes in liver tissue. qPCR on WT, Elovl5+/-, and EndoG+/- for A) FADS1 (***p=0.0006; +=0.0110; ++=0.0016; +++=<0.0001), B) FAD2 (**p=0.0023; ***p=<0.0001; +=0.0711; ++=0.0088; +++=<0.0001), C) Elovl5 (*p=0.0140; ***p=0.0006; +++=<0.0001), and D) Elovl2 (*p=0.0175; **p=0.0016; ++=0.0043; +++=<0.0001). Unpaired T-test used to compare significance within genotypes. Two-way ANOVA used to compare significance between genotypes across diet groups (WT n=7, Elovl5+/- n=6, EndoG+/- n=9). To measure specific proportions of various fatty acids, gas chromatography was used to analyze liver samples in all models. WT glutamine-fed mice demonstrated a significant decrease in α-linolenic (ALA, C18:3n3) and a significant increase in dihomo-γ-linolenic (DGLA, C20:3n6) (Table 2.12). On the other hand, Elovl5 KO mice showed no differences between fatty acid levels for either diet (Table 2.12). For EndoG+/- mice, the glutamine-fed group demonstrated significant increases in stearic acid (SA), γ-linolenic acid (GLA), DGLA, and eicosapentaenoic 37 acid (EPA), with a significant decrease in palmitic acid (PA) (Table 2.12). Genotypic differences were noted in FAs between alanine groups. SFA differences show decreased PA and increased SA in Elovl5+/- compared to EndoG+/-. For PUFA differences compared to WT, Elov5+/- mice had increased GLA and ARA, and EndoG+/- had decreased DGLA. These results highlight lipid changes that occurs as a result normal, deficient, or improved metabolic capabilities. Table 2.12. Relative proportions of certain FAs within WT and KO liver tissues, obtained by gas chromatography. Unpaired T-test used to calculate significance between diets for WT followed by Mann Whitney test (*p=0.0238; **p=0.0130), Elovl5 KO, and EndoG KO (*p=0.0043; **p=0.0020; #p=0.0103; ##p=0.0450). Two-way ANOVA used to determine baseline differences between all groups followed by Tukey’s test (a=0.01888; b=<0.0001; c=0.0463; d=0.0391; e=0.0356; f=0.0008; g=0.0025; h=0.0333; i=0.0024; j=0.0421; k=0.0493) (WT n=6; Elovl5+/- n=6; EndoG+/- n=9). Abbreviations: AVG (average); SD (standard deviation). Fatty Acid AVG (%) ± SD WT Elovl5 EndoG Alanine Glutamine Alanine Glutamine Alanine Glutamine Palmitic (C16:0) 25.08a±1.50 25.52±1.98 22.73b±2.80 23.36±0.96 28.43a,b±2.64 23.88**± 2.45 Stearic (18:0) 9.33c±0.86 10.31±1.19 9.63d±3.95 10.95±4.43 5.76c,d±2.51 10.14*± 2.58 Oleic (C18:1) 27.34±2.47 26.13±7.64 24.60±3.57 23.46±5.99 28.05±7.96 27.33±9.56 Linoleic (C18:2n6) 14.35±2.78 12.80±2.50 13.76±2.07 14.03±2.91 14.64±1.90 12.65±2.72 Alpha Linolenic (C18:3n3) 0.12±0.05 0.08*±0.04 0.07±0.04 0.06±0.01 0.09±0.05 0.07±0.03 Gamma-Linolenic (C18:3n6) 0.29e±0.08 0.24±0.04 0.75e,f±0.66 0.51±0.45 0.08f±0.04 0.22***± 0.10 Dihomo-γ-Linolenic (C20:3n6) 0.98g±0.17 1.18h,i,**± 0.24 0.65±0.23 0.79h±0.45 0.52g±0.14 0.83i,#±0.41 Arachidonic (C20:4n6) 11.57j±1.15 12.29±1.72 16.29j,k± 2.27 14.84±3.51 12.00k±3.74 13.59±4.48 Eicosapentaenoic (C20:5n3) 0.14±0.09 0.17±0.08 0.09±0.05 0.11±0.09 0.06±0.02 0.12##±0.07 Docosahexaenoic (C22:6n3) 5.31±0.81 6.48±1.51 5.77±0.86 5.22±1.92 5.31±1.52 6.17±2.73 38 2.7.4 Glutamine activated de novo lipogenesis genes in Elovl5+/- mouse livers To further investigate whether glutamine targets lipid pathways, qPCR was run for lipid synthesis genes. It is known that glutamine stimulates SREBP and its target genes.22 Our results confirm these findings, as SREBP1, the master transcriptional regulator of de novo lipogenesis, showed significantly increased gene levels in all genotypes. FAS, a target of SREBP1, demonstrated a significant increase in Elovl5+/- and a decrease in EndoG+/- mice, as shown below in Figure 11. Significant differences between glutamine and alanine groups across genotypes were also noted for SREBP1 and FAS. Figure 11. Glutamine increases SREBP1 gene levels in WT, Elovl5+/-, and EndoG+/- mice. qPCR gene expression on WT and Elovl5+/- mouse livers across diets for A) SREBP1 (*p=0.0175; **p=0.0070; ***p=<0.0001; +=0.0076; ++=0.0019; +++=<0.0001) and B) FAS (**p=0.0023; *p=0.0313; ++=0.0002; +++=<0.0001). Significance was analyzed within genotypes using an unpaired T-test and between genotypes via two-way ANOVA (WT n=6, Elovl5+/- n=6, EndoG+/- n=8). Western blots were assessed for acetyl-CoA carboxylase (ACC) and phospho-acetyl-CoA-carboxylase (pACC) to analyze glutamine stimulation of an alternate lipid pathway. ACC is a key enzyme involved in the biosynthesis and oxidation of fatty acids.119 It catalyzes the carboxylation of acetyl-CoA to malonyl-CoA.119 Phosphorylation of ACC at the Ser79 residue (p-ACC), inhibits its enzymatic activity.120 Thus by measuring the ratio of pACC to ACC, we demonstrated the inhibition or stimulation of acetyl-CoA conversion. No significant differences were noted for inhibition of ACC in any experimental group (Figure 12). 39 Figure 12. Glutamine does not alter pACC/ACC protein levels in WT, Elovl5+/-, or EndoG+/- mice. Western blot protein expression of pACC/ACC in A) WT mice, B) Elovl5+/-, and C) EndoG+/- mice. Significance between diets determined using an unpaired T-test and two-way ANOVA followed by Mann-Whitney and Tukey’s post hoc comparisons (WT n=6, Elovl5+/- n=5, EndoG+/- n=5). Triglycerides, glycerol and fatty acid moieties, are measured as a way of determining accumulated body fat.121 Liver triglycerides were evaluated for WT, Elovl5+/-, and EndoG+/- mice. No significant differences were observed in either the glutamine- or alanine-fed groups (Figure 13). 40 Figure 13. Triglyceride differences across diets in WT, and Elovl5 and EndoG KO livers. Unpaired T-test and two-way ANOVA followed by Mann-Whitney and Tukey’s post hoc comparisons were utilized to calculate significance (n=6). 2.8 Discussion It is known that lipid metabolism becomes increasingly dysregulated as we age.15,94,122,123 Specifically within PUFA metabolism, the decline in elongase enzymatic activity has been correlated with several age-related pathologies108,122 To further investigate this matter, we targeted elongase 5, which is responsible for the bioconversion of long-chain GLA to DGLA, to observe its dysregulation in middle-age. The Ghosh lab has previously shown that a heterozygous knockout model, which blocks fatty acid bioconversion, leads to an accumulation of LA, decreases antioxidant synthesis, and causes other associated problems.81 Elovl5-/- (complete KO) mice experience impaired reproductive abilities, while Elovl5+/+ (WT) mice have complete PUFA metabolic abilities.79 Hence, a partial Elovl5 KO model was used to study impaired lipid metabolism in middle-aged livers. Elovl5 elongates fatty acids through the addition of acetyl-CoA, which is generated in the mitochondria. The importance of mitochondria in PUFA metabolism prompted the use of an endonuclease G model. To recall, previous work by the Ghosh lab has demonstrated that the EndoG+/- genotype provides cardio-protective effects by decreasing mitochondrial stress and improving antioxidant capacities.81 Therefore, we utilized 41 a mitochondrially healthy model, partial EndoG KO mice, to identify the interactions of glutamine and lipids in middle-aged livers. As the aging process also involves declines in protein metabolism and a depletion of glutamine stores, the amino acid glutamine was utilized to assess whether supplementation could mitigate the progression of fatty liver and differences in mitochondrial health.29 To substantiate this hypothesis, glutamine and lipid metabolism pathways were analyzed in vivo. First, glutamine supplementation bore no effect on changes in weight between WT, Elovl5+/-, or EndoG+/- mice (Figure 8). There were genotypic and diet differences in glucose metabolism as demonstrated by IPGTT values (Figure 9). Glutamine-fed WT mice experienced significantly increased blood glucose levels overtime, which is similarly observed in the KO groups as compared to their alanine controls. These results support research identifying glutamine as a glucose precursor by contributing carbons to the glucose carbon pool.124 Thus, glutamine can induce gluconeogenesis, and within older models characterized by insulin resistance, it is possible to observe increased IPGTT values post-glutamine feeding.124 qPCR analysis concluded that glutamine did not drive PUFA bioconversion enzymes in WT mice, as no significant changes were noted for n-3 or n-6 metabolic enzymes: FADS1, FADS2, Elovl5, and Elovl2 (Figure 10). Interestingly, in Elovl5+/- mice, all PUFA conversion enzymes were upregulated except for Elovl2 (Figure 10). We resolve that glutamine increases gene expression for FADS1, FADS2, and Elovl5 as a probable compensatory response to a deletion in the Elovl5 gene. In tying mitochondrial health to glutamine and lipid metabolism, were able to show that EndoG+/- significantly decreased FADS2 and Elovl5 gene expression levels, increased Elovl2, and showed no differences in FADS1 (Figure 10). These trends are in exact opposition to what was observed in the Elovl5+/- model. It is rational to conclude that in this healthier EndoG KO model, the gene expression for PUFA bioconversion enzymes are downregulated due already-sufficient protein levels or improved mitochondrial metabolic abilities to metabolize fatty acids and produce energy. Changes in individual FAs were identified through gas chromatography. First, for PUFA metabolism, DGLA levels were highest in WT glutamine and alanine groups compared to Elovl5 or EndoG KOs. A significant decrease in ALA paired with an increase in DGLA was noted in 42 the WT glutamine group (Table 2.12). Therefore, although glutamine supplementation does not drive PUFA bioconversion genes, it may influence PUFA bioconversion via an Elovl5-mediated push to DGLA.79 This prompted a more concrete reason to investigate the effects of Elovl5 in the middle-aged model. Second, no observable differences were noted within the Elovl5 KO genotype for individual fatty acid levels (Table 2.12). Thus, we conclude that glutamine cannot drive PUFA bioconversion in a mouse with a genetic defect in part of the Elovl5 allele. However, baseline alanine comparisons between genotypes reveal highest levels of SA and GLA in the Elovl5+/- group. Accumulation of such C18 products can result from knocked-out elongase activity and promote progression of a fatty liver phenotype.79 Third, FA increases in the EndoG+/- genotype included GLA, DGLA, and EPA. GLA is the product of FADS2 bioconversion from LA (Figure 6).108 FADS2 is localized in the mitochondria, thus in the case of improved mitochondrial protection, GLA would increase as a result of effectively working mitochondria.85 DGLA results from Elovl5-mediated bioconversion of GLA, therefore in glutamine-fed mice increased DGLA could result from Elovl5-driven PUFA bioconversion, similar to what was observed in WT (Figure 6).79 Increased EPA levels in the EndoG glutamine group (Table 3.1) could stimulate the previously seen increase in Elovl2 gene levels (Figure 10) to allow for needed conversion of EPA to DHA, the terminal product of omega-3 metabolism.79 SFAs were notably different between the KO models. Within the EndoG+/- genotype, the glutamine diet caused a significant decrease in PA and increase in SA values compared to their alanine control. In Elovl5+/- alanine-fed mice, we measured a significant decrease in palmitic acid coupled with an increase in stearic acid compared to EndoG+/- (Table 2.12). However, no differences were seen with the addition of glutamine. Palmitic acid is the precursor to stearic acid, and it is converted via the enzymatic action of fatty acid elongase 6 (Elovl6).91 However, we did not estimate Elovl6 gene levels in our study. Within both these models, a universal elongation effect could be induced that includes both PUFAs and saturated fatty acids. Ultimately EndoG+/- mice experience more efficient PUFA bioconversion. To assert glutamine’s involvement in non-PUFA pathways, other lipid synthesis related enzymes were monitored via gene and protein analysis. SREBP are a group of transcription factors that regulate cholesterol (SREBP2) and fatty acid (SREBP1) metabolism.22 Its target genes include 43 FAS and ACC.22 In both the WT, and Elovl5 and EndoG KO mice, SREBP1 levels were significantly upregulated in the glutamine fed groups; a greater significance was noted in the KOs (Figure 11). FAS functions to synthesize palmitate (C16) de novo from acetyl-CoA (C2) and malonyl-CoA (C3).125 FAS was significantly increased in Elovl5+/- mice and decreased in EndoG+/- (Figure 11). Its upregulation in Elovl5+/- was a probable response to impaired lipid metabolism. Previous studies have shown that Elovl5 deficient mice increase SREBP1 and FAS gene levels, culminating in the development of fatty liver.79 Decreased FAS expression levels in EndoG+/- could result from negative feedback of the increased stearic acid, noted in Table 2.12.126,127 Glutamine mediated decrease in FAS thereby decreases palmitic fatty acid levels, which is shown in Table 2.12. Acetyl-CoA carboxylase (ACC) carboxylates acetyl-CoA to malonyl-CoA, thus regulating fatty acid synthesis.119 Phosphorylated ACC (pACC) inhibits ACC enzymatic activity, therefore by measuring the ratio of pACC to ACC, we can identify its activity status.119 pACC/ACC protein levels were similar between genotypes and diet groups (Figure 12). Together, these findings further confirm glutamine’s action on sterol regulatory binding elements and its ability to regulate its downstream gene targets.22 Lastly, triglycerides were measured to assess fat accumulation. Glutamine bore no effect on triglyceride accumulation in middle-aged mouse liver as compared to alanine control (Figure 13). In conclusion, glutamine played an Elovl5-mediated role in PUFA bioconversion, but not total lipids in the liver. In the WT liver model, glutamine supplementation drove PUFA bioconversion at least through Elovl5. In the Elovl5 KO model, it increased gene expression of PUFA conversion enzymes as an adaptive response to a deficiency in the Elovl5 gene. In the mitochondrially healthy EndoG KO model, glutamine supplementation abolished the increase for PUFA metabolism genes. Regarding individual fatty acids, glutamine supplementation resulted in a universal increase in elongation effects and FADS2 utilization. 44 Chapter 3: Impact of Glutamine on Mitochondrial and Immune Markers in Middle-Aged Mouse Livers 3.1 Rationale In chapter 2, impairing PUFA bioconversion and supplementing glutamine pushed fatty acid bioconversion. Hence, glutamine was able to alter FA profiles in WT, Elovl5 KO, and EndoG KO models. Dysregulated lipid metabolism and mitochondrial metabolic activity become prevalent in aging and may thus play a role in inflammaging. However, links between the three require further research. Current scientific literature lacks insight into the specific relationships between Elovl5 and EndoG in mitochondrial metabolism and inflammation. Improving or maintaining mitochondrial energy metabolism, via supplements such as glutamine, can mitigate the progression of age-related inflammation and lipid metabolism. 3.2 PUFAs, Mitochondria, and Inflammation PUFA metabolism and mitochondrial health decline with age. Polyunsaturated fatty acids are involved in various mitochondrial functions including apoptosis and the production of ROS. As mentioned, Elovl5 regulates PUFA metabolism, thus a connection stands to be made between Elovl5 and mitochondrial health. Elovl5+/- mice have impaired PUFA metabolism and develop fatty liver, which in turn can affect mitochondrial functioning. A study by Tripathy et al. demonstrated that elevated Elovl5 activity suppressed peroxisome PGC1α, and thus mitochondrial biogenesis.38 Other studies have demonstrated that depleted Elovl5 alter mitochondrial function and morphology via excess ROS production.128 PUFAs modulate immunity and overall inflammation. In this regard, Elovl5 can be linked to inflammatory status via its omega-6 products. 3.3 EndoG Transgenic Model, Mitochondria, and Inflammation Inflammaging and decreased mitochondrial health are concurrent and progressive. EndoG KO mice were used to determine the effects of glutamine supplementation on inflammation and mitochondria in metabolically healthier middle-old age livers. EndoG studies are predominately focused on its caspase-independent apoptotic effects. Cell apoptosis is a self-preservation mechanism used to prevent infections or further damage to tissues.102 Its homeostasis regulates 45 the progressive decline seen in aged cells.105 Studies have demonstrated that fibroblasts cells undergoing senescent cell arrest acquire resistance to apoptosis.129 Previous research by Ghosh et al. revealed that EndoG+/- mice simulate less oxidative stress and improved mitochondrial and heart functioning.81 Mitochondrial DNA can stimulate the immune system. Mitochondria harbour their own genome which acts as an immune antagonist. For this reason, apoptotic mechanisms, including EndoG, can stimulate an inflammatory response post fragmented DNA release.130 3.4 Background 3.4.1 Mitochondrial Markers in Middle-Aged Liver Dysregulated mitochondrial bioenergetics can cause oxidative stress and premature aging.131 Mitogenesis and mitochondrial complexes play a vast role in health. Examining mitochondrial enzymes can therefore serve as biochemical markers of aging. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) regulates energy metabolism including mitochondrial biogenesis and gluconeogenesis. PGC1α activity has been shown to decline with age correlating with mitochondrial dysfunction.131 NADH-ubiquinone oxidoreductase chain 5 (ND5) encodes the NADH dehydrogenase subunit, making up the largest of the five electron transport chain complexes. Genes for mitochondrial complexes similarly decline with age coupled with decreased ETC activity and respiration.131 3.4.2 Inflammatory Markers in Middle-Aged Liver Inflammation is an integral part of the body’s natural defenses, and it can be regulated by way of adipose and other organs. Research has demonstrated links between aging, fat metabolism, and inflammatory ARDs.132 Inflammaging broadly describes the upregulated inflammatory responses that occur with aging.21 Complex environmental and age-related factors govern an individual’s susceptibility to chronic inflammatory cytokine production.21 Factors such as cellular senescence, impaired autophagy, insulin resistance, and increased FFAs all contribute to inflammaging. Clinical measurements of inflammation involve several cytokine markers, namely tumor necrosis factor-α (TNFα), the interleukin family, and monocyte chemoattractant protein-1 (MCP1). TNFα is a proinflammatory cytokine responsible for regulating skeletal, neuronal, immune, and other cell homeostasis. TNFα has been markedly noted in diabetes. Increased levels 46 in adipose tissue are associated with chronic lipolysis, the process of hydrolyzing triglycerides to release lipids and FFAs.132 TNFα then moves to stimulate macrophage immune cells, leading to insulin resistance.132 Excessive muscle wasting, as seen in rheumatoid arthritis, has been associated with an uncontrolled TNFα-driven inflammatory response.132 Interleukins are known for their mediating role between white blood cells. They promote inflammation by signaling the liver to produce acute phase proteins which further induce protective effects, i.e., fever.133 Interleukin 6 (IL6) and interleukin 1B (IL1B) are clinically relevant. High glucose concentrations, as often seen in obese or diabetic patients, activate IL1B in cells and promote macrophage responses.133 Increased interleukin activity has also been correlated with increased risk of cancer.133 Next, MCP1 attracts or enhances the roles of other inflammatory factors and its function has been implicated in a variety of ARDs.134 Increased MCP1 involvement has been observed in fatty liver disease, cancer, and others.134 MCP1 activates hepatic stellate cells, which promote fibrosis in response to liver injury and inflammation.134 Cytokines, specifically TNFα and IL6, are strongly correlated to increased morbidity and mortality. With the involvement of cytokines in ARDs, they may act as diagnostic markers for investigation in aging and comorbidities. 3.5 Materials and Methods 3.5.1 Animal Model and Diet Wild-type, heterozygous knockout Elovl5, and heterozygous knockout EndoG mice were utilized as previously described in section 2.6.1. Feeding was performed as outlined in section 2.6.1, with either a) standard chow with alanine diet, or b) standard chow with glutamine diet. Food and water were provided ad libitum. Diet composition and nutritional information are provided in Table 2.1 and Table 2.2 in Chapter 2. 3.5.2 Total RNA Extraction and Quantitative PCR Total RNA extraction and qPCR experiments were conducted as explained in section 2.6.5. 3.5.3 Protein Extraction Protein extractions were performed as described in section 2.6.6. 47 3.5.4 Western Blot Western blot experiments were run as described in section 2.6.7. 3.5.5 Statistical Analysis Statistics were analyzed as described in section 2.6.8. 3.6 Results 3.6.1 Glutamine processing genes are upregulated in middle-aged Elovl5+/- livers and downregulated in EndoG+/- Liver tissues of WT, and Elovl5 and EndoG KO mice were analyzed via qPCR and western blotting methods to illuminate the effects of glutamine supplementation on glutamine metabolic enzymes. Glutamine synthesis requires glutamine synthetase (GS), while glutamine degradation and utilization involve glutaminase 2 (GLS2) and glutamate dehydrogenase (GDH). We demonstrated that there were no significant differences in GDH expression levels for WT, while GLS2 and GS were downregulated in the glutamine-fed group (Figure 14). On the contrary, Elovl5+/- expressed significant increases in all three metabolic enzymes, as shown in Figure 14. Between-group comparisons highlight high baseline GS levels in Elovl5+/- alanine compared to WT, as well as increased GDH in Elovl5+/- and EndoG+/- alanine compared to WT. Western blots portrayed GDH protein levels to be highest in alanine-fed EndoG+/- mice compared to Elovl5+/- and WT (Figure 16). Elovl5+/- demonstrates the highest GLS2, GS, and GDH gene expressions post glutamine feeding compared to WT and EndoG+/-. In EndoG+/- mice, glutamine synthesis (GS), breakdown (GLS2), and utilization (GDH) enzymes were significantly decreased post glutamine feeding, as opposed to the alanine control (Figure 14). Results were further supported through protein analysis and confirmed for EndoG+/- GDH levels in Figure 16. 48 Figure 14. Glutamine increases metabolic genes in Elovl5+/- mice while decreasing expression in EndoG+/-. qPCR gene expression for WT, Elovl5+/-, and EndoG+/- mouse livers for A) GLS2 (**p=0.0145; *p=0.0175; ***p=0.0003; +++=<0.0001), B) GS (*p=0.0296; ***p=0.0041; **p=0.0086; +=0.0433; +++=<0.0001), and C) GDH (*p=0.0367; ***p=<0.0001; +=0.0154; ++=0.0118; +++=<0.0001). Significance between diets determined using unpaired T-test and between genotypes using two-way ANOVA (WT n=6, Elovl5+/- n=6, EndoG+/- n=9). 49 Figure 15. Elovl5+/- and EndoG+/- glutamine-fed mice express increased GLS2 protein levels compared to WT. GLS2 western blot protein levels across diets in WT, Elovl5+/-, and EndoG+/- mouse livers (++=0.0006; +=0.0037). Unpaired T-test and two-way ANOVA followed by Mann-Whitney and Tukey’s post hoc comparisons used to compare significance between genotype and diet groups (n=6). 50 Figure 16. EndoG+/- mice express decreased GDH protein levels. GDH western blot protein levels across diets in mouse livers for WT, Elovl5+/-, and EndoG+/- (*p=0.0130) (+=0.0047; ++=0.0024). Unpaired T-test and two-way ANOVA followed by Mann-Whitney and Tukey’s post hoc comparisons used to compare significance between genotype and diet groups (n=6). To measure glutamine uptake and transport by cells, a western blot analysis was conducted for glutamine cell transporter alanine, serine, cysteine transporter 2 (ASCT2). No significant differences were observed between the glutamine or alanine diets for either WT, Elovl5+/-, or EndoG+/- mice, shown below in Figure 17. However, differences were noted between groups: Elovl5+/- glutamine-fed mice expressed significantly decreased levels of ASCT2, while both EndoG+/- glutamine- and alanine-fed mice demonstrate decreases in ASCT2 in comparison to WT (Figure 17). Figure 17. Elovl5+/- and EndoG+/- mouse livers express decreased ASCT2 protein levels. Western blot protein levels in for ASCT2 in WT, Elovl5+/-, and EndoG+/- mice (+=0.0263; ++=0.0052; +++=0.0005). Unpaired T-test and two-way ANOVA followed by Mann-Whitney and Tukey’s post hoc comparisons used to compare significance between genotype and diet groups (WT n=5, Elovl5+/- n=5, EndoG+/- n=6). 51 3.6.2 Glutamine reduces hepatic inflammation in EndoG+/- mice Inflammation is a hallmark of aging and fat metabolism.21 Thus, a qPCR gene panel was run to assess glutamine’s correlation with inflammation. WT, Elovl5+/-, and EndoG+/- mouse livers were used to analyze interleukin-6 (IL6), interleukin-1B (IL1B), monocyte chemoattractant protein-1 (MCP1), and tumor necrosis factor-α (TNFα). WT mice did not exhibit inflammation post glutamine supplementation, while Elovl5+/- mice had increased levels of IL6 (Figure 18). EndoG+/- mice displayed decreased levels of all inflammatory markers post glutamine feeding. Differences for glutamine and alanine diets between genotypes were noted as follows. Elovl5+/- glutamine-fed mice had increased TNFα and MCP1 compared to EndoG+/- and WT. The EndoG+/- alanine group demonstrated increased IL6 compared to WT and Elovl5+/-. The EndoG+/- glutamine group experienced the greatest decrease in IL6, TNFα, and MCP1 compared to Elovl5+/-. Significance for interactions shown below in Figure 18. 52 Figure 18. EndoG+/- glutamine-fed mice have decreased expression of inflammatory markers. qPCR gene expression across diets in WT, Elovl5+/-, and EndoG+/- mouse livers for A) IL6 (**p=0.0013; ***p=0.0003; +=0.0296; ++=0.0042; +++=0.0005; ++++=0.0003) B) IL1B (**p=0.0082), C) TNFα (*p=0.0175; +=0.0172), and D) MCP1 (***p=0.0003; +=0.0057; ++=0.0440). Unpaired T-test and two-way ANOVA used to compare significance between genotype and diet groups (WT n=5, Elovl5+/- n=5, EndoG+/- n=6). 3.6.3 Glutamine increases mitochondrial biogenesis genes in Elovl5+/- mice Mitochondrial health is vital for overall metabolism, thus we assessed glutamine’s relationship to mitochondrial biogenesis and copy number via qPCR analysis for PGC1α and ND5. PGC1α is the master transcriptional regulator for mitochondrial biogenesis. ND5 is measured as an indicator of mitochondrial abundance, as it encodes an essential subunit in the NADH dehydrogenase complex.135,136 No significant changes were noted in WT mice for either gene (Figure 19). Elovl5+/- glutamine-fed mice demonstrated significant increases in ND5 and PGC1α gene levels within their group and when compared to WT and EndoG+/-. EndoG+/- mouse livers expressed decreased levels of ND5 and PGC1α levels, graphed below. Figure 19. Elovl5+/- mice express increased mitochondrial biogenesis and copy number genes. qPCR for WT, Elovl5+/-, and EndoG+/- mice for A) ND5 (*p=0.0111; **p=0.0110; ++=0.0007; +++=0.0002) and B) PGC1α (*p=0.0217; **p=0.0111; +++=0.0007). Unpaired T-test and two-way ANOVA used to compare significance between genotype and diet groups (WT n=7; Elovl5+/- n=6; EndoG+/- n=9). 53 3.7 Discussion Aging involves impairments in lipid metabolism, decreases in mitochondrial health, and acute-to-chronic systemic inflammation. Mitochondrial health is an important factor in ensuring proper cell metabolism, specifically PUFA bioconversion. Impaired mitochondria have been implicated in a variety of age-related diseases.31 To recall, endonuclease G is encoded by mtDNA, and it directs DNA degradation and causes cell death.101 Theoretically, increased EndoG promotes mitochondrial stress, while the removal of EndoG exhibits protective effects. In Chapter 2, we examined the effects of amino acid (glutamine) supplementation on lipid metabolism. Using WT, Elovl5+/- fatty liver, and EndoG+/- mitochondrially healthy models, we concluded that glutamine may regulate PUFA bioconversion via Elovl5 activity, alter hepatic lipid profiles, and stimulate de novo lipogenesis. However, understanding its cellular mechanisms of action and links to inflammation required further investigation. To identify the pathway of glutamine utilization, glutamine metabolic enzymes were duly measured. WT mice only experienced decreases in the GLS2 gene, which drives the conversion of glutamine to glutamate, following glutamine feeding (Figure 14). The administration of glutamine may have been in excess therefore requiring the liver to intrinsically regulate these levels to prevent from overloading the mitochondria with excess glutamate.29 Opposingly, Elovl5+/- mice demonstrated increased gene expression levels of GLS2, GS (the conversion of glutamate to glutamine) and GDH (glutamate to α-ketoglutarate) (Figure 14). It is apparent that under these conditions, glutamine recycling is taking place via GS, and it is being pushed through GDH and GLS2 to the mitochondria where glutamine can then enter the TCA.19 EndoG+/- mice mimicked the trends in WT, but they were the opposite of what was observed in Elovl5+/- mice: GLS2, GS, and GDH gene levels were downregulated (Figure 14). EndoG+/- glutamine protein levels confirmed GDH findings (Figure 16). ASCT2 is a neutral amino acid transporter localized in the plasma membrane, and glutamine is its preferred substrate.137 Its expression levels increase in highly proliferative stem and immune cells, in order to keep up with glutamine demands.137 ASCT2 protein levels were similar between diet groups, indicating indifference between glutamine uptake and cell entry (Figure 17).138 However, significantly different diet values were noticed between the genotypes. The Elovl5+/- glutamine group had decreased ASCT2 protein levels compared to WT. EndoG+/- mice had decreased transporter 54 levels for both glutamine and alanine groups compared to WT. In these EndoG+/- livers post glutamine supplementation, the cells are restrictive of glutamine entering or being utilized by the mitochondria. In a healthy mitochondrion capable of efficiently producing citrate and oxidizing fatty acids, the entry of acetyl-CoA or other energy substrates into the TCA, renders glutamine as an unfavorable metabolic substrate. Glutamine is not preferred in the liver in the manner that fatty acids or glucose are.83 As inflammation plays a role in aging, ARDs, and is related to fat metabolism, a qPCR panel of inflammatory markers was used to monitor glutamine’s effects in WT, fatty liver and mitochondrially healthy models. MCP1 and TNFα are diverse cytokines. MCP1 is a key regulator of monocyte and macrophage immune cell migration; it is produced by cells post oxidative stress.139 TNFα is widely involved in turning on a myriad of inflammatory pathways.21 As part of the interleukin family, IL6 and IL1B promptly respond to infections and tissue injuries.21,133 IL1B specifically, is cleaved from its cytosolic precursor via caspase 1, and it regulates inflammatory responses along with cell apoptosis.133 No significant differences were measured in WT mice for any inflammatory gene (Figure 18). In contrast, glutamine increased the levels of the IL6 marker in Elovl5+/- mice (Figure 18). IL6 has pleiotropic properties depending on the secreting tissue. Previous studies have found increased exercise-induced plasma IL6 levels following glutamine supplementation to aid in the benefits of exercise.140 In the liver, increased IL6, in conjunction with other cytokines, is required for induction of a response to infection.21 ICU patients receiving parenteral glutamine have demonstrated increased IL6 and improved immune functioning.24 Other research has identified a possible effect of IL6 on increased fat metabolism and FAO in skeletal muscle and adipose tissue.141 Given our current understanding of the cytokine, within the context of an impaired PUFA metabolic model, glutamine could be further promoting multisystem fat metabolism effects through mechanisms explained in Chapter 2 and along with IL6 levels. As glutamine supplementation can stimulate IL6 to aid in metabolic processes, it may be inducing a protective effect in Elovl5+/-. The EndoG+/- mice displayed significantly reduced MCP1, IL6, IL1B, and TNFα values (Figure 18). The reduction of pro-inflammatory cytokines post glutamine feeding demonstrates a possible protective anti-inflammatory effect in mitochondrially healthier mice. These results are 55 consistent with previous studies elucidating glutamine’s anti-inflammatory effects via antioxidant defense upregulation, which is a characteristic of EndoG+/- mice.142 Mitochondrial health can be assessed via its functionality and ability grow and proliferate. ND5 is coded by mtDNA, and it is the largest of five complexes of the respiratory chain.135 In aged Elovl5+/- mouse livers, ND5 gene expression levels demonstrate a significant increase post glutamine feeding, while EndoG+/- mice display a significant decrease (Figure 19). Between genotypes, glutamine-fed Elovl5+/- mice exhibit the highest levels of ND5 compared to WT and EndoG+/-. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) is the master transcriptional regulator of mitochondria biogenesis; process by which mitochondria grow and divide.131 It is a useful indicator of mitochondrial health as well as disease progression. To assess glutamine and mitochondrial biogenesis, gene expressions levels were calculated for PGC1α. Gene levels were significantly increased in the Elovl5+/- glutamine-fed group, as opposed to the decreased levels seen in EndoG+/- mice (Figure 19). This was further supported with between-group glutamine analysis. Our Elovl5+/- model experiences defects in PUFA metabolism and can be detailed as experiencing overall decreased metabolic function. Glutamine supplementation may aid to improve mitochondrial health, and thus its functions in lipid and amino acid metabolism, by stimulating mitochondrial biogenesis.33 In our EndoG+/- model, we show that glutamine supplementation reduces mitochondrial copy number and biogenesis genes, as its heightened metabolic capabilities may create feedback to downregulate growth and proliferation.131 In conclusion, in the normal WT model, glutamine supplementation lowered glutaminase levels thus preventing excess glutamate formation, while it increased expressions of the glutamine to TCA pathway genes in the Elovl5+/- model, indicating a need for glutamine in the mitochondrial Krebs cycle. Mitochondrially healthier EndoG KO mice exhibited that glutamine supplementation abolished the increase for glutamine metabolism genes. Further, EndoG+/- mice experienced decreased inflammation in the liver post glutamine feeding. Mitochondrial biogenesis genes were stimulated in Elovl5+/- glutamine-fed mice, while downregulated in the EndoG+/- group. These findings highlight the conditionality of glutamine. Glutamine is sufficiently produced and used by the body except for in times of stress or disease where it is 56 depleted.54,24 In our models we can see that where they are healthier, WT and EndoG+/-, the mice do not require glutamine in the same manner as needed by the unhealthier Elovl5+/- model. Chapter 4: Conclusion 4.1 Limitations of Thesis Work The research presented in this thesis aims to provide a therapeutic strategy to mitigate the effects of dysregulated lipid and amino acid metabolism observed during aging. We conclude that glutamine is conditionally essential, therefore its supplementation is health-model dependent. However, these findings have several limitations. First, mitochondrial oxidative damage and antioxidant capabilities were not measured in any model. Second, it is difficult to study isolated systems in vivo, as there are biological redundancies in metabolism. Third, this study included mixed ratios of male and female mice of a broad age range between 7 to 19 months of age. This was unavoidable as COVID-related research curtailments eliminated our ongoing mouse colonies, leaving us with limited numbers of mice. To clarify such relationships further, future sex-specific studies are required to observe sex-related effects. 4.2 Conclusion With current knowledge indicating that dysregulated amino acid and lipid metabolism contribute to age-related diseases, it is vital to understand the mechanisms behind each. There is much evidence highlighting glutamine’s use as clinical care supplement, however conclusions on its benefits are contradictory. There is also much known about the changes that occur within lipid utilization and storage during aging, though specific enzymes, such as Elovl5 in PUFA metabolism, have yet to be understood. This thesis provides the following results, thus adding to the interconnected relationships between glutamine, lipid metabolism, and aging while highlighting mitochondrial and inflammatory effects: 1. In WT middle-aged mouse livers, glutamine supplementation increased blood glucose levels over time. Post glutamine feeding, GLS2 gene expression levels were decreased to prevent excess glutamate formation. WT mice also demonstrated changes in individual fatty acid levels, predominantly DGLA, possibly due to upregulated Elovl5-mediated 57 bioconversion. No other changes in triglycerides, inflammation, or mitochondrial enzymes were noticed in these mice. 2. In the Elovl5 KO fatty liver model, glutamine supplementation drove PUFA bioconversion genes FADS1, FADS2, and Elovl5 as a compensatory response to a deficient Elovl5 allele, however no individual FA differences were noted. There were also increases in GS, GLS2, and GDH gene expressions indicating the need for glutamine synthesis, breakdown, and utilization mitochondria. These results highlight the conditionality of glutamine and its beneficial use during an impaired lipid metabolic state. Lastly, glutamine induced expression of ND5 and PGC1α, indicating a possible need to promote mitochondrial metabolic activity. 3. In the mitochondrially healthier EndoG KO model, glutamine supplementation abolished gene increases in both glutamine and PUFA conversion pathways. Increases in GLA, DGLA, and EPA FAs were seen as a result of improved elongase and desaturase activity. These results mimicked what was observed in the WT but were opposite to those of Elovl5 KO. Interestingly, glutamine decreased gene expression for inflammatory cytokines, suggesting that glutamine may have anti-inflammatory properties in healthy mice. Finally, glutamine decreased mitochondrial biogenesis genes, which indicates a feedback mechanism of already highly efficient mitochondria. 4.3 Significance Though many studies can attest to the important functions of glutamine supplementation, contradictory results have been reported regarding exactly how beneficial it can be. This increases the demand for further glutamine supplementation studies in order to narrow down the likelihood of glutamine working as a treatment in humans. Even less understood are the mechanisms of action by which glutamine functions for a majority of biochemical pathways. The rapidly growing older population poses a threat to both individual humans and healthcare; exponential growth in the aging population will correspondingly result in a greater number of age-related illnesses, such as liver disease, and overload healthcare systems. With this, more studies targeting age-related diseases need to be conducted to decrease the suffering and dependability of these patients. This research will contribute to literature suggesting a more 58 holistic approach to tackling aging and disease while providing more knowledge on biological properties of glutamine. 4.4 Future Directions This thesis further adds to the breadth of understanding of glutamine and lipid metabolism in a middle-aged model. With these conclusions, further research questions have arisen which require additional study. 1. Measure glutamine and glutamate ratios inside tissues to obtain indication as to the utilization of the two within the mitochondria for energy and fat production. 2. Provide in vitro data to assess the effects of glutamine in an isolated system: measure genetic over-expressions of glutaminase 2 and glutamate dehydrogenase to highlight glutamine’s mechanisms of action. 3. 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Thesis/Dissertation
eng
Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Ghosh, Sanjoy
The role of glutamine homeostasis in the middle-aged liver
Text