Pharmaceutical Sciences, Faculty of
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UBCV
Wang, Hualin
Master of Science - MSc
University of British Columbia
Diabetes affects over half a billion people worldwide, with cardiovascular disease (CVD) as the leading cause of death among diabetes patients. Heart failure can result from diabetic cardiomyopathy (DCM). Altered cardiac metabolism contributes significantly to the development of DCM via augmenting fatty acid (FA) delivery and metabolism in the heart, resulting in oxidative stress and lipid accumulation in the heart. Inhibition of vascular endothelial growth factor B (VEGFB) has been reported to counter these factors associated with abnormal cardiac metabolism by inducing metabolic flexibility and preventing FA flux to the heart. In this study, the role of VEGFB in diabetes has been investigated as a potential therapy for DCM. Knockout (KO) of VEGFB globally in rats had subtle phenotype and transcriptome changes under normal conditions including decreased white adipose tissue (WAT) lipolysis, and decreased cardiac lipoprotein lipase (LPL) activity probably by hindering LPL translocation from cardiomyocyte (CMO) to coronary vasculature. With the induction of diabetes with streptozotocin (STZ), VEGFB KO rats displayed increased cardiac LPL activity compared to wildtype (WT) rats. This increase in LPL activity prevented animals from dyslipidemia and provided more LPL-derived FA, which inhibited excess FA oxidation, sustained angiogenesis and alleviated cell death in the heart. Inhibiting VEGFB could be a promising therapeutic strategy to address the current lack of mechanism-based treatments for DCM. Future studies on cardiomyocytes are anticipated to further explore the signaling pathways involved in VEGFB's regulation of LPL translocation, which will be crucial for advancing drug development.
Role of Vascular Endothelial Growth Factor B in the Cardiovascular Complications of Diabetes by Hualin Wang B.Sc., University of Toronto, 2022 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2024 © Hualin Wang, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Role of Vascular Endothelial Growth Factor B in the Cardiovascular Complications of Diabetes submitted by Hualin Wang in partial fulfilment of the requirements for the degree of Master of Science in Pharmaceutical Sciences Examining Committee: Dr. Brian Rodrigues, Professor, Pharmaceutical Sciences, UBC Supervisor Dr. Colin Ross, Professor, Pharmaceutical Sciences, UBC Supervisory Committee Member Dr. Nicole Krentz, Assistant Professor, Pharmaceutical Sciences, UBC Supervisory Committee Member Dr. Thomas Pulinilkunnil, Professor, Department of biochemistry and molecular biology, Dalhousie University Supervisory Committee Member Dr. Ying Wang, Assistant Professor, Department of Pathology and Laboratory Medicine, Faculty of Medicine, UBC Additional Examiner iii Abstract Diabetes affects over half a billion people worldwide, with cardiovascular disease (CVD) as the leading cause of death among diabetes patients. Heart failure can result from diabetic cardiomyopathy (DCM). Altered cardiac metabolism contributes significantly to the development of DCM via augmenting fatty acid (FA) delivery and metabolism in the heart, resulting in oxidative stress and lipid accumulation in the heart. Inhibition of vascular endothelial growth factor B (VEGFB) has been reported to counter these factors associated with abnormal cardiac metabolism by inducing metabolic flexibility and preventing FA flux to the heart. In this study, the role of VEGFB in diabetes has been investigated as a potential therapy for DCM. Knockout (KO) of VEGFB globally in rats had subtle phenotype and transcriptome changes under normal conditions including decreased white adipose tissue (WAT) lipolysis, and decreased cardiac lipoprotein lipase (LPL) activity probably by hindering LPL translocation from cardiomyocyte (CMO) to coronary vasculature. With the induction of diabetes with streptozotocin (STZ), VEGFB KO rats displayed increased cardiac LPL activity compared to wildtype (WT) rats. This increase in LPL activity prevented animals from dyslipidemia and provided more LPL-derived FA, which inhibited excess FA oxidation, sustained angiogenesis and alleviated cell death in the heart. Inhibiting VEGFB could be a promising therapeutic strategy to address the current lack of mechanism-based treatments for DCM. Future studies on cardiomyocytes are anticipated to further explore the signaling pathways involved in VEGFB's regulation of LPL translocation, which will be crucial for advancing drug development. iv Lay Summary Diabetes is a major global health issue. Following diabetes, the heart decreases the ability to use glucose and increases FA use. This alteration in energy source during diabetes causes lipid accumulation and stress in the heart. This contributes significantly to the development of DCM, which progresses to heart failure. Inhibiting VEGFB has been shown to have beneficial effects by enhancing glucose utilization and reducing FA delivery to the heart. In my study we found that whole-body VEGFB inhibition during diabetes is protective by decreasing plasma lipid, reducing oxidative stress and preventing cell death in the heart. Therefore, VEGFB inhibition could be a viable therapeutic strategy against DCM. Future research should focus on the underlying mechanism to help the drug development against DCM. v Preface All the work presented in this thesis was conducted in the Faculty of Pharmaceutical Sciences at the University of British Columbia. Our animal protocols were approved by the University of British Colombia Animal Committee (A22-0014 and A21-0052). All animal procedures performed conform to guidelines and regulations published by the Canadian Council on Animal Care. Lipid analysis was performed at the Analytical Core for Metabolomics and Nutrition, British Columbia Children's Hospital. RNA sequencing was processed at the BRC Sequencing Core, University of British Columbia. I performed the rest of the experiments, data analysis and figure generation with the help of Dr. Brian Rodrigues, Bahira Hussein, Dr. Rui Shang, and Dr. Chae Syng Lee. This thesis is written by me with the review and editing from Dr. Brian Rodrigues. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Figures ............................................................................................................................... ix List of Abbreviations ................................................................................................................... xi Acknowledgements .................................................................................................................... xiv Dedication .....................................................................................................................................xv Chapter 1: Introduction ................................................................................................................1 1.1 Diabetes ........................................................................................................................... 1 1.1.1 Complication of diabetes ............................................................................................ 1 1.2 Diabetic cardiomyopathy ................................................................................................ 1 1.2.1 Etiology of diabetic cardiomyopathy .......................................................................... 2 1.3 Normal cardiac metabolism ............................................................................................ 2 1.3.1 Glucose ....................................................................................................................... 3 1.3.2 Fatty acid ..................................................................................................................... 4 1.3.2.1 White adipose tissue ...................................................................................................... 6 1.3.2.1.1 Regulation of lipolysis ...................................................................................... 6 1.3.2.2 Cardiac intracellular TG ............................................................................................... 7 1.3.2.3 Lipoprotein lipase .......................................................................................................... 8 1.3.2.3.1 Regulation of LPL ............................................................................................. 9 vii 1.4 Cardiac metabolism following diabetes ........................................................................ 10 1.4.1 WAT lipolysis during diabetes ................................................................................. 11 1.4.2 Intracellular TG during diabetes ............................................................................... 12 1.4.3 LPL under diabetes ................................................................................................... 12 1.4.4 Consequence of increased FA supply during diabetes ............................................. 13 1.5 Vascular endothelial growth factor B ........................................................................... 14 1.5.1 Function of VEGFB .................................................................................................. 15 1.5.1.1 VEGFB overexpression ............................................................................................. 15 1.5.1.1.1 Angiogenesis ................................................................................................... 15 1.5.1.1.2. Metabolism ...................................................................................................... 16 1.5.1.1.3. Cell survival .................................................................................................... 17 1.5.1.2 VEGFB inhibition ...................................................................................................... 18 1.5.1.2.1 Angiogenesis ................................................................................................... 18 1.5.1.2.2. Metabolism ...................................................................................................... 18 1.5.1.2.3. Cell survival .................................................................................................... 19 1.6 Hypothesis..................................................................................................................... 20 1.7 Specific aims ................................................................................................................. 20 Chapter 2: Methods .....................................................................................................................21 2.1 Experimental animals .................................................................................................... 21 2.1.1 VEGFB KO rat generation ........................................................................................ 21 2.1.2 Streptozotocin-induced diabetes ............................................................................... 21 2.2 LPL activity assay ......................................................................................................... 22 2.3 Ex vivo WAT lipolysis .................................................................................................. 22 viii 2.4 Pancreatic Insulin extraction ......................................................................................... 23 2.5 Metabolic analysis ........................................................................................................ 23 2.6 Quantitative Real-Time PCR and RNA sequencing ..................................................... 24 2.7 Western blot ................................................................................................................. 25 2.8 Statistical analysis ......................................................................................................... 25 Chapter 3: Results ........................................................................................................................26 3.1 Generation of the global VEGFB KO rat ..................................................................... 26 3.2 Effect of VEGFB KO on overall glucose homeostasis ................................................. 27 3.3 Coronary vascular LPL following VEGFB KO ............................................................ 28 3.4 Effect of VEGFB KO on cardiac lipid uptake .............................................................. 33 3.5 Induction T1D in WT and VEGFB KO rat ................................................................... 36 3.6 Effect of VEGFB KO on cardiac LPL following diabetes ........................................... 39 3.7 Effect of VEGFB KO on plasma lipid profile after diabetes ........................................ 42 3.8 Effect of VEGFB KO on cardiac lipid accumulation under diabetes ........................... 46 3.9 Effect of VEGFB KO on transcriptome related to cardiac lipid metabolism during diabetes ....................................................................................................................................... 49 3.10 Effect of VEGFB KO on cardiac cell death after diabetes ........................................... 58 Chapter 4: Discussion ..................................................................................................................60 4.1 Limitations .................................................................................................................... 70 4.2 Future directions ........................................................................................................... 71 Chapter 5: Conclusion .................................................................................................................72 References .....................................................................................................................................73 ix List of Figures Figure 1. Substrate utilization in the cardiomyocyte ...................................................................... 4 Figure 2. Three main sources of FA for cardiac use ....................................................................... 5 Figure 3. LPL translocation and regulation .................................................................................. 10 Figure 4. Augmented FA supply to the heart during diabetes and implication for DCM ............ 11 Figure 5. Global VEGFB KO rat model ....................................................................................... 26 Figure 6. Effect of VEGFB KO on overall glucose homeostasis ................................................. 28 Figure 7. Effect of VEGFB KO on coronary vascular LPL ......................................................... 29 Figure 8. The role of VEGFB in LPL translocation from the CMO where it is produced to EC where it is functional ..................................................................................................................... 30 Figure 9. Lipid regulation in WT and VEGFB KO animals ......................................................... 32 Figure 10. Plasma FA profile in VEGFB KO animals ................................................................. 33 Figure 11. FA uptake in WT and VEGFB KO hearts ................................................................... 34 Figure 12. Cardiac TG along with their individual FAs in WT and VEGFB KO hearts .............. 35 Figure 13. Transcriptomic analysis of WT and VEGFB KO hearts ............................................. 36 Figure 14. STZ diabetes in WT and VEGFB KO rats .................................................................. 38 Figure 15. Pancreatic and plasma insulin and cardiac glucose utilization in WT and VEGFB KO rats made diabetic with STZ ......................................................................................................... 39 Figure 16. Effect of VEGFB KO on coronary vascular LPL after diabetes ................................. 41 Figure 17. Potential mechanism by which VEGFB KO affects coronary vascular LPL .............. 42 Figure 18. Plasma TG profile in WT and VEGFB KO control and diabetic animals .................. 44 x Figure 19. WAT lipolysis along with plasma FA in WT and VEGFB KO control and diabetic animals .......................................................................................................................................... 44 Figure 20. Plasma FA profile in WT and VEGFB KO animals during diabetes .......................... 45 Figure 21. Cartoon explaining how NEFA and LPL-derived FA contribute towards TG storage or oxidation ....................................................................................................................................... 47 Figure 22. FA uptake in WT and VEGFB KO hearts following diabetes .................................... 47 Figure 23. Cardiac FAs in WT and VEGFB KO hearts following diabetes ................................. 48 Figure 24. Cardiac TG along with their individual FAs in WT and VEGFB KO hearts following diabetes ......................................................................................................................................... 49 Figure 25. Ventricular transcriptome of rats with diabetes depicting the differentially expressed genes ............................................................................................................................................. 52 Figure 26. Dotplots of ventricular transcriptome clustered based on biological processes of rats with diabetes ................................................................................................................................. 53 Figure 27. Category netplots of ventricular transcriptome clustered based on biological processes of rats with diabetes ...................................................................................................................... 55 Figure 28. Circos plots of ventricular transcriptome clustered based on biological processes associated with diabetes ................................................................................................................ 57 Figure 29. Ventricular transcriptome with emphasis on FA metabolism and angiogenesis in rats with diabetes ................................................................................................................................. 58 Figure 30. Ventricular transcriptome and protein expression related to cell death in rats with diabetes ......................................................................................................................................... 59 xi List of Abbreviations AAV AGEs AMPK ATGL ATP BAT CMO CPT CVD DAG DCM EC ER FA FABPpm FAT/CD36 FATP G6P GLUT GPIHBP1 HFD Adeno-associated virus Advanced glycation end products AMP-activated protein kinase Adipose triglyceride lipase Adenosine triphosphate Brown adipose tissue Cardiomyocyte Carnitine palmitoyl-transferase Cardiovascular disease Diacylglycerol Diabetic cardiomyopathy Endothelial cell Endoplasmic reticulum Fatty acid Plasma membrane fatty acid binding protein Fatty acid translocase/cluster differentiation 36 Fatty acid transport protein Glucose-6-phosphate Class I facilitative glucose transporters Glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 High-fat diet xii HPLC-MS HSL Hsp25 HSPG KO LacZ LD LPL MAG MGL mTOR NEFA NRP1 PARP PDH PDK4 PKA PUFA RAAS ROS SFA SR STZ High-performance liquid chromatography-mass spectrometry Hormone-sensitive lipase Heat shock protein 25 Heparan sulphate proteoglycans Knockout Beta-glucosidase gene Lipid droplet Lipoprotein lipase Monoacylglycerol Monoacylglycerol lipase Mammalian target of the rapamycin Non-esterified fatty acids Neuropilin 1 Poly (ADP-ribose) polymerase Pyruvate dehydrogenase complex Pyruvate dehydrogenase kinase 4 Protein kinase A Polyunsaturated fatty acid Renin-angiotensin-aldosterone system Reactive oxygen species Saturated fatty acid Sarcoplasmic reticulum Streptozotocin xiii T1D T2D TCA TG VEGF VEGFB VEGFR VLDL WAT WT Type 1 diabetes Type 2 diabetes Tricarboxylic acid cycle Triglyceride Vascular endothelial growth factor Vascular endothelial growth factor B Vascular endothelial growth factor receptor Very low-density lipoprotein White adipose tissue Wildtype xiv Acknowledgements First, I would like sincerely to thank my supervisor, Dr. Brian Rodrigues, for his support and mentorship throughout my projects. His patience, availability, and dedication to my development have been a constant source of encouragement. He has not only helped me grow as a professional researcher but also taught me the importance of humility, kindness, and integrity in both professional and personal life. His passion for science and relentless work ethic inspire me to strive for excellence and to aspire to become the same type of person as you. Working under your supervision has been an incredibly enriching experience that I will carry with me throughout my future. I would also like to extend my heartfelt thanks to my lab members. Thank you, Bahira, Dr. Rui Shang, and Dr. Chae Syng Lee for your guidance, for sharing your knowledge and lab techniques, and for being both mentors and friends. Especially Bahira, our incredible lab manager, who always knows everything both in research and in life, and helped me complete my project smoothly. I am also grateful to Gala, Trevor, and Parisa for their continuous encouragement and kindness. I would like to thank my supervisory committee members, Dr. Corey Nislow, Dr. Colin Ross, Dr. Nicole Krentz, Dr. Thomas Pulinilkunnil and Dr. Ying Wang for their enlightening guidance and valuable support throughout my research. Last, I am deeply grateful to my family for their unconditional love and to my friends, Liu Li, Even, Charlie, Sylus, and Nitasha, for your constant encouragement and for bringing joy into my life. xv 献给知道我不完美但依然爱着我的家人们 王业友, 王春艳 和王华一 1 Chapter 1: Introduction 1.1 Diabetes Diabetes is one of the most prevalent metabolic diseases and causes a significant medical burden worldwide. According to the International Diabetes Federation, there were approximately 537 million people globally living with diabetes in 2021, resulting in healthcare expenditures amounting to 966 billion USD; however, the diabetic population is projected to increase significantly to 783 million by 2045, with potential costs exceeding 1054 billion USD 1. Diabetes primarily exists in two forms: Type 1 (T1D) and Type 2 diabetes (T2D). T1D accounts for 5-10 % of all diabetes, while T2D constitutes 90-95%. T1D is characterized by the destruction of pancreatic beta cells due to autoimmunity, resulting in an inability to produce insulin. In contrast, T2D is characterized by insulin resistance. However, as T2D progresses, the beta cells will also progressively lose insulin production primarily due to inflammation and metabolic stress 2,3. 1.1.1 Complication of diabetes Diabetes is associated with multiple complications, such as angiopathy, nephropathy, retinopathy, and neuropathy. Among these complications, CVD is the leading cause of death, which causes over 60 % of deaths in patients with diabetes 1,4,5. Although macrovascular disease such as atherosclerosis is a primary cause of CVD, heart failure can also result from muscle malfunction (DCM) 6-10. 1.2 Diabetic cardiomyopathy DCM is characterized by abnormal heart morphology (left ventricular remodeling and hypertrophy) and impaired heart function (ventricular dysfunction) that happens in the absence of hypertension, coronary artery disease, or valvular or congenital heart disease. Although the 2 etiology of T1D and T2D is different, DCM is observed in both T1D and T2D patients 10-13. Evidence of DCM is also reported in animal models of diabetes. These animals exhibited reduced diastolic and systolic function, alongside decreased CMO contraction with alterations in mitochondrial energetics and sarcoplasmic reticulum protein defects 9,14-16. 1.2.1 Etiology of diabetic cardiomyopathy Several factors have been associated with the development of DCM. For instance, hyperglycemia enhances the generation of advanced glycation end products (AGEs), which cause the cross-linking of connective tissue, which increases cardiac fibrosis and cardiac stiffness and impairs heart contraction. AGEs can also directly bind the various surface receptors on CMO and induce oxidative stress, inflammation and fibrosis 10,17,18. The renin-angiotensin-aldosterone system (RAAS) is also involved in the development of DCM. Angiotensin II levels increase in tissues during diabetes, resulting in the activation of the mammalian target of the rapamycin (mTOR)-S6 kinase 1 pathway, consequently causing cardiac hypertrophy and ventricular remodeling in addition to promoting insulin resistance 10,13,19-23. However, we, and others, have also suggested that alterations in cardiac metabolism during diabetes also contribute to the etiology of DCM 6,24-27. 1.3 Normal cardiac metabolism Heart is a high energy-demand organ, consuming kilograms of adenosine triphosphate (ATP) per day to maintain normal function 28. Due to its high demand for ATP, cardiac muscle utilizes multiple energy sources, primarily glucose (30%) and FA (70%). Lactate, ketones, and branched-chain amino acids are considered minor contributors to energy generation under normal conditions 29,30. As the cardiac ATP storage only supports heart pumping for a short duration, a coordinated substrate uptake and metabolism is critical for normal heart function 31. 3 1.3.1 Glucose Glucose cannot permeate through the cell membrane and must be taken up via glucose transporters. Class I facilitative glucose transporter (GLUT) family is a major transporter of glucose. GLUT1 and GLUT4 are the most predominant glucose transporters in the heart, with GLUT4 being the most dominant glucose transporter in normal adult hearts. GLUT1 is primarily present at the plasma membrane and responsible for basal cardiac glucose uptake, while GLUT4 is mainly present in intracellular vesicles and translocated to the cell surface in response to insulin stimulation 32. Once glucose enters CMO, glucose is first phosphorylated to glucose-6-phosphate (G6P) by hexokinase. Subsequently, G6P primarily goes through glycolysis in the cytosol to produce two pyruvate and two ATP 33. However, the contribution of glycolysis to whole heart ATP generation is minor under normal conditions (less than 10%) 28,34. Pyruvate then enters the mitochondria for oxidation to form acetyl-CoA under the control of the pyruvate dehydrogenase complex (PDH). Then, the acetyl-CoA produced enters the tricarboxylic acid (TCA) cycle to be fully oxidized to CO2 releasing electron carriers, NADH and FADH2. These electron carriers pass electrons to the electron transport chain to generate 34 ATP through oxidative phosphorylation 35. Besides being metabolized, glucose can also be converted to glycogen for storage for future energy demands. Glycogen exists in low amounts in the heart but is still an important energy source for the heart under aerobic conditions 36. Under normal conditions, there is rapid glycogen turnover in the heart. Glucose from glycogen is preferentially oxidized compared with exogenous glucose, and this glycogen-derived glucose contributes almost a similar amount of ATP compared to exogenous glucose 37 (Fig. 1). 4 Figure 1. Substrate utilization in the cardiomyocyte 1.3.2 Fatty acid The heart cannot synthesize FA and relies on obtaining it from other sources like i) release from adipose tissue and transport to the heart, ii) breakdown of endogenous intracellular cardiac triglyceride (TG), and iii) lipolysis of circulating TG-rich lipoproteins by cardiac LPL 6,38 (Fig. 2). Of these, LPL-mediated lipolysis of lipoproteins is suggested to be the key source of FA for cardiac use 6. Under normal conditions, exogenous FA is the preferred energy substrate, which contributes more than 50% towards ATP generation 37. 5 Figure 2. Three main sources of FA for cardiac use From the plasma, there are two ways for FAs to move into the heart. The first method is flip-flop, where FA can be passively flipped from the outer leaflet of the cell membrane to the inner leaflet without a receptor. However, this process contributes minimally to FA uptake 39,40. The second and dominant method involves receptor-mediated uptake via various FA transporters, including plasma membrane fatty acid binding protein (FABPpm), fatty acid transport protein (FATP) and fatty acid translocase/cluster differentiation 36 (FAT/CD36) 41. Of these, CD36 mediate 50-80% of the non-esterified fatty acid (NEFA) uptake 42. Once FAs enter CMO, they are converted into fatty acyl CoA in the cytosol. These fatty acyl CoA then enter mitochondria by binding carnitine to form acyl-carnitine with the help of carnitine palmitoyl-transferase (CPT) I. Once acyl-carnitine enters mitochondria, it will be converted back to fatty acyl CoA by carnitine CPT II. The fatty acyl CoA then undergoes beta-oxidation in mitochondria to form acetyl-CoA, which enters the TCA cycle and interacts with the electron transport chain to generate ATP 43. Compared with glucose, FA metabolism provides almost 5-fold more ATP, albeit with a higher oxygen requirement 43 (Fig. 1). 6 1.3.2.1 White adipose tissue In mammals, adipose tissue is made up of WAT and brown adipose tissue (BAT). The main function of WAT is to store TG and provide FA to other tissues in response to energy demand 44-46. TG makes up over 90% of the composition of white adipocytes 47. WAT can also secrete hormones and cytokines to regulate metabolic hemostasis. WAT is further classified as visceral and subcutaneous WAT based on its location. Increased visceral WAT is associated with an increased risk of metabolic and CVD 46,48,49. Lipolysis is defined as the hydrolysis of TGs catalyzed by endogenous lipases to generate FA and glycerol. It involves multiple steps with the participation of three major lipases: adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL). Initially, TG is hydrolyzed by both ATGL and HSL to release diacylglycerol (DAG) and FA. Subsequently, DAG is broken down by HSL to release monoacylglycerol (MAG) and another FA. Finally, MAG is broken down by MGL to release the third FA. The combined action of these lipases results in the generation of FAs that are released into the circulation for use by other organs 44,50. It should be noted that HSL is the rate-limiting step 51, and that the combined action of ATGL and HSL accounts for over 95% of the hydrolysis of TG in mice 52. 1.3.2.1.1 Regulation of lipolysis Multiple factors are responsible for regulating lipolysis with the most important being catecholamines, insulin and natriuretic peptides 53. Catecholamines (epinephrine, norepinephrine) induce lipolysis via β-adrenergic receptors to stimulate ATGL and HSL. On binding to β-adrenergic receptors, activation of adenylate cyclase increases cAMP to cause protein kinase A (PKA) to be active. PKA phosphorylates HSL to ensure HSL translocation to the surface of the lipid droplet (LD). PKA also phosphorylates LD-associated proteins to increase the exposure of 7 LD to lipases. Insulin has an anti-lipolytic effect by binding to adipocyte insulin receptors to initiate activation of phosphodiesterase 3B. This reduces cAMP and thus PKA activity, causing inhibition of HSL activity and thus lipolysis 44,50,51. Both catecholamines and insulin are rapid-acting factors. Natriuretic peptides are also a potent lipolytic factor via the natriuretic peptide receptor A to activate HSL, but this occurs mainly in humans 54. There are other activating factors (thyroid hormone, glucocorticoids, growth hormone and cytokines) that can also induce lipolysis mainly through altering gene transcription and protein modification of ATGL and HSL 50. Sex, age, exercise and nutrition are also important regulators of lipolysis 53. 1.3.2.2 Cardiac intracellular TG Myocardial TGs are stored within LD and distributed throughout the sarcoplasm, serving as a dynamic FA reserve for CMO to respond to energy fluctuation 55. These myocardial TG are primarily synthesized through the acylation of glycerol-3-phosphate in mitochondria as well as sarcoplasmic (SR) and endoplasmic reticulum (ER) membrane, before being packed into LD 47,55. Although heart contains relatively low ATGL and HSL, the breakdown of intracellular TG is similar to the lipolysis in adipocytes. However, the regulation of myocardial TG lipolysis is less understood compared to adipocyte TG lipolysis. Even though healthy CMOs typically contain relatively low intercellular TG, CMOs exhibit very rapid TG turnover 56. The turnover rate of intercellular TG surpasses the FA oxidation rate, indicating that in CMO, most FAs that are taken up first participate in intracellular TG generation before being oxidized. This rapid turnover is sustained by continuous cycling between TG and lipid intermediates (DAG or MAG) 57. Additionally, LDs in CMO are relatively small and located near the mitochondria, which increases the surface area and enhances contact with mitochondria, thereby ensuring rapid intracellular TG turnover 55. 8 1.3.2.3 Lipoprotein lipase (LPL) 90% of plasma FA is contained within lipoprotein-TG, which needs to be released by the lipolytic action of LPL before they are used by other organs 58. LPL is an enzyme that is located at the vascular lumen and hydrolyzes TG in circulating plasma lipoprotein. LPL is expressed in multiple organs, including the heart, skeletal muscle, and adipose tissue, with the heart having the highest expression of LPL 59. Although LPL functions at the vascular endothelial cell (EC) lumen, ECs have no capacity to produce LPL. Instead, LPL is produced by parenchymal cells such as CMO and adipocytes. Initially, LPL was thought to be synthesized as an inactive monomer, which then enters the ER to undergo dimerization. The dimerized LPL subsequently proceeds to the Golgi, where it is sorted into secretory vesicles58. However, Young’s group has also detected active LPL monomers in humans 60. Once LPL is secreted, it is anchored to heparan sulphate proteoglycans (HSPG) on the CMO surface due to ionic interaction. Subsequently, heparanase releases LPL from HSPG, which crosses the interstitial space and binds to glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) at the basolateral side of EC via an ionic interaction. GPIHBP1 helps LPL cross EC and move to the vascular lumen. At this location, LPL hydrolyzes lipoprotein-TG to FA with GPIHBP1 serving as a platform at this location 58. A recent study has also shown that LPL can stay at the EC glycocalyx on the luminal side of blood vessels without interacting with GPIHBP1 61. It should be noted that LPL plays a crucial role in regulating plasma TG and controlling tissue FA uptake. Cardiac-specific LPL KO mice exhibited hyperlipidemia and heart dysfunction; neither increased glucose utilization nor NEFA uptake can compensate for the loss of LPL-derived FA for cardiac function 62. 9 1.3.2.3.1 Regulation of LPL Since LPL is an important determinant of plasma TG clearance and tissue FA uptake, LPL activity is closely regulated via both transcriptional and post-translational mechanisms. Although multiple factors alter LPL gene transcription, most rapid changes in LPL activity, especially during fasting and exercise, are driven by post-translational mechanisms 63. It should be noted that in the heart, approximately 80 % of LPL is found in CMO with less than 20 % in capillary endothelium 64. LPL vesicles in CMO serve as a rapid-release reserve for the heart in response to energy demand 65. During energy demands, such as fasting and exercise, rather than passively relying on NEFA from WAT, AMP-activated protein kinase (AMPK), an energy sensor, is activated in CMO. The activated AMPK pathway results in the phosphorylation of heat shock protein 25 (Hsp25), leading to the dissociation of Hsp25 from actin monomers. This results in actin cytoskeleton formation and transport of LPL vesicles to the CMO surface. Eventually, this LPL moves to the vascular lumen to increase coronary LPL activity and provide adequate FA for heart contraction 66. FA has also been suggested to modulate LPL activity. Increased plasma FA induces Angptl4 expression in both plasma and CMO 67,68. Angptl4 promotes the unfolding of monomeric LPL, with subsequent loss of catalytic activity 69. In addition, FA has been shown to inhibit heparanase secretion, thus hindering LPL translocation 70. Excess FA also induces proteolysis of protein kinase D, a protein that regulates LPL vesicle fission, thus impairing LPL translocation to the EC luminal surface 71. These intricate regulatory mechanisms underscore the critical role of LPL in maintaining cardiac energy homeostasis (Fig.3). Fluctuations in LPL activity have also been associated with disruptions in cardiac metabolism 62,72, potentially leading to DCM. 10 Figure 3. LPL translocation and regulation 1.4 Cardiac metabolism following diabetes As glucose uptake and utilization are impaired due to impaired insulin signaling in diabetes, the heart is obliged to use FA almost exclusively 6,29. Multiple mechanisms operate to make this achievable including augmented adipose tissue lipolysis, greater hepatic very low-density lipoprotein (VLDL)-TG availability 73, increase in cardiac FA transporters 41, enhanced ATGL breakdown of stored cardiomyocyte TG to FA 74 and increase (depending on diabetes severity) in luminal LPL that initiates lipolysis of lipoproteins 75 (Fig. 4). 11 Figure 4. Augmented FA supply to the heart during diabetes and implication for DCM 1.4.1 WAT lipolysis during diabetes WAT responds differently depending on the type of diabetes. With T2D or obesity, there is WAT hypertrophy due to excess fat accumulation, while in T1D, WAT undergoes shrinking 76. However, both types of diabetes exhibit augmented lipolysis and increased plasma FA due to loss of insulin suppression on various adipose tissue lipases 77. Lipolysis in WAT releases various FAs, including palmitic acid, a saturated FA (SFA). Palmitic acid has been reported to contribute to increased ceramide and reactive oxygen species (ROS) generation and impaired insulin signaling 28,78. It also alters membrane composition and flexibility, leading to ER stress and heart dysfunction 79. WAT not only modulates plasma FA but also regulates plasma TG. Hypertriglyceridemia in diabetic patients is primarily driven by increased hepatic liver TG production. These hepatic TGs mainly come from dietary FA and WAT-derived FA 80. 12 Furthermore, there is a positive association between WAT lipolysis rate and insulin resistance 76. Global inhibition of ATGL or HSL in high-fat diet (HFD) mice has been shown to improve glucose uptake and utilization and alleviate lipid accumulation-induced damage in peripheral tissues, notably the heart and liver 81,82. Similar results have been shown in clinical trials of pre-diabetic or T2D patients with adipogenesis activators such as pioglitazone, which have shown improved insulin sensitivity and cardioprotective effects 83,84. 1.4.2 Intracellular TG during diabetes Myocardial TG breakdown is increased in the CMO from acute diabetic rats due to increased ATGL and HSL expression and activity 55. Although there is increased intracellular TG breakdown, there is still increased lipid accumulation in the heart. This is because besides the increased myocardial TG breakdown, there is excess FA delivery to the heart during diabetes due to loss of insulin signaling. Unlike glucose, FA oxidation requires more O2 to produce ATP 85. However, diabetic heart has a mismatch between FA delivery and O2 supply (due to diabetic microangiopathy). FA supply exceeds the mitochondrial oxidative capacity of CMOs, subsequently increasing FA conversion to TG and toxic lipid intermediates like DAG and ceramides in the CMO. These toxic lipid intermediates are reported to diminish insulin signaling and induce inflammation and heart dysfunction 64,86,87. 1.4.3 LPL under diabetes During diabetes, LPL responds in a tissue-specific manner. LPL contributes substantially to facilitating FA storage in WAT. During diabetes, WAT has decreased LPL activity 88. However, the heart shows either increased or decreased LPL activity based on the severity of diabetes 72. In moderate diabetes (STZ 55 mg/kg), Wistar rats have enhanced cardiac LPL activity and normal 13 plasma TG 72. The increased LPL activity is not due to increased LPL mRNA level or protein synthesis but is due to an increased transfer and occupation of LPL on EC binding sites 89. Wistar rats with severe diabetes (STZ 100 mg/kg) have decreased cardiac LPL activity and hyperlipidemia 72. In severe diabetes, excess plasma NEFA inhibits LPL translocation and also facilitates the inactivation and degradation of LPL 67,68,70,71. Cardiac LPL activity also influences the plasma lipid profile during diabetes. It has been shown that STZ-induced diabetic mice displayed hypertriglyceridemia without alteration in hepatic de novo lipogenesis and TG secretion. Moreover, these diabetic animals had delayed TG clearance after oil gavage due to decreased LPL activity in peripheral tissues, including the heart 90. This suggested that compared to hepatic lipogenesis, peripheral TG clearance contributes more substantially to modulating plasma TG during diabetes. As KO of LPL specifically in skeletal muscle and adipose tissue in mice had no effect on plasma TG and high-density lipoprotein cholesterol levels under normal conditions 91, this further highlights the significant role of cardiac LPL in moderating plasma lipid as compared to other tissues. In fact, simply rescuing cardiac LPL in mice that had global KO of LPL was able to protect the animals against hypertriglyceridemia and death 92. 1.4.4 Consequence of increased FA supply during diabetes Although increased FA supply is initially beneficial, it is potentially catastrophic over time. A downside to the increased FA supply, added to diabetic microangiopathy 22,93,94, is the increased accumulation of toxic lipids in the heart, which has been reported to decrease insulin sensitivity and correlate with cardiac dysfunction 64,86. Additionally, augmented FA oxidation increases ROS, which paired with the lipid metabolite accumulation, could be potentially detrimental, leading to cell death 38,95-99. In this environment, protection against excessive utilization of FA and restoring 14 metabolic flexibility in the diabetic heart would be advantageous. Targeting VEGFB has been shown to influence all the above approaches 77,100-102, and may contribute to the prevention of diabetic heart disease. 1.5 Vascular endothelial growth factor B VEGFB is a glycoprotein that belongs to the vascular endothelial growth factor (VEGF) family. VEGF family consists of 7 growth factors: VEGF A-F, and placental growth factor 103,104. Unlike VEGFA, the most extensively studied member of this family, the function of VEGFB still remains contradictory due to conflicting results. Among the VEGF family, VEGFB is most structurally close to VEGFA 164, with approximately 47% identity in the VEGF homology domain. Besides, VEGFB is highly conserved across mammals, particularly in mice and humans, sharing about 88% protein similarity 105. VEGFB has 7 exons. Due to alternative splicing in exon 6, there are two isoforms, VEGFB 167 and VEGFB 186. VEGFB is usually secreted as a homodimer of VEGFB 167 or VEGFB 186 with molecular weights 42 and 60 kDa respectively. VEGFB 186 can also be cleaved, giving a 34 kDa dimer. However, in vitro, VEGFB can also form a heterodimer with VEGFA 105. VEGFB 167 is the predominant isoform (over 80%) in normal mouse tissues, while VEGFB 186 is more prevalent in cancer cell lines 106,107. VEGFB 167 only contain partial exon 6 (exon 6b) and has a heparin-binding carboxy-terminal, helping it bind tightly to HSPG on the cell surface 104. VEGFB 186 contain the entire exon 6 and has a hydrophobic carboxy-terminal, which is freely diffusible. Both VEGFB isoforms bind to either transmembrane or soluble vascular endothelial growth factor receptor (VEGFR)1 and co-receptor neuropilin 1 (NRP1) and activate downstream signaling, but only VEGFB 186 with proteolytic processing can bind NRP1 108,109. However, Lee and colleagues 15 have recently proposed that VEGFB 167 can also bind to fibroblast growth factor receptor 1 both in EC culture and multiple mouse tissues 110. 1.5.1 Function of VEGFB One interesting aspect of VEGFB is that it seems to have no effects under normal conditions, posing challenges to understanding its role under normal conditions. Genetic KO or overexpressed VEGFB animal models are healthy and fertilized under normal conditions 103,111. The functional role of VEGFB has been determined largely through genetic modifications that either increase or decrease its expression under pathological conditions. 1.5.1.1 VEGFB overexpression Overexpressing VEGFB can i) expand the coronary vasculature (it increases capillary diameter and arterial number) 112,113, ii) regulate metabolic reprogramming (augments glucose uptake and glycolytic rates and decreases FA utilization) 112 and iii) promote cell survival (related to its antioxidant properties 114 and ability to downregulate cell death-related genes 115). 1.5.1.1.1 Angiogenesis VEGFA is considered a key regulator of angiogenesis through its binding and activation of VEGFR2 116. Although VEGFB structure is very similar to VEGFA, unlike VEGFA, VEGFB alone did not promote angiogenesis 117. VEGFB uncharacteristically targets angiogenesis by binding VEGFR1. VEGFA also binds to VEGFR1 with a 10-fold higher affinity 118 but with limited angiogenic effect 119. VEGFB overexpression increases the availability of VEGFA to bind to VEGFR2 and initiate angiogenesis 113,120-122. Compared with WT rats, VEGFA administration stimulated higher VEGFA/VEGFR2 downstream signaling in VEGFB overexpression rats 112. In rats with cardiac-specific overexpression of VEGFB, an expanded coronary vasculature (increases 16 the capillary diameter and arterial number) was observed and these transgenic hearts were protected from ischemic damage 103,104. Using an AP2/FABP4 promoter to specifically overexpress VEGFB in adipose tissue, augmented vasculature was also observed in this tissue 122. Similar results were observed in HFD fed mice transduced with adeno-associated virus (AAV)-VEGFB that demonstrated an increase in WAT vasculature. Besides, the deletion of VEGFR1 also induced angiogenesis in a similar degree to AAV-VEGFB 123. Furthermore, blocking of VEGFR2 abolished the angiogenesis effect of AAV-VEGFB in both heart and WAT 122,123. However, there are some contradictory results. Expressed VEGFB via AAV failed to provoke an angiogenesis response in the ischemia limb muscle, rabbit carotid arteries and mouse eye 115,124,125. These contradictory results are probably due to the different VEGFB isoforms used; most early studies mainly used VEGFB 167 115,124,125. It is actually VEGFB 186 that induces angiogenesis rather than VEGFB 167. AAV transfers different VEGFB isoforms in pig myocardium; only VEGFB186 had an effective increase of capillaries 126,127. Previous studies from our lab and our collaborators also supported that cardiac-specific overexpression of VEGFB in rats predominately increases VEGFB186 expression, and these rats have increased cardiac vasculature 86,112. However, Lee and colleagues have recently proposed another novel explanation that VEGFB can also be anti-angiogenic in mouse retinae via suppressing fibroblast growth factor 2 mediated vessel formation 110. Collectively, overexpressing VEGFB has been largely proven to be able to indirectly induce angiogenesis by increasing the binding of VEGFA to VEGFR2 103,104,113,120-122. However, further studies are required to investigate the exact mechanism of VEGFB-induced angiogenesis. 1.5.1.1.2 Metabolism VEGFB is mainly expressed in highly metabolically active organs like the heart, skeletal muscle and adipose tissue 112. Besides, the expression of VEGFB and mitochondrial genes and proteins 17 are coordinately regulated 128. These data imply that VEGFB may play an important role in metabolism. In animals that overexpress VEGFB in adipose tissue, improved glucose and insulin tolerance were observed along with an inhibition of WAT lipolysis despite HFD. These effects were suggested to be a consequence of improved vascular perfusion and thus augmented insulin delivery 122. Similarly, in rats with cardiac-specific overexpression of VEGFB, improved insulin sensitivity and glucose uptake with higher intermediate products related to glycolysis and glycogenolysis under normal conditions were observed, indicating that these hearts were using more glucose 86,112. Unlike its effect on glucose metabolism, there was no alteration of cardiac FA uptake, and a decreased gene expression of proteins involved in FA metabolism 112. Additionally, these hearts had a decrease in cardiac LPL activity at the vascular lumen, the rate-limiting enzyme for exogenous FA delivery to the heart 86. It should be noted that conflicting results have also been reported with VEGFB treatment and overexpression. For instance, in transwell 2 chamber systems, treating EC with VEGFB has been reported to reduce glucose delivery to underlying tissue cells via inhibiting GLUT1, resulting in impaired glucose utilization 129. Furthermore, VEGFB treatment on cardiac EC has been shown to upregulate FATP3 and FATP4 expression with subsequent trans-endothelial FA transfer, resulting in an increased risk of cardiac lipotoxicity 100. Overall, VEGFB overexpression has been generally accepted to induce insulin sensitivity and switch the heart from FA to glucose utilization 86,112,122,130. 1.5.1.1.3 Cell survival Animal studies have acknowledged a favorable role for VEGFB in cell survival 112,121,130-134. Administering recombinant VEGFB protein or expressing VEGFB via AAV protects the CMO against damage from H2O2-induced oxidative stress 131 and doxorubicin-induced cardiomyopathy 133. VEGFB therapy also protects the heart against transverse aortic constriction 132, pacing-18 induced dilated heart failure 134, and hypoxia/reoxygenation apoptosis 130. Additionally, rats with cardiac-specific overexpression of VEGFB have smaller infarct size and preserved heart function in the ischemia and reperfusion damage 112. However, pathological angiogenesis, together with inflammation, has been reported in VEGFB overexpression mouse WAT 77, questioning the cell protection effect of VEGFB. 1.5.1.2 VEGFB inhibition Although overexpression of VEGFB is thought to be largely beneficial, there is also evidence indicating that inhibiting VEGFB is also helpful. 1.5.1.2.1 Angiogenesis Adipose tissue-specific VEGFB knockdown mice exhibit comparable vascularization in WAT and decreased inflammation induced by HFD compared to WT mice 77. Similarly, VEGFB KO mice or rats are viable and fertile, with smaller heart sizes but normal cardiac vasculature and heart function under normal conditions 112,135. However, conflicting results from two independent VEGFB KO mice stains indicated that VEGFB KO has no impact on heart size 136. One explanation for these diverse results could be the different animals used along with the diverse methodology employed. 1.5.1.2.2 Metabolism Intriguingly, inhibition of VEGFB has no obvious metabolic change unless these animals were challenged 77,112,136. When fed a HFD, VEGFB KO mice exhibited enhanced glucose and insulin tolerance and ameliorated dyslipidemia 102,137. Interestingly, db/db VEGFB KO diabetic mice also showed increased glucose uptake and decreased lipid accumulation in the heart, skeletal muscle and pancreatic islet 102. Additionally, an improved cardiac glucose uptake was also observed in 19 HFD mice treated with VEGFB neutralizing antibodies (2H10) 129. Reduced expression of FATP3 and FATP4 in cardiac EC and decreased lipid droplets in cardiomyocytes were also observed in VEGFB KO mice 100. With the decreased lipid delivery to the heart, VEGFB KO mice demonstrated increased GLUT4 expression and glucose uptake to the heart 100, indicating that VEGFB KO has the potential for re-establishing the physiological utilization of glucose. Collectively, VEGFB KO improves insulin sensitivity, which is probably due to the decreased lipid delivery to peripheral tissues. Adipocyte-specific VEGFB deficient mice showed decreased WAT lipolysis, decreased HSL activation, decreased plasma FA level, and increased glucose uptake to WAT under HFD, compared with control mice 77. Additionally, VEGFB KO mice had decreased lipid uptake in the heart, skeletal muscle and BAT with increased fat deposition in WAT 100. VEGFB has also been reported to protect the liver 77 and kidney 101 against lipotoxicity during diabetes. These results suggested that inhibition of VEGFB could prevent lipid accumulation in non-adipose tissues by increasing the sensitivity of WAT to insulin. 1.5.1.2.3 Cell survival VEGFB inhibition is also a potential survival factor. VEGFB is upregulated in various tumors, and this upregulation is associated with tumor progression by promoting tumor vasculature 111,138,139. However, tumors with VEGFB inhibition tended to have a larger size independent of tumor vasculature, inflammation and alteration of lipid metabolism 140, suggesting that VEGFB KO also promote cell survival and this improved tumor survival was probably due to a direct survival effect of VEGFB KO. However, the molecular mechanism underlying the cell survival related to VEGFB inhibition was enigmatic. A recent novel study from Zhang’s group gave insight into the potential molecular mechanism: melatonin ameliorates DCM by upregulating autophagy through 20 suppressing VEGFB. Overexpression of VEGFB in mice eliminated the melatonin-induced cardioprotective effect 141, suggesting that VEGFB inhibition augments cell survival by autophagy. Altogether, suppression of VEGFB has largely proven to be supportive of heart function through effects related to metabolism under HFD. As excessive FA flux and metabolic inflexibility are major contributors towards DCM 142, and given the role of VEGFB inhibition to counter these effects, blocking VEGFB action may be a promising therapeutic approach to protect against the progression of heart failure following diabetes. However, the current beneficial effects of VEGFB inhibition are predominantly observed in mice under HFD, and no significant differences were reported in rats. Additionally, HFD supports T2D development. Whether VEGFB KO would be helpful in the heart under T1D is currently unknown. 1.6 Hypothesis My hypothesis is that inhibition of VEGFB will help defend the myocardium against diabetes-induced susceptibility to cardiomyopathy. 1.7 Specific aims My specific aims are to: i) elucidate the phenotype associated with global KO of VEGFB, ii) determine whether VEGFB suppression can affect FA delivery to the heart, and iii) reveal the metabolic changes that are associated with VEGFB KO and diabetes induction. 21 Chapter 2: Methods 2.1 Experimental animals Our animal protocols were approved by the University of British Colombia Animal Committee (A22-0014 and A21-0052). All animal procedures performed conform to guidelines and regulations published by the Canadian Council on Animal Care. In this study, all animals had free access to food (LabDiet PicoLab Rodent 20 No. 5053) and reverse osmosis water and were housed in standard cages with 12 hours of light/dark cycles. VEGB KO and WT littermate male rats 7-12 weeks old were used for various studies. Rats were euthanized using a single dose of 100 mg/kg intraperitoneal injection of sodium pentobarbital (Euthanyl). Thoracotomy was performed for organ collection when the toe pinch and corneal reflexes were absent. 2.1.1 VEGFB KO rat generation VEGFB KO rats on the Sprague Dawley background were generated with zinc-finger nuclease technology by our collaborator, Dr. Alitalo 112. Beta-glucosidase gene (lacZ) was inserted into the exon1 region of VEGFB to disrupt the VEGFB gene. PCR was run on rats’ ear notch samples with the primers 5’-CCTGCTCCGTCGCTTGCTG-3’, 5’GGTCTGCTTTCTGACAAACTCG-3’ (VEGFB) and 5’-GGGGCCATCAAACTGGGAC-3’(lacZ) to check the genotyping of rats. 2.1.2 Streptozotocin (STZ)-induced diabetes The selective beta-cell toxin STZ (Sigma) is used to lower insulin and generate a rat model of poorly controlled T1D 68,75. With 55 mg/kg STZ (D55; i.v.), animals are hyperglycemic and hypoinsulinemic. D55 animals were monitored for 4 days after injection of STZ. D55 animals mimic insufficient glycemic management in T1D where multiple finger pricks and daily insulin 22 injections could mean poor patient compliance and repeated exposure to bouts of hyperglycemia. Given the sex differences in the susceptibility to STZ diabetes 143, only male rats were used. 2.2 LPL activity assay LPL activity was performed as previously described 86,144. Briefly, the heart was retrogradely perfused with Ca2+-free Krebs-Ringer HEPES buffer to clear blood from the coronary vasculature. Subsequently, buffer was switched to fresh Ca2+-free Krebs-Ringer HEPES buffer containing 5 U/mL heparin (Sandoz) via the Langendorff technique to displace LPL from the coronary vasculature at a flow rate of 8 ml/min. The coronary perfusates were collected for 15 seconds over 10 minutes. The LPL activity was measured through the hydrolysis of a [3H] triolein (Perkin-Elmer) substrate emulsion. The perfusate samples mixed with the reaction mix (piperazine-N,N’-bis(2-ethanesulfonic acid), albumin, MgCl2 and chicken serum) were incubated at 30°C for 30 min. Sodium [3H] oleate was extracted via centrifugation at 4°C and 3500 rpm for 30 min, and measured by liquid scintillation counting. 2.3 Ex vivo WAT lipolysis Fresh pararenal WAT was collected and incubated in prewarmed DMEM (Thermo Fisher Scientific). During the ex vivo lipolysis assay, WAT was dissected into small pieces (approximately 25 mg) and immediately incubated in 200 μl DMEM containing 2% FA-free BSA (Sigma) for 1h in the cell culture incubator (37°C, 5% CO2). After incubation, the medium was collected, and NEFA in the medium were determined by a commercial kit (Abcam). The lipolysis rate was also normalized by the weight of WAT used (NEFA μM/mg tissue). 23 2.4 Pancreatic insulin extraction Pancreatic insulin was extracted by the acid-ethanol method. Briefly, frozen pancreas powder was homogenized and incubated in Acid-Ethanol (1.5% HCl in 70% Ethanol) solution overnight at -20 °C. Aqueous layer was obtained after centrifuging at 2000 rpm for 15 minutes, followed by neutralizing Acid-Ethanol aqueous extract with 1M Tris (pH 7.5) in 1:1 ratio. 2.5 Metabolic analysis Tail vein blood glucose was measured by a glucometer (AccuSoft) and glucose test strips (Accu-Chek Advantage). Blood was collected from the thoracic cavity by an EDTA-coated tube (Sigma) and centrifuged (1500 rpm at 4°C for 15 minutes ) to collect plasma. Plasma NEFA (Abcam) and TG (Stanbio) were measured by commercial kits using the manufacturer’s protocols. Plasma insulin and extracted pancreatic insulin were detected by the insulin ELISA kit (ALPCO). The pancreatic insulin concentration was normalized by the protein concentration of neutralized insulin extraction. Plasma and cardiac individual FAs and TG were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS; Waters 2690 Alliance HPLC, Milford, MA) as previously described 72,86. Briefly, plasma and cardiac lipids were extracted and solubilized by the modified Folch method with chloroform:methanol:acetone:hexane (4:6:1:1 v/v/v/v). The chromatographic separation of TG and FA was performed on a YMC DIOL column (4.6×250 mm, YMC, Asan). The liquid chromatography flow was divided, with approximately 80% directed to an FCII fraction collector (Waters, Milford, MA), and the remaining 20% directed to the evaporative light scattering detector (Waters, Milford, MA). Peak areas were quantified using Chemstation software (Agilent Technologies). Lipid analysis was performed at the Analytical Core for Metabolomics and Nutrition, British Columbia Children's Hospital. 24 2.6 Quantitative Real-Time PCR and RNA sequencing RNA from the frozen VEGFB KO and WT heart ventricles was extracted via TRIzol (Invitrogen). Briefly, 20-50 mg frozen ventricle tissue powder was homogenized with 1 ml TRIzol reagent via glass homogenizer. Extracted RNA was washed with ethanol three times and dissolved in DEPC water. The quality and concentration of mRNA for quantitative Real-Time PCR were determined via the nanodrop machine. Qualified mRNA was first reversely transcribed into cDNA, followed by cDNA amplification via TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and Taqman primer of targets (FATP4: Rn01438951_m1, CD36: Rn00580728_m1, Rat VEGFB: Rn01454585_g1 and B2M: Rn00560865_m1) in StepOnePlus Real-Time PCR machine (Applied Biosystem). Gene expression was calculated based on comparative cycle threshold (ΔΔCT) with B2M as reference genes. The integrity of RNA samples for RNA sequencing was checked for quality by Bioanalyzer (Agilent Technologies) and quantified using a Qubit fluorometer (Thermo Fisher Scientific). The sequencing library was prepared by using NEBNextâ Ultra II directional RNA library Prep Kit (Illumina) and sequenced using Illumina Nextseq500 (Illumina), collecting a total of 20 million paired reads. Multiple pipelines have been applied to align to the rat reference transcriptome genome (Rattus_norvegicus.mRatBN7.2.112) and quantify for count reads at the transcript level for each gene for all samples as previously described 72,145. Differential expression analysis was performed with DESeq2 and edgeR packages in RStudio. No outliers were identified during sample clustering, so all samples were included in the differential expression analysis. The output from each pipeline was filtered for multiple tests with a cutoff: adjusted p-value less than 0.05 to ensure statistical significance. The filtered data list was then ranked by the adjusted p-value for 25 differential expression. A combined output from all pipelines was then created by ranking the genes based on their median values. The differential expression was also determined by log2 fold change between control and diabetic rats within their own genotyping group. The secondary cut-off is an absolute log2 fold change larger than 0.27 to ensure biological significance. The network analysis and functional analysis were conducted via the clusterProfiler in RStudio with hypergeometric testing to calculate the adjusted p-values for each category. RNA sequencing activities were performed at the BRC Sequencing Core, University of British Columbia. 2.7 Western blot Western blot analysis was performed as previously described 131. Protein extracted from the heart ventricle was visualized by Li-COR Odyssey CLX digital imaging system, and bands were quantified by Image Studio™. Antibodies used in this project are listed: anti-α-Tubulin DM1A (Millipore, 05-829), anti-vinculin (Cell signaling, 13901), anti-LPL 5D2 (Santa Cruz, sc-73646), PDK4 (Abcam, ab214938) and Poly (ADP-ribose) polymerase (PARP) (Cell signaling, 9542). 2.8 Statistical analysis All statistical analysis was generated via Graph Prism 10.0 (GraphPad Inc, San Diego, CA, USA). The Shapiro–Wilk test was performed to determine the normality of the data. For normalized data, unpaired Student’s t test, or Two-way ANOVA followed by Bonferroni’s post-hoc comparisons test was performed to determine the statistical significance. Otherwise, nonparametric analysis with the Mann-Whitney test or Wilcoxon test was performed. The results are presented as mean ± SEM with a P value < 0.05 against the negative control pair, which was considered significant. 26 Chapter 3: Results 3.1 Generation of the global VEGFB KO rat To investigate the function of VEGFB, our collaborator, Dr. Alitalo generated a global VEGFB KO rat using zinc finger nucleases with beta-galactosidase knock-in to disrupt the VEGFB gene (Fig. 5A). The gel electrophoresis image illustrated genotyping results of gDNA extracted from heterozygote, WT and VEGFB KO rat ear notch samples. Our results confirmed that the VEGFB gene was indeed deleted from VEGFB KO rats (Fig. 5B). Quantitative RT-PCR on the VEGFB KO rat heart ventricle (Fig. 5C) and skeletal (Fig. 5D) muscle also showed no VEGFB gene expression as compared with the WT group. Our data successfully validated the global KO of VEGFB in the rat. Figure 5. Global VEGFB KO rat model A: Process of generating a systemic VEGFB gene knockout rat by zinc finger nucleases with beta-galactosidase knock-in. B: PCR amplification of gDNA extracted from heterozygote, WT and VEGFB KO rat ear notch samples, confirming VEGFB gene deletion via beta-galactosidase WT VegfbKO0.00.51.01.5VEGFB mRNA level (fold change)✱✱✱✱WT VegfbKO0.00.51.01.52.0VEGFB mRNA level (fold change)✱✱✱✱AB C D Heart Ventricle Skeletal Muscle27 knock-in. C&D: mRNA expression of VEGFB in WT and VEGFB KO rat heart ventricle (n=7-25) and skeletal muscle (n=5-6) as determined using Quantitative RT-PCR, confirming VEGFB gene deletion. Data are presented as mean ± SEM. Unpaired t-tests were used to compare the two groups, n=5-25, **** p < 0.0001. 3.2 Effect of VEGFB KO on glucose homeostasis VEGFB has been reported to alter glucose metabolism in the heart 86,102. We investigated whether deletion of VEGFB influences basic glucose metabolism in rats under normal conditions. There was no difference in body weights between WT and VEGFB KO rats up to 9 weeks of age (Fig. 6A). Measurement of blood glucose showed no difference between the two groups of rats (Fig. 6B). Insulin plays a crucial role in regulating glucose metabolism 146. Previously, beta cell-specific VEGFB KO in mice has been demonstrated to increase insulin production and secretion 147. In our experiment, measurement of pancreatic (Fig. 6C) and plasma (Fig. 6D) insulin indicated that global KO of VEGFB did not influence insulin production or secretion. We also measured cardiac glucose metabolism by determining pyruvate dehydrogenase kinase 4 (PDK4) protein, a key inhibitor of glucose utilization 148. PDK4 levels in the ventricle were comparable between WT and VEGFB KO rats under normal conditions, demonstrating that VEGFB KO was unable to influence glucose utilization in the heart (Fig. 6E). Together, our data suggested that deleting VEGFB in rats had no significant influence on the overall glucose homeostasis under normal conditions. 28 A: Body weight of male rats at 9 weeks, n=5-8. B: Tail vein blood glucose, n=10-16. C: Pancreatic insulin, n=4-5. D: Plasma insulin levels, n=8-12. E: PDK4 level in heart ventricle. The inset represents the Western Blot of the heart samples, n=10-20. Data are presented as mean ± SEM. Unpaired t-tests or Mann-Whitney tests were used to analyze the data. 3.3 Coronary vascular LPL following VEGFB KO An important enzyme that regulates lipid metabolism is LPL 6. To address the influence of VEGFB KO on lipid metabolism, isolated hearts were retrogradely perfused with heparin. The LPL displaced from the luminal surface of coronary blood vessels into the perfusion buffer was collected, and its activity was subsequently measured (Fig. 7A). The addition of heparin induced a rapid increase in LPL activity in the coronary perfusate, with peak LPL activity appearing within the first minute and quickly returning to basal levels within two minutes (Fig. 7B). Interestingly, hearts from KO animals exhibited a significant reduction in peak LPL activity compared to WT (Fig. 7C). This reduction in activity occurred in the absence of any change in LPL protein from WT VegfbKO012345Plasma insulin (ng/mL)WT VegfbKO020406080100Pancreatic insulin(ng /µg of protein)A BWT VegfbKO0.00.51.01.52.02.5PDK4 protein expression (fold change) DWT VegfbKO051015Blood glucose (mM)WT VegfbKO0100200300400500Body weight (g)CEFigure 6. Effect of VEGFB KO on overall glucose homeostasis 29 ventricle muscle (Fig. 7D). This discrepancy between vascular lumen surface LPL activity and LPL protein level suggested that VEGFB may play an important role in LPL translocation, and its KO obstructs LPL movement from CMO to the vascular lumen (Fig. 8). Figure 7. Effect of VEGFB KO on coronary vascular LPL A: Vascular LPL activity was determined by perfusing the isolated heart with the highly negatively charged heparin and collecting the coronary perfusate over time for the determination of LPL activity. B: Representative example of heparin-releasable LPL activity from WT control heart following 10 minutes of heparin (5 U/mL; arrow) perfusion. Fractions of perfusate (over 15 seconds) were collected at the indicated times and analyzed for LPL activity. C: Peak heparin-releasable LPL activity of each animal was normalized to the peak of WT control rats. Only the fold change is presented, n=4. D: Western blot of LPL protein in intact hearts isolated from different groups of rats, n=10-14. The inset represents a representative example of WT and WT VegfbKO0.00.51.01.52.02.5LPL protein expression(fold change)-1 0 1 2 3 5 6 7 8 9 1001000200030004000Time (min)LPL activity (nmol/h/mL) heparinA BC DWT VegfbKO0.00.51.01.5Peak LPL activity(fold change) ✱✱✱✱30 VEGFB KO heart samples. Data are presented as mean ± SEM. Unpaired t-tests or Mann-Whitney tests were used to analyze the data, **** p < 0.0001. Figure 8. The role of VEGFB in LPL translocation from the CMO where it is produced to EC where it is functional As the main role of LPL is to clear plasma TG, we investigated whether the reduced LPL activity in VEGFB KO rats leads to increased plasma TG due to a reduction in TG clearance. Surprisingly, we were unable to detect any difference in plasma TG between the WT and VEGFB KO groups (Fig 9A). It is possible that this effect is a consequence of a reduction in the hepatic secretion of TG, thus providing a mechanism to explain this normal plasma TG (Fig. 9B). One factor that controls the liver secretion of TG is the amount of FA taken up by the liver. As the majority of plasma FA comes from WAT lipolysis 149, ex vivo WAT lipolysis was conducted to investigate 31 the ability of WAT to release FA. WAT from VEGFB KO rats released significantly less FA compared to WAT from WT (Fig. 9C), suggesting that the substrate required for TG synthesis and its subsequent secretion from the liver is reduced. Thus, we propose that the reduced TG clearance is balanced by a decrease in hepatic TG secretion, such that total plasma TG remains unchanged. Total plasma FA was also assessed as an indirect indicator of WAT lipolysis. It was quite surprising that there was no difference in both total plasma FA (Fig. 10A) and individual plasma FAs (Fig.10 B-D) between WT and VEFGB KO rats. Additionally, plasma FA composition also showed no change between the two groups (Fig.10E). Thus, the plasma FA profile did not match WAT lipolysis data. At this point, it remains unclear why decreased WAT lipolysis did not result in decreased plasma FA. It is possible that VEGFB KO probably reduces FA uptake in peripheral tissues such as skeletal muscle and BAT, a mechanism to explain the unchanged plasma FA. In summary, our data suggest that deleting VEGFB in rats reduces WAT lipolysis under normal conditions but does not affect the plasma lipid profile. 32 Figure 9. Lipid regulation in WT and VEGFB KO animals A: Plasma TG in the two groups of animals, n=14-20. B: Schematic image describing how adipose tissue-derived FA contributes towards hepatic TG synthesis. C: Ex vivo WAT lipolysis using perirenal WAT and determination of FA released into the incubation medium after a one-hour incubation, n=3. Data are presented as mean ± SEM. Unpaired t-tests were used to analyze the data, ** p < 0.005. 33 Figure 10. Plasma FA profile in VEGFB KO animals A: Total plasma FA level in the two groups, n=9-10. B-D: Percentage of key FAs in the plasma, n=5-6. E: Overall contribution of key FAs that make up the plasma composition, n=5-6. Data are presented as mean ± SEM. Unpaired t-tests were used to analyze the data. 3.4 Effect of VEGFB KO on cardiac lipid uptake Besides LPL and plasma lipids, we were also interested in the relationship between VEGFB and cardiac lipid uptake. It has been reported that VEGFB also plays a role in cardiac FA uptake via its regulation of FA transporters 100,112. We determined FATP4 and CD36 in the heart to investigate whether VEGFB KO affects cardiac FA uptake. We did not see a change in either of the FA transporters measured. Thus, despite VEGFB KO animals having decreased cardiac LPL activity, both WT and VEGFB KO rats had similar cardiac FATP4 and CD36 levels (Fig. 11 B&C). Since the expression of FA transporters may not accurately reflect FA uptake in the heart, we also measured cardiac FA content via HPLC-MS. In line with FA transporter data, there was no WT VegfbKO05101520% Palmitic acid16:0 WT VegfbKO010203040% Linoleic acid18:2n6 WT VegfbKO051015% Oleic acid18:1n9 WT VegfbKO050100Plasma FA composition %SFAMUFAPUFAAD EB CWT VegfbKO020406080100Plasma FA (µM)34 difference in the accumulation of individual cardiac FAs (Fig. 11 D) or overall cardiac FA composition (Fig. 11E). Since FAs can be converted to intracellular TG in the heart, we also measured cardiac TG. Cardiac TG was extracted, separated, and evaluated using HPLC-MS. Comparable total cardiac TG content and FA percentage within these cardiac TGs between the two groups further supported that although VEGFB KO rats had a decreased LPL activity, it did not affect cardiac lipid take and storage under normal conditions (Fig. 12A&B). Altogether, loss of VEGFB has no influence on lipid metabolism under normal conditions. Figure 11. FA uptake in WT and VEGFB KO hearts A: Schematic of different mechanisms that are responsible for FA uptake into the heart. B: FATP4, (n=6-12). C: CD36, (n=5-10). D: Key cardiac FAs percentage in both groups of hearts, n=5-6. E: Overall contribution of key FAs that make up the cardiac FA composition. Data are presented as mean ± SEM. Unpaired t-tests or Mann-Whitney tests were used to analyze the data. DWT VegfbKO01234FATP4 mRNA level (fold change)BWT VegfbKO0.00.51.01.52.0CD36 mRNA level (fold change)CBEAPalmitic acid Stearic acid Oleic acid Linoleic acid Arachidonic acid0102030Percentage of fatty acids WTVegfbKOWT VegfbKO050100%FA composition in heartMUFAPUFASFA35 Figure 12. Cardiac TG along with their individual FAs in WT and VEGFB KO hearts A: Cardiac TG content, n=5-6. B: The percentage of individual FAs that make up cardiac TG, n=5-6. Data are presented as mean ± SEM. Data are presented as mean ± SEM. Unpaired t-tests were used to analyze the data. Overall, in rats with global KO of VEGFB, no major phenotype changes were observed in both glucose and FA metabolism, except for the decreased cardiac LPL activity. Nevertheless, we conducted RNA sequencing for heart ventricle muscle from WT and VEGFB KO rats. Based on the principal component analysis, WT and VEGFB KO points were intermixed with no clear separation. This suggested that VEGFB KO had only a subtle effect across the two groups. The gene expression of WT and KO groups was similar, and the differences might not be significant enough to be captured by the principal component analysis (Fig. 13A). The Spearman correlation heatmap also showed similar results. In the top right and bottom left corners of the heatmap, most of the cells were orange and yellow, suggesting that the deletion of VEGFB under normal WT VegfbKO0246ugTG/mg tissueBC 16:0 C 18:0 C 18:2n6 C 20:4n6 C 22:6n3010203040Percentage of fatty acids WTVegfbKOA36 conditions did not cause a significant change in gene expression. Additionally, upon examining individual cells, we found that the impact of VEGFB on gene expression was not as pronounced as the batch effect or other interfering factors (Fig. 13B). In summary, global VEGFB deletion did not affect cardiac gene expression under normal conditions. It remains unknown whether the absence of VEGFB will impact the heart following diabetes. Figure 13. Transcriptomic analysis of WT and VEGFB KO hearts A: Principal component analysis, n=5. B: Spearman’s correlation heatmap, n=5. 3.5 Induction T1D in WT and VEGFB KO rat Interestingly, previous research has focused predominantly on the VEGFB KO mice. They also found VEGFB KO animals had no phenotype changes until exposed to HFD, a model of insulin resistance model and T2D 77,101,136,150,151. We induced T1D via STZ in rats. STZ can specifically kill beta cells, which are insulin-secreting cells, resulting in a hypoinsulinemic model (similar to the T1D model). Rats injected with 55 mg/kg STZ exhibited hyperglycemia within 24 hours. This level of hyperglycemia was maintained up to 4 days after STZ injection (Fig. 14A). At this time, A B−5.0−2.50.02.55.0−4 0 4PC1: 27% variancePC2: 23% varianceGenotypeKOWT37 the body weights from the two diabetic groups were significantly reduced after 4 days of diabetes compared to their respective controls. Moreover, the percentage drop in body weight in WT and VEGFB KO diabetic rats was the same (Fig. 14B). When pancreatic insulin was determined, both diabetic groups had significantly decreased levels compared with their own controls (Fig. 15A), an effect that was mirrored by the significant drop of plasma insulin in these diabetic groups (Fig. 15B). The reduction in pancreatic insulin and plasma insulin levels were comparable in both diabetic groups (Fig. 15 A & B). Upon assessing cardiac PDK4 levels, it was found that both diabetic groups exhibited significantly higher PDK4 levels compared to their respective controls, suggesting that both groups had a comparable reduction in cardiac glucose utilization during diabetes (Fig. 16C). Altogether, these data indicate that WT and VEGFB KO rats are equally susceptible to STZ, demonstrating a similar intensity of diabetes. 38 Figure 14. STZ diabetes in WT and VEGFB KO rats A: Blood glucose change over the 4 days of STZ diabetes. n=4-9. B: Body weights of WT and VEGFB KO rats at day 0 and after 4 days of STZ diabetes. Two-way ANOVA with repeated measures was used to analyze the data, n=10-12. The inset represents the percentage of body weight loss after 4 days of STZ diabetes in both groups of animals. Unpaired t-tests were used to analyze the data, n=10-12. Data are presented as mean ± SEM，****p < 0.0001. 0 4 0 40100200300400500Body weight (g) WT VegfbKODay✱✱✱✱ ✱✱✱✱WT VegfbKO051015Body weight loss %nsSTZ0 1 4010203040Blood glucose (mM)WTWTVegfbKOVegfbKOSTZConDays after STZ injectionSTZA B39 Figure 15. Pancreatic and plasma insulin and cardiac glucose utilization in WT and VEGFB KO rats made diabetic with STZ A: Pancreatic insulin levels in the different groups were extracted and measured by an insulin ELISA, n=4-8. B: Plasma insulin in the different groups as measured using an insulin ELISA. n=8-15. C: PDK4 level in heart ventricle. The inset represents the Western Blot of the heart samples, n=5-20. Data are presented as mean ± SEM. Two-way ANOVA was used to analyze data, **** p < 0.0001. 3.6 Effect of VEGFB KO on cardiac LPL following diabetes Insulin plays an important role in regulating LPL activity. When insulin decreases, cardiac LPL activity also decreases during severe diabetes 152. As indicated in Figure 7C, we found that VEGFB KO decreased LPL activity at the vascular lumen surface. Since insulin levels were similarly Con STZ Con STZ012345Plasma insulin (ng/mL)WT VegfbKO✱✱✱✱✱✱✱✱Con STZ Con STZ024620406080Pancreatic insulin(ng /µg of protein)WT VegfbKO✱✱✱✱ ✱✱✱✱ACCon STZ Con STZ01234PDK4 protein expression (fold change) WT VegfbKO✱✱✱✱ ✱✱✱✱B40 reduced during diabetes, we expected a similar trend of LPL activity between the two groups. As expected, WT diabetic rats significantly reduced peak LPL activity compared to their own control. Intriguingly, the opposite effect was observed with the VEGFB KO diabetic rats, which showed an increase of peak LPL activity compared to their own controls (Fig. 16A). In fact, when the two diabetic groups were compared, VEGFB KO diabetic group had a significantly higher peak LPL activity compared to the WT diabetic rats (Fig. 17A). This difference in LPL activity was not matched by LPL protein in heart ventricle muscle, which remained the same in all 4 groups (Fig. 17B). Our data suggests that in VEGFB KO animals, the low LPL activity in control animals is because the LPL protein accumulates inside the CMO with limited movement to the EC surface. However, following diabetes, these accumulated LPL proteins start to move to the EC lumen surface to increase luminal LPL activity (Fig. 17). 41 Figure 16. Effect of VEGFB KO on coronary vascular LPL after diabetes A: Peak heparin-releasable LPL activity of each animal was normalized to the peak LPL of WT control rats. Only the fold change is presented, n=4-5. B: Western blot of LPL protein in intact hearts isolated from different groups of rats, n=5-14. The inset is a representative example of WT and VEGFB KO heart samples following diabetes. Two-way ANOVA was used to analyze data. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.005 Con STZ Con STZ0.00.51.01.52.02.5LPL protein expression(fold change)WT VegfbKOA BCon STZ Con STZ0.00.51.01.52.0Peak LPL activity(fold change) WT VegfbKO✱✱✱✱✱✱42 Figure 17. Potential mechanism by which VEGFB KO affects coronary vascular LPL 3.7 Effect of VEGFB KO on plasma lipid profile after diabetes As described, cardiac LPL is an important contributor towards managing plasma TG 6. With higher LPL activity in the VEGFB KO diabetic rat coronary lumen, we expected greater plasma TG clearance. To test this, we measured plasma TG, which was increased in the WT diabetic group. Intriguingly, this change in plasma TG in the VEGFB KO diabetic rats was not as evident as in WT groups (Fig. 18), indicating that deletion of VEGFB had beneficial effects on plasma TG clearance likely by affecting cardiac LPL and protecting these animals from dyslipidemia observed during diabetes. WAT-specific VEGFB knockdown mice had decreased WAT lipolysis and increased insulin sensitivity under HFD 77. When we applied the same logic to our STZ rats, we found that the ex VegfbKONormal VegfbKODiabetes 43 vivo WAT lipolysis rate was indistinguishable between the two diabetic groups (Fig. 19A). Our results implied that VEGFB KO did not affect WAT lipolysis following T1D. Although there was no difference in WAT lipolysis, plasma FA was altered. Induction of diabetes caused a significant increase in total plasma FA in WT diabetic groups. Interestingly, the VEGFB KO diabetic group exhibited normal plasma FA. Additionally, the WT diabetic group had significantly higher plasma FA compared to the KO diabetic rats (Fig. 19B). This suggested that VEGFB KO did not appear to affect WAT lipolysis but alleviated total plasma FA under T1D conditions (Fig. 19C). Besides the quantity of plasma FA measured, the quality of plasma FA was also assessed. Although there were not many changes in individual plasma FAs (Fig. 21B, C, E&F), both diabetic groups had significantly increased percentages of plasma palmitic acid and linoleic acid. Additionally, WT diabetic animals had a higher palmitic acid and a lower linoleic acid percentage compared to KO diabetic rats (Fig. 20A&D). This difference in plasma FA was more evident in the overall plasma FA composition. Compared to VEGFB KO diabetic rats, the plasma SFA was augmented in WT diabetic rats (Fig. 20F). My data demonstrates that induction of diabetes increases SFA, but VEGFB KO can alleviate this change. In summary, ablation of VEGFB did not impact WAT lipolysis under T1D conditions, but it did improve the plasma lipid profile. 44 Figure 18. Plasma TG profile in WT and VEGFB KO control and diabetic animals Plasma TG, n=13-17. Data are presented as mean ± SEM. Two-way ANOVA was used to analyze the data, ** p < 0.005，**** p < 0.0001. Figure 19. WAT lipolysis along with plasma FA in WT and VEGFB KO control and diabetic animals Con STZ Con STZ02004006008001000Plasma TG (mg/dL)WT VegfbKO✱✱✱✱ ✱✱✱✱ABCon STZ Con STZ02468eWAT ex vivolipolysis (NEFAs uM/mg tissue)WT VegfbKO✱ ✱✱Con STZ Con STZ050100150Plasma FA (µM)WT VegfbKO✱✱ ✱✱C45 A: Ex vivo WAT lipolysis using perirenal WAT and determination of FA released into the incubation medium after a one-hour incubation, n=3-4. B: Total plasma FA levels following diabetes, n=7-12 C: Schematic describing how VEGFB KO influences adipose tissue lipolysis and plasma FA. Data are presented as mean ± SEM. Two-way ANOVA was used to analyze the data, * p < 0.05，** p < 0.005. Figure 20. Plasma FA profile in WT and VEGFB KO animals during diabetes A-E: Percentage of key FAs in the plasma, n=5-6. F: Overall contribution of key FAs that make up the plasma composition. The inset represents only the percentage of SFA, n=5-6. Data are presented as mean ± SEM. Two-way ANOVA was used to analyze the data, * p < 0.05，** p < 0.005，**** p < 0.0001. Con STZ Con STZ10152025% Palmitic acid16:0 WT VegfbKO✱✱✱✱ ✱✱✱✱Con STZ Con STZ01020304050% Linoleic acid18:2n6 WT VegfbKO✱✱ ✱✱✱✱✱✱Con STZ Con STZ051015% Stearic acid18:0 WT VegfbKO✱✱Con STZ Con STZ051015% Arachidonic acid20:4n6 WT VegfbKOCon STZ Con STZ051015% Oleic acid18:1n9 WT VegfbKOConSTZConSTZ050100Plasma FA composition %SFAMUFAPUFAWT VegfbKOAD EB CFCon STZ Con STZ010203040% Plasma SFAWT VegfbKO✱✱✱✱ ✱✱✱46 3.8 Effect of VEGFB KO on cardiac lipid accumulation under diabetes As previously mentioned, diabetic animals cannot utilize glucose properly, and thus have an increased demand for FA to generate energy 38. VEGFB KO diabetic animals had higher LPL activity and normal total plasma FA compared with WT diabetic rats (Fig. 16A & 19B), which implies that VEGFB KO diabetic animals use more LPL-derived FA and less NEFA. Conversely, WT diabetic rats obtain less LPL-derived FA. In order to compensate for the missing LPL-derived FA, WT diabetic rats may use more NEFA (Fig. 21). As NEFA uptake mainly depends on CD36 42, the alteration of FA sources may change FA transporter expression in the heart under diabetic conditions. Although Hagberg’s group has shown that VEGFB KO affected FATP4 level 100, this was not the case with my results. There is no difference in the cardiac FATP4 level among all four groups (Fig. 22A). Interestingly, we found both diabetic animals had increased CD36 levels (Fig. 22B). In fact, VEGFB KO diabetic hearts had a significantly lower CD36 level compared to the WT diabetic rats, supporting that WT diabetic rats may use more NEFA, whereas KO diabetic animals use more LPL-derived FA and less NEFA. However, when we investigated cardiac FA, there was no difference in the accumulation of major cardiac FAs under diabetic conditions, except that both diabetic rats had a significantly higher percentage of cardiac stearic acid and a lower percentage of linoleic acid (Fig. 23A-E). This difference in cardiac FAs was better illustrated when the overall cardiac FA composition was determined. Polyunsaturated FA (PUFA) significantly declined in both WT and VEGFB KO diabetic rats (Fig. 23F). Cardiac FA mainly has two fates: they can either be converted into cardiac TG or be oxidized. Total cardiac TG content and FA percentage within these cardiac TGs were assessed. Both were indistinguishable among the 4 groups, except for a decreased C20:4n6 observed in both diabetic animals (Fig. 24A&B). This suggested that VEGFB KO did not affect FA uptake and lipid 47 accumulation in the heart during short-term diabetes (4-day STZ). Whether extending the duration of diabetes influences cardiac lipid accumulation remains unknown. Figure 21. Cartoon explaining how NEFA and LPL-derived FA contribute towards TG storage or oxidation Figure 22. FA uptake in WT and VEGFB KO hearts following diabetes Expression of A: FATP4, (n=5-12) and B: CD36, (n=5-10) in the heart. Data are presented as mean ± SEM. Two-way ANOVA was used to analyze the data, ** p < 0.005，**** p < 0.0001. Con STZ Con STZ01234FATP4 mRNA level (fold change)WT VegfbKOCon STZ Con STZ0123CD36 mRNA level (fold change)WT VegfbKO✱✱✱✱✱✱✱✱✱✱A B48 Figure 23. Cardiac FAs in WT and VEGFB KO hearts following diabetes A-E: Percentage of key cardiac FAs in the different groups of rats, n=5-6. F: Overall contribution of key FAs that make up the cardiac FA composition. The inset represents only the polyunsaturated FA. Data are presented as mean ± SEM. Two-way ANOVA was used to analyze the data, * p < 0.05，** p < 0.005，*** p < 0.0005. Con STZ Con STZ051015% Palmitic acid16:0 WT VegfbKOCon STZ Con STZ0102030% Linoleic acid18:2n6 WT VegfbKO✱✱ ✱✱Con STZ Con STZ0510152025% Stearic acid18:0 WT VegfbKO✱✱✱ ✱Con STZ Con STZ05101520% Arachidonic acid20:4n6 WT VegfbKOCon STZ Con STZ051015% Oleic acid18:1n9 WT VegfbKO✱ConSTZConSTZ050100Cardiac FA composition %MUFAPUFAWT VegfbKOSFAAD EB CFCon STZ Con STZ0204060% Cardiac PUFAWT VegfbKO✱✱✱ ✱49 Figure 24. Cardiac TG along with their individual FAs in WT and VEGFB KO hearts following diabetes A: Cardiac TG content, n=5-6. B: The percentage of individual FAs that make up cardiac TG, n=5-6. Data are presented as mean ± SEM. Two-way ANOVA was used to analyze the data, * p < 0.05. 3.9 Effect of VEGFB KO on transcriptome related to cardiac lipid metabolism during diabetes To study cardiac FA metabolism, heart ventricle transcriptome in WT and VEGFB KO groups following diabetes were compared. Two cut-offs (adjusted p-value less than 0.05 and gene fold change larger than 1.2) were set to filter genes that were both statistically significant and biologically meaningful, improving the robustness and interpretability of our results. Interestingly, WT animals exhibited dramatic transcriptomic changes with 2271 differentially expressed genes Con STZ Con STZ0510152025ugTG/mg tissueWT VegfbKOAC 16:0 C 18:0 C 18:2n6 C 20:4n6 C 22:6n301020304050Percentage of fatty acids ConSTZConSTZWTVegfbKO✱ ✱B50 when encountering diabetes, whereas VEGFB KO animals showed less severe transcriptomic changes with 1426 differentially expressed genes (Fig. 25 A&B). When clustering genes that were differentially expressed based on the biological process via Gene Ontology and ranked based on gene ratio, the top 20 most significantly modified biological processes in both WT and KO groups under diabetes were largely related to metabolic processes (Fig. 26 A&B). A better illustration was shown in the category netplots. These metabolism processes can be simply separated into lipid metabolism & nucleotide metabolism in both diabetic groups. However, for the WT group, there were also alterations in heart function and hypoxia-related biological processes in the top 20 most modified biological processes (Fig. 27 A&B). Although the top 20 most significant altered biological processes in both WT and KO groups were largely related to the metabolism process, diabetes is a such severe disease that also affects multiple biological processes. Under diabetes, both WT and KO had significant modifications in angiogenesis, apoptosis, FA metabolism, glucose metabolism, cardiac TG synthesis and heart contraction process. However, the WT diabetic group had a higher number of genes involved in the abovementioned biological process that changed (Fig. 28). When looking specifically into FA metabolism, changes in genes were more pronounced in the WT group compared with the KO group during diabetes. More genes related to TG breakdown and FA oxidation were upregulated, and more genes related to TG biosynthesis were downregulated (Fig. 29 A). This was probably a result of the oxidation of NEFA. Interestingly, LPL-derived FA is a safer source of substrate as: i) CMO can regulate LPL-derived FA uptake via altering LPL activity and ii) NEFA can be immediately oxidated after uptake and may induce oxidative stress 144. Furthermore, unlike glucose, FA oxidation requires more oxygen delivered via blood vessels 85. It has been reported that diabetes will cause angiopathy 3. Although both diabetic 51 animals tried to upregulate certain pro-angiogenesis regulators to meet the demand for oxygen, classic pro-angiogenesis regulators and blood vessel markers were downregulated (Fig. 29 B). However, the WT diabetic group had more angiogenesis related genes altered than the VEGFB KO group during diabetes (Fig. 25 B). My data implies that there is excess fatty acid metabolism and inadequate oxygen supply in the WT group during diabetes, resulting in more severe lipotoxicity in the WT group compared to VEGFB KO rats. 52 Figure 25. Ventricular transcriptome of rats with diabetes depicting the differentially expressed genes Ventricle RNA from the different groups of rats was sequenced (n=5). The volcano plot describes the profile of differentially expressed genes in A: WT and B: VEGFB KO following diabetes. The x axis represents Log2FC expression of genes vs Log2CPM on the y axis. Red dots represent genes upregulated and blue dots represent genes that are downregulated. CPM indicates counts per million. The insets describe the total number of genes whose expression was either up‐ or downregulated. Angptl4Dpysl5Hmgcs2Chrna2Pik3c2gGlp2rAsic5Acot1GalPimreg Dnah6TtkCenptPdk4Ampd1Pnpla3Kif14Cntnap2Phactr3Nr1h4Habp2TxnipGpihbp1Acot2Hk2Hcn2 Cntnap1Slc2a4 Pnpla2LipeNesFabp4051015−2 0 2 4log2 fold changelog 2 CPM diffexpressedaaaDownregulatedNot significantUpregulatedVegfb_KO con vs. Vegfb_KO STZAngptl4AABR07015057.1Rn45sAABR07000398.1AABR07012592.1Asic5Chrna2Hmgcs2PbkGlp2rPik3c2gAcot1Fhad1Pdk4PrndSlc6a13Atp12aMyo16B3gat1 Nr1h4Ampd1RhouDpysl5Lrrn4TtkH2bc18DdnCdca2Hist1h4m051015−4 −2 0 2 4log2 fold changelog 2 CPM diffexpressedaaaDownregulatedNot significantUpregulatedWT con vs. WT STZTotal=2271UpregulatedDownregulated11811090UpregulatedDownregulatedTotal=1426770665ABDifferentially Differentially expressed 53 Figure 26. Dotplots of ventricular transcriptome clustered based on biological processes of rats with diabetes Dotplots display the top 20 most enriched GO biological processes terms by gene ratio (the number of genes related to GO term / total number of significant genes). The adjusted p value is represented by colour, and the size of the circle represents the counts involved in each biological process. A: WT control versus WT diabetic rats. B: VEGFB KO control versus VEGFB KO diabetic rats. 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Category netplots of ventricular transcriptome clustered based on biological processes of rats with diabetes The category netplot shows the relationships between the top 20 most significant GO biological process terms (Yellow Nodes). Grey nodes represent significant genes associated with given GO biological process terms. Lines represent associations between genes and GO terms. Coloured circles indicate which category these GO terms belong to. The organ colour represents metabolism, pink is related to hypoxia and purple represents heart contraction. A: WT control versus WT diabetic rats. 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lPpargrTecrr Insig1I iI iI iLpgat1tttUcp3Nr1h3rHacd2Edn1Gstp1tttCav1GnpattttPla2g15lllFads2Prkab1rRgnEif2ak3ifififLiasiiiAngpt1tttSuoxGamttttChst1tttGlcelllFmo3Nox4Hspa9MpsttttSpock2Hs3st1tttMical2i li li lExtl3tltltlExt1tttDcnB4galt4ltltltSqorrAbcb7size306090B56 CRHR2APLNCCN2TGFB2ZC3H12AGATA4CAV1NOS3VEGFBDNM1LJAK2TNFSREBF1EDN2KCNQ1ADRA1BPIK3R1PIK3CADMPKNOS2SGCGTMEM38ASCN5AACTC1MYH6SMTNSCN3BRPS6KA2SNTA1CALM1HRCTHRASLC9A1TNNI3MEF2AADRA1DIRX3POPDC2KCND3TBX5SCN1AGNAO1ATP2A2PPP1R13LSLC1A1CACNB2GJA1TNNI3KTPM1SLC8A1KCNE4PRKG1KCNJ5TNNT2ADRB1SLC4A3MAP2K3AKAP9MYH7MYL2ATP1B1NEDD4LIRX5CACNA2D2NOS1APNPPATMEM161BCAV3GPD1LADORA1JUPKCNN2ANGPTL4HIF3AADTRPFGF2HK2EPHB1SERPINE1ESM1HTATIP2NRP2HMOX1CYSLTR1FGF16FGF18HIPK2PIK3C2ASTARD13MCAMCXCL12FZD5AGTR1APLXND1COL4A2AKT1VEGFCGLULPPARGSRFMEOX2PDGFAFGF9NRP1NRARPFOXO4ANGPT1FBXW7VEGFAHIF1AIL15TIE1HEY1PTPRBWNT5ACD34OTULINECSCRSMOC2CYBBCOL4A1IGF2SOX17ELK3EMCNLRG1ID1SP100LGALS3CX3CL1ROCK2ITGB8PTK2BABCC8CDH13FLNASASH1SPRY2JAM3EPN1PXNPIK3R3SEMA5ADAB2IPNRASERAP1PAK4BTG1DAG1EMC10TPD52L1ACAA2PPIFBOKMPV17LSGMS1DAPK1TMBIM1HIGD1ATP53INP1BCL2L1TXNDC12RNF34CTSSLMNAHERPUD1ITGA6IVNS1ABPSLC25A5PARP1RALBP1P4HBLRRK2EYA2CD74OPA1TESTINNACC2TMC8CTSLURI1MYCDAPK2TNFSF10PRKNSFPQUNC5BSERINC3INHBBKDM6AACSL5RBCK1NOL3HYOU1UBBPEA15MCL1PYCARDBMFPLEKHF1HSPH1UBQLN1RPL26TP53TIMP3WWOXSTEAP3IGBP1SH3RF1GHITMCTSCRACK1NDUFS3SH3GLB1BUB1PDK4LIPEACOT1ACOT2ECH1DECR1PNPLA3NUDT7CD36ABHD1ACSL6PCCAGPAMSCD2FMO2ACOT7FABP4PPARAACSS1ECI1HADHBHADHAETFBKMTACOT4SLC27A1LONP2PLA2G4AACOT3EPHX2C1QTNF9PORHSD17B4UCP3GSTM7MMUTACAD11PRKAB1CRATDBIABCD3ACADLCPT1AELOVL5INSIG1LPLELOVL6AKT2PHYHTECRLPGAT1GSTA1HACD1NUCB2PLA2G5ACOX3GSTM1CPT1BFMO1NFE2L1ACSF3PLA2G15ACOT11MLXIPLERLIN2FASNFADS6FADS2NUDT8RGNLPIN2NR1H3DLDLPIN1CROTACAA1ADGAT1IVDOLAHCYP4V3ALOX15ANKRD23EPHX1ACADMACADVLMLYCDSCDMGLLDAGLAGGT5THEM4PCCBPRKAG2CYP1A1MAPK14MAPK3ACBD5CYP2E1ACADSBCYP2T1ETFDHPPARDGPAT3NR1H4KAT5CTDNEP1ENO3CBR2PFKFB2PCPGAM2DLATGCKRGPTPRKACARORCIGFBP3KCNJ11GAPDHINPPL1ENO2MIDNTIGARSLC37A4SLC25A13G6PC3PGAM1GPD2PFKLPFKMDYRK2H6PDLDHAALDOCFOXK2TPI1ALDOASLC25A11RGD1563601IGFBP4BCKDKGPT2PKMDDB1SCAMP3GO TermsAngiogenesis Apoptosis FA metabolism Triglyceride synthesisGlucose metabolism Heart contractionshape NAAWT con vs WT STZ 57 Figure 28. Circos plots of ventricular transcriptome clustered based on biological processes associated with diabetes Circos plot was used to display the association between differentially expressed genes in A: WT and B: VEGFB KO hearts following diabetes (n=5). CCN2APLNEDN1PRKCACAV1DNM1LTNFJAK2KCNQ1ACTC1CACNB2KCNJ5CALM1GNAO1TPM1DMPKSLC8A1TNNI3KTCAPATP1A3MYL2MYH6MYH7MYL3TNNT2ADORA1POPDC2SLC1A1MAP2K3TMEM38ATNNI3MEF2ANOS1APEXT1NOS2ATP1A2PEBP1SGCGKCNJ8ADRA1DSNTA1GAAADRA1AGSNHOPXATP2A2ANGPTL4ADTRPSERPINE1HK2EPHB1CYSLTR1VEGFDANGPT1FGF18PPARGCCL5CELA1NRP2SMOC2NFATC4STARD13HSPB6PIK3R3LGALS3SOX17HIPK2AKT1SRFLRG1COL4A2PGK1FOXO4HEY1ITGB8TIE1PKMHIF1AMCAMFBXW7VEGFASP100COL4A1JMJD6CD34NRP1DCNGATA2CDH13PDCL3BOKAIFM1PERPOPA1HERPUD1CD74PPIFS100A8SOD2PRKRAPCBP4PARP1ENO1CEBPBUACACDKN1ANBNUSP28P4HBRALBP1ATP2A3PRKNTXNDC12EIF2AK3SH3GLB1PPP2R5CIVNS1ABPMTCH2SERINC3SELENOSS100A9LIPEPDK4PNPLA3ACOT1ACOT2ECH1FABP4GPAMDECR1SLC27A1PCCACD36ACSL6ACOT7ACOT3SCD2ABHD1PPARAECI1HADHBCPT1ANUDT7ACOT4PHYHPORMGLLCRATMMUTACAA2C1QTNF9LPLACADSBACAD11GSTM7HADHAACAA1AACSS1FMO2MLYCDAKT2MLXIPLEPHX2ALOX15ACOT11EPHX1CYP2E1HACD1LPIN1IVDLONP2ABHD2CPT1BTHEM4IRS2DLDEHHADHETFBKMTNFE2L1FMO1ECHS1ECHDC2ELOVL5TECRINSIG1LPGAT1HSD17B4NUDT8ACSF3UCP3NR1H3HACD2GSTP1FASNGNPATPLA2G15FADS2PRKAB1ACACBRGNTREX1LIASLPIN3CNEP1R1GPAT3NR1H4PFKFB2CBR2DLATPGAM2PCENO3SLC25A13GCKRRORCPRKACAPGAM1SLC25A12GAPDHTIGARPDHA1PDHBLDHAENO2PGM1GPTH6PDGOT1COX11TPI1LOC500959ALDOARGD1563601MDH2PFKMSLC25A11GO TermsAngiogenesis Apoptosis FA metabolism Triglyceride synthesisGlucose metabolism Heart contractionshape NABVEGFBKO con vs VEGFBKO STZ 58 Figure 29. Ventricular transcriptome with emphasis on FA metabolism and angiogenesis in rats with diabetes The volcano plot describes the profile of differentially expressed genes associated with A: FA metabolism and B: Angiogenesis in WT and VEGFB KO following diabetes. The x axis represents Log2FC expression of genes vs Log2CPM on the y axis. CPM indicates counts per million. 3.10 Effect of VEGFB KO on cardiac cell death after diabetes Increased oxidative stress due to excess FA metabolism, increased lipotoxicity due to impaired angiogenesis, and increased plasma SFA have all been linked to ER stress and ROS generation 22,64,78,86,93-98. Thus, the WT group may experience more cell death than the VEGFB KO group following diabetes. Indeed, there were more genes related to apoptosis altered in the WT group -5 0 5051015Log2 fold changeLog2CPMWTC vs WTD_ FA metabolism-5 0 5051015Log2 fold changeLog2CPMKOC vs KOD_ FA [binary]-5 0 5051015Log2 fold changeLog2CPMWTC vs WTD_Angiogensis-5 0 5051015Log2 fold changeLog2CPMKOC vs KOD_AngiogensisAngptl4 Angptl4Fgf2Angptl1Hif3aAdtrpNos3Cd34Hif1aNrp1VegfaPdgfaHif1aVegfaCd34Nrp1AdtrpAngptl159 than in the KO group during diabetes (Fig. 20 A). Taken together with an increase of cardiac cleaved PARP levels in WT diabetic rats compared to KO diabetic rats (Fig. 30B), my results revealed that VEGFB KO may protect the heart from cell death during diabetes. Figure 30. Ventricular transcriptome and protein expression related to cell death in rats with diabetes A: The volcano plot describes the profile of differentially expressed genes associated with cell death in WT (top panel) and VEGFB KO (bottom panel) following diabetes. The x axis represents Log2FC expression of genes vs Log2CPM on the y axis. CPM indicates counts per million. B: The cleaved PARP over full length PARP in hearts from the different groups, n=5-13 The inset is a Western Blot representing WT and VEGFB KO hearts following diabetes. Two-way ANOVA was used to analyze data. Data are presented as mean ± SEM, * p < 0.05. Con STZ Con STZ01234cleaved PARP/ full PARPprotein expression (fold change) WT VegfbKO✱ ✱-5 0 5051015Log2 fold changeLog2CPM-5 0 5051015Log2 fold changeLog2CPMA BWTC vs WTD_Cell deathKOC vs KOD_Cell death60 Chapter 4: Discussion Following diabetes, the heart has a limited capacity to utilize glucose for energy generation and primarily relies on FAs. This shift in cardiac metabolism significantly contributes to the development of DCM, which can progress to heart failure, one of the leading causes of death in patients with diabetes. KO of VEGFB on animals has a minimal effect on phenotype changes under normal conditions. Noticeable changes were only observed when animals underwent environmental stress, like diabetes. VEGFB KO plays a protective effect against DCM by alleviating plasma lipid profile and reducing cardiac lipid accumulation and oxidative stress, ultimately enhancing cell survival during DCM. This study emphasizes the protective role of VEGFB inhibition in T1D. There was no difference in overall glucose metabolism observed between WT and VEGFB KO rats under normal conditions. Other studies have also observed that inhibition of VEGFB has limited basal glucose metabolic changes in various VEGFB KO mice and rat models 77,112,136,147. VEGFB KO animals only exhibited decreased blood glucose and body weight when they were exposed to HFD 77,112,147. However, contradictory results were also reported with VEGFB KO mice demonstrating increased body weight with equal or lower blood glucose under HFD 100,153. Similarly, VEGFB KO db/db mice did not have elevated blood glucose like WT db/db mice. This normalized blood glucose was not due to an alteration in plasma insulin 102. Related to insulin profile, mice with beta cell-specific VEGFB KO had increased insulin expression in islets without altering glucose homeostasis under normal conditions, and improved glucose and insulin tolerance were only observed under HFD 147. In terms of glucose uptake, Hagberg’s group has shown that KO of VEGFB in mice enhanced cardiac glucose uptake by regulating GLUT4 levels 100. Additionally, VEGFB treatment inhibited endothelial glucose transcytosis by decreasing GLUT1 61 levels. Either treating these mice with VEGFB neutralizing antibodies (2H10) or KO VEGFB in mice improved glucose uptake to the heart and increased cardiac glycogen level 129. These conflicting results of VEGFB KO on glucose metabolism could be due to different animal models along with diverse measurements being used. Cardiac-specific VEGFB overexpressing rats had decreased LPL activity levels despite an increase in total LPL protein in the ventricle muscle, demonstrating that VEGFB overexpression obstructs LPL translocation 144. This inhibition of LPL translocation is probably because of improved insulin perfusion due to the expanded vasculature associated with VEGFB overexpression 86,122. Enhanced insulin action reprograms the heart to favor glucose utilization, thereby decreasing FA utilization. In the current study, cardiac LPL activity was significantly decreased without altering the total LPL protein in VEGFB KO rat hearts, suggesting that VEGFB KO also impedes LPL translocation. This disruption, however, is unlikely a result of a change in angiogenesis as RNA sequencing results indicated that VEGFB KO did not impact genes related to angiogenesis (data not shown). Alitalo’s group also reported no differences in vessel diameter or density between VEGFB KO and WT rats 112. Unlike rats with VEGFB overexpression, VEGFB KO rats did not exhibit alteration in the canonical machinery involved in LPL translocation, specifically heparanase and GPIHBP1 144. Whether VEGFB KO impacts insulin sensitivity under normal conditions remains unknown. In future, the insulin action and surface LPL activity in primary CMOs from WT and KO rats should be detected following insulin treatment. Cardiac LPL activity is critical for regulating plasma TG 6. With decreased LPL activity in VEGFB KO rats under normal conditions, we expected reduced TG clearance in KO animals. However, comparable plasma TG was observed between WT and KO groups. There are two main factors that regulate plasma TG: TG clearance by tissue LPL and hepatic TG secretion 90. Hepatic TG 62 synthesis requires FAs, that originate from either endogenous or exogenous sources. When there are extra carbohydrates, liver converts glucose into FA via de novo lipogenesis. Related to exogenous FA sources, dietary TGs absorbed by the intestines are packaged into chylomicrons and released into the plasma. Most chylomicron TGs are taken up by muscle and adipose tissue with the help of LPL. The remaining TGs in the chylomicron remnants are uptake by the liver via a receptor-mediated process, where FAs are released during the lysosomal breakdown of these remnants 80. Additionally, FAs directly taken up by the liver also contribute significantly to hepatic TG synthesis. These FAs mainly come from WAT lipolysis 80,149. It is possible that a reduction in WAT lipolysis could affect hepatic FA uptake and TG synthesis. Indeed, VEGFB KO rats had decreased WAT lipolysis, suggesting that the liver has less substrate for hepatic TG synthesis, an explanation for the unchanged plasma TG. The inhibitory effect of VEGFB on WAT lipolysis could be attributed to various possibilities. First, VEGFB is highly expressed in WAT, and WAT is highly vascularized with VEGFR1 mainly expressed on vascular ECs. VEGFB may directly regulate lipolysis through VEGFB/VEGFR1 signaling via the crosstalk between adipocyte and EC. Knockdown VEGFB in mice induced BAT whitening, and inhibition of VEGFA restored it 154. Both VEGFB and VEGFA can bind to VEGFR1, with VEGFA having a higher affinity 118. These results suggest that VEGFA and VEGFB have opposing actions to regulate adipose tissue development through VEGFR1. However, neither VEGFB/VEGFR1 nor the interaction between adipocytes and ECs has been extensively studied. Another possibility is that VEGFB indirectly regulates WAT lipolysis by modulating insulin sensitivity. A recent study reported that although adipocyte-specific VEGFB deficient mice have no difference in WAT lipolysis and activation of HSL under control conditions, these mice displayed improved insulin sensitivity under HFD associated with reduced 63 WAT lipolysis 77. Additionally, oral gavage of radioactive FAs to VEGFB KO mice resulted in a significant increase of FA accumulation in WAT instead of other peripheral tissues, suggesting inhibition of VEGFB promotes lipid storage in WAT under normal conditions 100. Future experiments are anticipated to determine whether WAT from VEGFB KO rats exhibits improved insulin sensitivity. WAT lipolysis plays an important role in regulating plasma FA 80,149. With reduced WAT lipolysis in VEGFB KO rats under normal conditions, we expected decreased plasma FA in KO animals. However, plasma FA was indistinguishable between WT and KO groups. One potential mechanism could be that there is decreased FA uptake in peripheral tissues. It has been reported that VEGFB treatment induced various FA transporter expressions in diverse primary human ECs and mouse EC lines 100. Similarly, inhibiting VEGFB signaling via neutralizing antibodies against VEGFR1 or NRP1 reduced FA transporters and decreased FA transport across primary EC from skeletal muscle 154. In addition, KO of VEGFB in mice decreased FA transporter expression in both skeletal muscle and BAT 100. Moreover, caloric restriction followed by refeeding in rats (catch-up growth model) induced excess lipid accumulation and insulin resistance in skeletal muscle through the activation of VEGFB signaling. Knockdown of VEGFB via AAV transfection in the mice tibialis anterior muscle decreased FATP3, FATP4 and subsequent FA uptake during catch-up growth 154. Thus, future studies examining FA uptake in tissues like skeletal muscle and BAT are necessary to validate this suggestion. VEGFB inhibition has also been reported to decrease FA uptake in the heart. Hagberg’s group reported that deletion of VEGFB in mice decreased FATP3 and FATP4 levels in whole heart and cardiac EC without impacting FATPs expression in cardiac non-EC fraction. These VEGFB KO mice also displayed a reduction of cardiac FA uptake during FA gavage 100. In addition, global 64 VEGFB KO rats have also been reported to decrease FATP4 in the whole heart without affecting other FA transports 112. However, we did not observe any change in either FA transporters (especially FATP4 and CD36) or key cardiac FAs in rat ventricle muscle, implying that global VEGFB KO in rats did not influence FA uptake in the heart under normal conditions. Without an effect on FA uptake, VEGFB KO is unlikely to impact cardiac TG content. Indeed, no difference in cardiac TG was observed between WT and KO rats. In accordance with our results, Alitalo’s group found that there was a similar cardiac TG level in both WT and KO rats 112. However, whether ablation of VEGFB changes TG content in other peripheral tissues is not known and should be examined. RNA sequencing data indicated that there were no apparent transcriptome changes following KO of VEGFB; only 6 out of 15k genes were significantly altered (data not shown), suggesting that VEGFB is not a transcription factor. Interestingly, overexpressing VEGFB in the heart induced a remarkable transcriptome change with over 2000 genes significantly differentially expressed 86. This transcriptome change following VEGFB overexpression is probably associated with angiogenesis. Increased angiogenesis improves insulin action, which alters tissue metabolism. In addition, previous research related to VEGFB inhibition found that VEGFB KO had no phenotype changes until exposed to HFD or constructed onto the db/db model 77,101,102,136,150,151, implying that VEGFB KO has an impact on phenotype in the context of environmental factors such as diabetes. Various studies have found that increased plasma VEGFB was observed in obesity 155 and early onset patients with diabetes 156. Patients with diabetic kidney disease have also increased VEGFB and VEGFR1 expression in kidney samples 151. These results imply VEGFB has a correlation with diabetes and associated complications. Hence, in later studies, we mainly focused on diabetes 65 instead of the genotype. Both the HFD model and db/db mice model belong to T2D. The effect of VEGFB KO in T1D is poorly understood. Thus, we induced T1D via STZ in VEGFB KO rats. There was no significant difference in the severity of diabetes between WT and KO groups. However, changes in LPL activity were observed. Reducing insulin through overnight fasting or pharmacological suppression with diazoxide has been reported to increase cardiac LPL translocation through the AMPK pathway 144,157. Consistent with previous findings, decreased circulating insulin following STZ-diabetes induction enhanced LPL activity in VEGFB KO animals. In contrast, decreased LPL activity was observed in WT diabetic rats. Intriguingly, there was no difference in total LPL protein level among the four groups, suggesting increased LPL activity in KO diabetic groups is probably because the increased LPLs accumulated in CMO translocate to the coronary vessels. A decline in LPL activity in WT diabetic rats is likely due to inadequate LPL reserve paired with the inhibitory effect of the elevated circulating FA on LPL activity 67-71. Notably, this study is the first to comprehensively investigate the effect of VEGFB KO on LPL activity during diabetes. Further experiments are necessary to test my suggestion related to the accumulation of LPL in CMO following VEGFB KO and this movement to the vascular lumen after diabetes. Given the association between cardiac LPL activity and plasma TG 90,91, it was not surprising that the WT diabetic rats exhibited hypertriglyceridemia. Intriguingly, VEGFB KO diabetic rats maintained relatively normal plasma TG, which could be explained by the increase in LPL activity. WAT lipolysis also indirectly regulates plasma TG via the liver, with FA derived from WAT lipolysis contributing substantially to hepatic TG synthesis during diabetes 80. Ex vivo WAT lipolysis rates were similar between the two diabetic groups. Given the lower lipolysis rates observed under control conditions, VEGFB KO rats showed a higher percentage increase in WAT 66 lipolysis during diabetes compared to the WT groups. Currently, the effect of VEGFB KO on WAT lipolysis during T1D is unknown. It should be noted that inflammation has been reported to inhibit lipolysis 158. Thus, it is possible that the inflammatory process may be more severe in the WAT of WT diabetic rats. Indeed, an increase in inflammation markers, upregulation of pro-inflammatory genes, and pathological angiogenesis were observed in adipocyte-specific VEGFB overexpressing mice under HFD conditions, effects that were not observed in VEGFB deficient mice 77. In parallel, administering anti-VEGFR-1 antibodies was found to decrease the incidence of arthritis, synovial inflammation and atherosclerosis in mice. Notably, the suppression of VEGFR2 did not yield an anti-inflammation effect in mice with arthritis or atherosclerosis 159, implying that this anti-inflammation effect is probably through VEGFB signaling. Moreover, a correlation between hypertriglyceridemia and C-reactive protein levels (an inflammation marker) has been reported in humans 160. Hypertriglyceridemia probably also causes inflammation in WAT in the WT diabetic group, where inflamed WAT exhibited resistance to catecholamine-stimulated activation, a known positive regulator for WAT lipolysis 158. Given that VEGFB KO rats have normal plasma TG together with the anti-inflammation effect associated with VEGFB KO, the decreased inflammation in VEGFB KO WAT may enhance its sensitivity to catecholamine, resulting in a higher degree of lipolysis compared to WT rats. Despite increased WAT lipolysis in VEGFB KO diabetic rats, these rats still displayed normal plasma FA. It is possible that excess FA was cleared by peripheral tissues such as the liver. There are three major FA supplies that contribute to hepatic TG synthesis following diabetes and obesity: i) de novo lipogenesis, ii) dietary FA intake and iii) FA derived from WAT 80. Inhibition of VEGFB through genetic KO or pharmacological inhibition in mice demonstrated decreased expression of genes involved in de novo lipogenesis without altering food intake or intestine lipid absorption 77. This implies that liver 67 will uptake more FA derived from WAT during diabetes, resulting in greater clearance of plasma FA. Future investigations into WAT inflammation and FA uptake in peripheral tissues like the liver are required to support these hypotheses regarding WAT lipolysis and FA uptake. Plasma FAs were grouped according to the number of double bonds. SFA does not contain double bonds, monounsaturated FA have one, and PUFA possess two or more double bonds. Palmitate acid, a typical SFA, is known to have deleterious effects on cardiac function 79. Palmitic acid can bind to toll-like receptor 4, triggering pro-inflammation gene expression and contributing to insulin resistance 161. Mice fed with SFA-rich HFD have elevated SFA composition in both plasma and ER membranes in the heart, causing ER stress and left ventricle diastolic dysfunction 162. In contrast, increasing intake of linoleic acid, a PUFA, is linked to a decreased risk of CVD 163. In VEGFB KO diabetic rats, a lower palmitic acid and higher linoleic acid percentages were observed compared to WT diabetic rats, suggesting that VEGFB KO rats have less toxic lipids and more beneficial lipids. This may help the VEGFB KO heart against dysfunction during diabetes. The reasons behind why VEGFB KO diabetic rats demonstrate this alteration in plasma FA composition remain unknown and are worthy of further investigation. Based on the FA source, external FAs can be classified as LPL-derived FA and NEFA. Both plasma FA and LPL-derived FA need to be taken up mainly through transporters. CD36 is critical for NEFA uptake. When radioactive FA was injected into both WT and CD36 null mice, CD36 deficient mice had a significant decrease in FA uptake into the multiple tissues including the heart 42. Similar results were also observed in healthy and CD36-deficient humans 164. However, KO of CD36 in mice did not impact the uptake of LPL-derived FA into CMO following the injection of radiolabelled VLDL 165. This indicates that CD36 is unlikely responsible for the uptake of LPL-derived FA. With decreased CD36 expression and elevated LPL activity in the heart compared to 68 WT diabetic groups, VEGFB KO diabetic animals may rely less on NEFA, and more on LPL-derived FA, which is considered a better FA source for the following reasons. CMO can regulate LPL translocation by modulating LPL-derived FA delivery through the AMPK pathway in response to energy demand 66,157. Furthermore, our lab showed that the NEFA uptake rate was almost equivalent to the NEFA oxidation rate in the fasting rat hearts 144. These findings imply that NEFA is preferentially oxidized following uptake, which may contribute to oxidative stress 144. Moreover, lipoproteins contain substantial FA content, thus offering a richer FA supply than other sources for ATP generation 58. It should be noted that there was no change in major cardiac FAs among the four groups, suggesting that VEGFB KO did not impact cardiac FA uptake. However, cardiac FAs can either be stored as TG or be oxidized for energy generation. In 4-day diabetes, the cardiac TG content was similar among the four groups. We suggest the possibility that the increase of TG content during 4-day diabetes is too small to be detected. Alternatively, these cardiac FAs can also be present in lipid intermediates such as DAG and ceramide. Future studies using µPET imaging to visualize and quantify FA uptake are essential to validate our findings regarding cardiac FA uptake. Furthermore, a chronic diabetes study is anticipated to show whether VEGFB affects lipid accumulation in the heart, including both TG and lipid intermediates. Alteration in FA substrates influences lipid metabolism 42,79. Even though the severity of diabetes based on glucose homeostasis was similar in both WT and VEGFB KO groups, there were fewer genes differentially expressed in VEGFB KO groups compared to WT groups, implying diabetes causes less deleterious effects in VEGFB KO rats. In WT group, a greater number of genes associated with FA metabolism were altered, particularly those genes related to FA oxidation that were upregulated. NEFA are preferentially oxidized following uptake 144. This further implies that 69 WT diabetic animals may use more NEFA for energy. In addition, the WT group also had more genes related to angiogenesis altered with classic angiogenesis genes that were downregulated, which may induce incomplete lipid oxidation, ultimately causing the accumulation of toxic lipid intermediate 85,93,166. Increased FA oxidation induces oxidative stress, and decreased angiogenesis gene induces lipotoxicity; both have been linked to the development of DCM 38,95-99. Given that fewer genes related to cell death were differentially expressed and fewer protein cell death markers were detected in the VEGFB KO diabetic animals than in the WT group, it suggests that inhibition of VEGFB has a protective effect against diabetes induced cell death. This finding is consistent with the pro-survival effect of VEGFB KO on pancreatic tumors. Mice overexpressing VEGFB have approximately a 40% decrease in tumor size, while VEGFB deficient mice have an almost 2-fold increase in tumor size compared with their own control 140. However, RNA sequencing results do not always correlate with protein level and activity due to the post-translational regulation. When classical genes involved in each biological process were visualized in a heatmap, no obvious differences were observed between the two diabetic groups (data not shown). This could be because there are multiple genes involved in different biological processes. Even though the expression of classical genes appears similar, the actual activity could vary. In this regard, the following experiments could be considered in future: i) measuring cardiac glucose and FA oxidation rate and mitochondria respiration capacity in isolated CMOs (via Seahorse), heart tissue (oxygraphy) and rats (µPET imaging), ii) determining the capillary size and density via immunohistochemistry (heart section) and μCT (whole heart), iii) directly quantifying the ROS level in the heart and iv) assessing toxic lipid accumulation via HPLC-MS. 70 4.1 Limitations In addition to the abovementioned limitations and future experiments, only ventricle muscles were used for analysis in this project. The heart comprises 11 cell types. Although CMO is the predominant cell type and EC is the major non-CMO cells in the ventricle tissue 167, other non-CMO cells still act as confounding factors in our analysis. In the future, isolated CMOs from four groups are required to repeat similar measurements that have been done in the ventricle tissue. Epinephrine serves as a transient positive regulator of WAT lipolysis 44,50,51. Dose-dependent epinephrine treatment should be considered in WAT ex vivo lipolysis experiments to investigate whether VEGFB KO impacts the sensitivity of WAT to epinephrine-induced lipolysis. Furthermore, the measurements of LPL activity and WAT lipolysis rate were done ex vivo, which lack the influence of circulating factors like hormones, FA, enzymes and cytokines, which are known to influence these processes. Future in vivo studies using radiolabeled TG in either VLDL or adipocyte LD would be beneficial for assessing LPL activity and WAT lipolysis rate under physiological conditions. Moreover, the contradictory plasma lipid profile observed in KO rats has led to several hypotheses that require further validation. Assessing hepatic TG secretion and genes related to de novo lipogenesis in the liver could provide evidence to support my explanations. Additionally, investigating the molecule mechanism regarding how inhibition of VEGFB modulates LPL translocation is also essential for drug development targeting DCM. Intrinsic LPL activity in primary CMOs from 4 groups should be assessed to substantiate my hypothesis. Single-cell RNA sequencing in CMOs could be helpful in identifying differentially expressed genes and altered signaling pathways. 71 4.2 Future directions Effect of VEGFB KO on WAT Since LPL is also expressed in WAT and responds in a tissue-specific manner following diabetes 88, and given that VEGFB is also highly expressed in WAT 112, the effect of global VEGFB KO on WAT LPL activity and lipid storage is also worth studying. LPL facilitates lipid storage in WAT 88. Measuring LPL activity in the vasculature of WAT is challenging. Nevertheless, heparin-releasable LPL activity in the medium of WAT segments should be attempted and correlated to adipocyte size determined using H&E staining. Effect of VEGFB KO on VEGFR1 VEGFR1 is the primary receptor for VEGFB, but VEGFR1 is also implicated in additional signaling beyond VEGFB 123. Notably, VEGFR1 has been reported to increase in heart following both acute and chronic diabetes 131. Understanding the influence of VEGFB KO on VEGFR1 expression and associated signaling under physiological and diabetic conditions could provide valuable insights into diseases related to VEGFR1 dysregulation. Timing of VEGFB inhibition following diabetes. In our study, VEGFB KO attenuated cell death in the heart during short-term diabetes. However, VEGFB also has an antioxidant effect. VEGFB KO rats displayed larger infarct size in response to ischemia compared with WT rats 112. Since patients with diabetes have elevated mortality due to heart attacks and heart failure, determining the optimal timing for VEGFB inhibition following diabetes is also important for providing a therapeutic intervention to improve the survival rate of diabetic patients. 72 Chapter 5: Conclusion During diabetes, increased FA utilization for energy generation due to limited capacity in glucose utilization causes oxidative stress and lipid accumulation in the heart, which contributes significantly to the development of DCM. VEGFB KO had minimal phenotype and transcriptome changes under normal conditions. However, following diabetes, KO of VEGFB in rats is beneficial to mitigate cell death through i) alleviation of hyperlipidemia by upregulating cardiac LPL activity, ii) prevention of excess FA oxidation by providing more LPL-derived FA to the heart, and iii) inhibition of pathological lipid accumulation in the heart by improving angiogenesis. Inhibiting VEGFB may offer a promising therapeutic approach to protect the heart from diabetes-induced cardiomyopathy. Notably, this study is the first to examine the impact of VEGFB KO on LPL activity during diabetes. Further investigation of the mechanism behind how VEGFB inhibition modulates LPL translocation could provide valuable insights for drug development and help fill the gap in mechanism-based treatments for DCM. 73 References 1. Magliano D, Boyko EJ. IDF diabetes atlas. Tenth edition. ed. Brussels: International Diabetes Federation; 2021. 2. American Diabetes Association Professional Practice C. 2. Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes-2022. Diabetes Care. 2022;45:S17-S38. doi: 10.2337/dc22-S002 3. Sapra A, Bhandari P. Diabetes. In: StatPearls. Treasure Island (FL); 2024. 4. Forbes JM, Cooper ME. Mechanisms of diabetic complications. Physiological reviews. 2013;93:137-188. doi: 10.1152/physrev.00045.2011 5. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;414:813-820. doi: 10.1038/414813a 6. An D, Rodrigues B. Role of changes in cardiac metabolism in development of diabetic cardiomyopathy. American journal of physiology Heart and circulatory physiology. 2006;291:H1489-1506. doi: 10.1152/ajpheart.00278.2006 7. Rodrigues B, McNeill JH. The diabetic heart: metabolic causes for the development of a cardiomyopathy. Cardiovascular research. 1992;26:913-922. 8. Seferovic PM, Paulus WJ. Clinical diabetic cardiomyopathy: a two-faced disease with restrictive and dilated phenotypes. European heart journal. 2015;36:1718-1727, 1727a-1727c. doi: 10.1093/eurheartj/ehv134 9. Bugger H, Abel ED. Rodent models of diabetic cardiomyopathy. Disease models & mechanisms. 2009;2:454-466. doi: 10.1242/dmm.001941 10. Jia G, Hill MA, Sowers JR. Diabetic Cardiomyopathy: An Update of Mechanisms Contributing to This Clinical Entity. Circ Res. 2018;122:624-638. doi: 10.1161/CIRCRESAHA.117.311586 11. Regan TJ, Ahmed S, Haider B, Moschos C, Weisse A. Diabetic cardiomyopathy: experimental and clinical observations. New Jersey medicine : the journal of the Medical Society of New Jersey. 1994;91:776-778. 12. Boudina S, Abel ED. Diabetic cardiomyopathy, causes and effects. Reviews in endocrine & metabolic disorders. 2010;11:31-39. doi: 10.1007/s11154-010-9131-7 13. Fang ZY, Prins JB, Marwick TH. Diabetic cardiomyopathy: evidence, mechanisms, and therapeutic implications. Endocrine reviews. 2004;25:543-567. doi: 10.1210/er.2003-0012 14. Severson DL. Diabetic cardiomyopathy: recent evidence from mouse models of type 1 and type 2 diabetes. Canadian journal of physiology and pharmacology. 2004;82:813-823. doi: 10.1139/y04-065 15. Penpargkul S, Fein F, Sonnenblick EH, Scheuer J. Depressed cardiac sarcoplasmic reticular function from diabetic rats. J Mol Cell Cardiol. 1981;13:303-309. doi: 10.1016/0022-2828(81)90318-7 16. Trost SU, Belke DD, Bluhm WF, Meyer M, Swanson E, Dillmann WH. Overexpression of the sarcoplasmic reticulum Ca(2+)-ATPase improves myocardial contractility in diabetic cardiomyopathy. Diabetes. 2002;51:1166-1171. doi: 10.2337/diabetes.51.4.1166 17. Khalid M, Petroianu G, Adem A. Advanced Glycation End Products and Diabetes Mellitus: Mechanisms and Perspectives. Biomolecules. 2022;12. doi: 10.3390/biom12040542 74 18. Jia G, Whaley-Connell A, Sowers JR. Diabetic cardiomyopathy: a hyperglycaemia- and insulin-resistance-induced heart disease. Diabetologia. 2018;61:21-28. doi: 10.1007/s00125-017-4390-4 19. Shehadeh A, Regan TJ. Cardiac consequences of diabetes mellitus. Clinical cardiology. 1995;18:301-305. 20. Fein FS, Sonnenblick EH. Diabetic cardiomyopathy. Progress in cardiovascular diseases. 1985;27:255-270. 21. Dhalla NS, Liu X, Panagia V, Takeda N. Subcellular remodeling and heart dysfunction in chronic diabetes. Cardiovascular research. 1998;40:239-247. 22. Laakso M. Heart in diabetes: a microvascular disease. Diabetes Care. 2011;34 Suppl 2:S145-149. doi: 10.2337/dc11-s209 23. Kim JA, Jang HJ, Martinez-Lemus LA, Sowers JR. Activation of mTOR/p70S6 kinase by ANG II inhibits insulin-stimulated endothelial nitric oxide synthase and vasodilation. Am J Physiol Endocrinol Metab. 2012;302:E201-208. doi: 10.1152/ajpendo.00497.2011 24. Taha M, Lopaschuk GD. Alterations in energy metabolism in cardiomyopathies. Annals of medicine. 2007;39:594-607. doi: 10.1080/07853890701618305 25. Sung MM, Hamza SM, Dyck JR. Myocardial metabolism in diabetic cardiomyopathy: potential therapeutic targets. Antioxidants & redox signaling. 2015;22:1606-1630. doi: 10.1089/ars.2015.6305 26. Wan A, Rodrigues B. Endothelial cell-cardiomyocyte crosstalk in diabetic cardiomyopathy. Cardiovascular research. 2016;111:172-183. doi: 10.1093/cvr/cvw159 27. Chong CR, Clarke K, Levelt E. Metabolic Remodeling in Diabetic Cardiomyopathy. Cardiovascular research. 2017. doi: 10.1093/cvr/cvx018 28. Da Dalt L, Cabodevilla AG, Goldberg IJ, Norata GD. Cardiac lipid metabolism, mitochondrial function, and heart failure. Cardiovascular research. 2023;119:1905-1914. doi: 10.1093/cvr/cvad100 29. Lopaschuk GD, Ussher JR, Folmes CD, Jaswal JS, Stanley WC. Myocardial fatty acid metabolism in health and disease. Physiological reviews. 2010;90:207-258. doi: 10.1152/physrev.00015.2009 30. Ritterhoff J, Tian R. Metabolic mechanisms in physiological and pathological cardiac hypertrophy: new paradigms and challenges. Nat Rev Cardiol. 2023;20:812-829. doi: 10.1038/s41569-023-00887-x 31. Kolwicz SC, Jr., Purohit S, Tian R. Cardiac metabolism and its interactions with contraction, growth, and survival of cardiomyocytes. Circ Res. 2013;113:603-616. doi: 10.1161/CIRCRESAHA.113.302095 32. Shao D, Tian R. Glucose Transporters in Cardiac Metabolism and Hypertrophy. Compr Physiol. 2015;6:331-351. doi: 10.1002/cphy.c150016 33. Tran DH, Wang ZV. Glucose Metabolism in Cardiac Hypertrophy and Heart Failure. J Am Heart Assoc. 2019;8:e012673. doi: 10.1161/JAHA.119.012673 34. Chanda D, Luiken JJ, Glatz JF. Signaling pathways involved in cardiac energy metabolism. FEBS Lett. 2016;590:2364-2374. doi: 10.1002/1873-3468.12297 35. Martinez-Reyes I, Chandel NS. Mitochondrial TCA cycle metabolites control physiology and disease. Nat Commun. 2020;11:102. doi: 10.1038/s41467-019-13668-3 36. Daghlas SA, Mohiuddin SS. Biochemistry, Glycogen. In: StatPearls. Treasure Island (FL); 2024. 75 37. Henning SL, Wambolt RB, Schonekess BO, Lopaschuk GD, Allard MF. Contribution of glycogen to aerobic myocardial glucose utilization. Circulation. 1996;93:1549-1555. doi: 10.1161/01.cir.93.8.1549 38. Kim MS, Wang Y, Rodrigues B. Lipoprotein lipase mediated fatty acid delivery and its impact in diabetic cardiomyopathy. Biochim Biophys Acta. 2012;1821:800-808. doi: 10.1016/j.bbalip.2011.10.001 39. Schaffer JE. Fatty acid transport: the roads taken. Am J Physiol Endocrinol Metab. 2002;282:E239-246. doi: 10.1152/ajpendo.00462.2001 40. Bharadwaj KG, Hiyama Y, Hu YY, Huggins LA, Ramakrishnan R, Abumrad NA, Shulman GI, Blaner WS, Goldberg IJ. Chylomicron- and VLDL-derived Lipids Enter the Heart through Different Pathways. Journal of Biological Chemistry. 2010;285:37976-37986. doi: 10.1074/jbc.M110.174458 41. van der Vusse GJ, van Bilsen M, Glatz JF. Cardiac fatty acid uptake and transport in health and disease. Cardiovascular research. 2000;45:279-293. 42. Coburn CT, Knapp FF, Jr., Febbraio M, Beets AL, Silverstein RL, Abumrad NA. Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice. J Biol Chem. 2000;275:32523-32529. doi: 10.1074/jbc.M003826200 43. Talley JT, Mohiuddin SS. Biochemistry, Fatty Acid Oxidation. In: StatPearls. Treasure Island (FL); 2024. 44. Ahmadian M, Duncan RE, Sul HS. The skinny on fat: lipolysis and fatty acid utilization in adipocytes. Trends Endocrinol Metab. 2009;20:424-428. doi: 10.1016/j.tem.2009.06.002 45. Recazens E, Mouisel E, Langin D. Hormone-sensitive lipase: sixty years later. Prog Lipid Res. 2021;82:101084. doi: 10.1016/j.plipres.2020.101084 46. Leszek S, Leszek S, Szablewski L. Adipose tissue. London, England: IntechOpen; 2018. 47. Coleman RA, Mashek DG. Mammalian triacylglycerol metabolism: synthesis, lipolysis, and signaling. Chem Rev. 2011;111:6359-6386. doi: 10.1021/cr100404w 48. Nishimoto S, Fukuda D, Higashikuni Y, Tanaka K, Hirata Y, Murata C, Kim-Kaneyama JR, Sato F, Bando M, Yagi S, et al. Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance. Sci Adv. 2016;2:e1501332. doi: 10.1126/sciadv.1501332 49. Raheem J, Sliz E, Shin J, Holmes MV, Pike GB, Richer L, Gaudet D, Paus T, Pausova Z. Visceral adiposity is associated with metabolic profiles predictive of type 2 diabetes and myocardial infarction. Commun Med (Lond). 2022;2:81. doi: 10.1038/s43856-022-00140-5 50. Grabner GF, Xie H, Schweiger M, Zechner R. Lipolysis: cellular mechanisms for lipid mobilization from fat stores. Nat Metab. 2021;3:1445-1465. doi: 10.1038/s42255-021-00493-6 51. Althaher AR. An Overview of Hormone-Sensitive Lipase (HSL). ScientificWorldJournal. 2022;2022:1964684. doi: 10.1155/2022/1964684 52. Schweiger M, Schreiber R, Haemmerle G, Lass A, Fledelius C, Jacobsen P, Tornqvist H, Zechner R, Zimmermann R. Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism. J Biol Chem. 2006;281:40236-40241. doi: 10.1074/jbc.M608048200 76 53. Arner P. Human fat cell lipolysis: biochemistry, regulation and clinical role. Best Pract Res Clin Endocrinol Metab. 2005;19:471-482. doi: 10.1016/j.beem.2005.07.004 54. Lafontan M, Moro C, Berlan M, Crampes F, Sengenes C, Galitzky J. Control of lipolysis by natriuretic peptides and cyclic GMP. Trends Endocrinol Metab. 2008;19:130-137. doi: 10.1016/j.tem.2007.11.006 55. Kienesberger PC, Pulinilkunnil T, Nagendran J, Dyck JR. Myocardial triacylglycerol metabolism. J Mol Cell Cardiol. 2013;55:101-110. doi: 10.1016/j.yjmcc.2012.06.018 56. Paul A, Chan L, Bickel PE. The PAT family of lipid droplet proteins in heart and vascular cells. Current hypertension reports. 2008;10:461-466. doi: 10.1007/s11906-008-0086-y 57. Swanton EM, Saggerson ED. Effects of adrenaline on triacylglycerol synthesis and turnover in ventricular myocytes from adult rats. Biochem J. 1997;328 ( Pt 3):913-922. doi: 10.1042/bj3280913 58. Chiu AP, Wan A, Rodrigues B. Cardiomyocyte-endothelial cell control of lipoprotein lipase. Biochim Biophys Acta. 2016;1861:1434-1441. doi: 10.1016/j.bbalip.2016.03.016 59. Lee CS, Zhai YJ, Rodrigues B. Changes in Lipoprotein Lipase in the Heart Following Diabetes Onset. Engineering-Prc. 2023;20:19-25. doi: 10.1016/j.eng.2022.06.013 60. Beigneux AP, Allan CM, Sandoval NP, Cho GW, Heizer PJ, Jung RS, Stanhope KL, Havel PJ, Birrane G, Meiyappan M, et al. Lipoprotein lipase is active as a monomer. Proceedings of the National Academy of Sciences of the United States of America. 2019;116:6319-6328. doi: 10.1073/pnas.1900983116 61. Song W, Beigneux AP, Weston TA, Chen K, Yang Y, Nguyen LP, Guagliardo P, Jung H, Tran AP, Tu Y, et al. The lipoprotein lipase that is shuttled into capillaries by GPIHBP1 enters the glycocalyx where it mediates lipoprotein processing. Proceedings of the National Academy of Sciences of the United States of America. 2023;120:e2313825120. doi: 10.1073/pnas.2313825120 62. Noh HL, Okajima K, Molkentin JD, Homma S, Goldberg IJ. Acute lipoprotein lipase deletion in adult mice leads to dyslipidemia and cardiac dysfunction. Am J Physiol Endocrinol Metab. 2006;291:E755-760. doi: 10.1152/ajpendo.00111.2006 63. Kersten S. Physiological regulation of lipoprotein lipase. Biochim Biophys Acta. 2014;1841:919-933. doi: 10.1016/j.bbalip.2014.03.013 64. Blanchette-Mackie EJ, Masuno H, Dwyer NK, Olivecrona T, Scow RO. Lipoprotein lipase in myocytes and capillary endothelium of heart: immunocytochemical study. Am J Physiol. 1989;256:E818-828. doi: 10.1152/ajpendo.1989.256.6.E818 65. Zaha VG, Young LH. AMP-activated protein kinase regulation and biological actions in the heart. Circ Res. 2012;111:800-814. doi: 10.1161/CIRCRESAHA.111.255505 66. Kim MS, Wang F, Puthanveetil P, Kewalramani G, Hosseini-Beheshti E, Ng N, Wang Y, Kumar U, Innis S, Proud CG, et al. Protein kinase D is a key regulator of cardiomyocyte lipoprotein lipase secretion after diabetes. Circ Res. 2008;103:252-260. doi: 10.1161/CIRCRESAHA.108.178681 67. Yu X, Burgess SC, Ge H, Wong KK, Nassem RH, Garry DJ, Sherry AD, Malloy CR, Berger JP, Li C. Inhibition of cardiac lipoprotein utilization by transgenic overexpression of Angptl4 in the heart. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:1767-1772. doi: 10.1073/pnas.0409564102 77 68. Wang Y, Puthanveetil P, Wang F, Kim MS, Abrahani A, Rodrigues B. Severity of Diabetes Governs Vascular Lipoprotein Lipase by Affecting Enzyme Dimerization and Disassembly. Diabetes. 2011;60:2041-2050. doi: 10.2337/db11-0042 69. Kristensen KK, Leth-Espensen KZ, Mertens HDT, Birrane G, Meiyappan M, Olivecrona G, Jorgensen TJD, Young SG, Ploug M. Unfolding of monomeric lipoprotein lipase by ANGPTL4: Insight into the regulation of plasma triglyceride metabolism. Proceedings of the National Academy of Sciences of the United States of America. 2020;117:4337-4346. doi: 10.1073/pnas.1920202117 70. Wang F, Kim MS, Puthanveetil P, Kewalramani G, Deppe S, Ghosh S, Abrahani A, Rodrigues B. Endothelial heparanase secretion after acute hypoinsulinemia is regulated by glucose and fatty acid. American journal of physiology Heart and circulatory physiology. 2009;296:H1108-1116. doi: 10.1152/ajpheart.01312.2008 71. Kim MS, Wang F, Puthanveetil P, Kewalramani G, Innis S, Marzban L, Steinberg SF, Webber TD, Kieffer TJ, Abrahani A, et al. Cleavage of protein kinase D after acute hypoinsulinemia prevents excessive lipoprotein lipase-mediated cardiac triglyceride accumulation. Diabetes. 2009;58:2464-2475. doi: 10.2337/db09-0681 72. Puri K, Lal N, Shang R, Ghosh S, Flibotte S, Dyer R, Hussein B, Rodrigues B. Diabetes Mellitus Severity and a Switch From Using Lipoprotein Lipase to Adipose-Derived Fatty Acid Results in a Cardiac Metabolic Signature That Embraces Cell Death. J Am Heart Assoc. 2019;8:e014022. doi: 10.1161/JAHA.119.014022 73. Pulinilkunnil T, Rodrigues B. Cardiac lipoprotein lipase: metabolic basis for diabetic heart disease. Cardiovascular research. 2006;69:329-340. doi: 10.1016/j.cardiores.2005.09.017 74. Zechner R, Kienesberger PC, Haemmerle G, Zimmermann R, Lass A. Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores. Journal of lipid research. 2009;50:3-21. doi: 10.1194/jlr.R800031-JLR200 75. Rodrigues B, Cam MC, Jian K, Lim F, Sambandam N, Shepherd G. Differential effects of streptozotocin-induced diabetes on cardiac lipoprotein lipase activity. Diabetes. 1997;46:1346-1353. 76. Morigny P, Boucher J, Arner P, Langin D. Lipid and glucose metabolism in white adipocytes: pathways, dysfunction and therapeutics. Nat Rev Endocrinol. 2021;17:276-295. doi: 10.1038/s41574-021-00471-8 77. Falkevall A, Mehlem A, Folestad E, Ning FC, Osorio-Conles O, Radmann R, de Hollanda A, Wright SD, Scotney P, Nash A, et al. Inhibition of VEGF-B signaling prevents non-alcoholic fatty liver disease development by targeting lipolysis in the white adipose tissue. J Hepatol. 2023;78:901-913. doi: 10.1016/j.jhep.2023.01.014 78. Palomer X, Pizarro-Delgado J, Barroso E, Vazquez-Carrera M. Palmitic and Oleic Acid: The Yin and Yang of Fatty Acids in Type 2 Diabetes Mellitus. Trends Endocrinol Metab. 2018;29:178-190. doi: 10.1016/j.tem.2017.11.009 79. Yamamoto T, Sano M. Deranged Myocardial Fatty Acid Metabolism in Heart Failure. Int J Mol Sci. 2022;23. doi: 10.3390/ijms23020996 80. Alves-Bezerra M, Cohen DE. Triglyceride Metabolism in the Liver. Compr Physiol. 2017;8:1-8. doi: 10.1002/cphy.c170012 81. Schweiger M, Romauch M, Schreiber R, Grabner GF, Hutter S, Kotzbeck P, Benedikt P, Eichmann TO, Yamada S, Knittelfelder O, et al. Pharmacological inhibition of adipose 78 triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice. Nat Commun. 2017;8:14859. doi: 10.1038/ncomms14859 82. Girousse A, Tavernier G, Valle C, Moro C, Mejhert N, Dinel AL, Houssier M, Roussel B, Besse-Patin A, Combes M, et al. Partial inhibition of adipose tissue lipolysis improves glucose metabolism and insulin sensitivity without alteration of fat mass. PLoS Biol. 2013;11:e1001485. doi: 10.1371/journal.pbio.1001485 83. Cusi K, Orsak B, Bril F, Lomonaco R, Hecht J, Ortiz-Lopez C, Tio F, Hardies J, Darland C, Musi N, et al. Long-Term Pioglitazone Treatment for Patients With Nonalcoholic Steatohepatitis and Prediabetes or Type 2 Diabetes Mellitus: A Randomized Trial. Ann Intern Med. 2016;165:305-315. doi: 10.7326/M15-1774 84. DeFronzo RA, Inzucchi S, Abdul-Ghani M, Nissen SE. Pioglitazone: The forgotten, cost-effective cardioprotective drug for type 2 diabetes. Diab Vasc Dis Res. 2019;16:133-143. doi: 10.1177/1479164118825376 85. Kessler G, Friedman J. Metabolism of fatty acids and glucose. Circulation. 1998;98:1351. 86. Shang R, Lal N, Lee CS, Zhai Y, Puri K, Seira O, Boushel RC, Sultan I, Rasanen M, Alitalo K, et al. Cardiac-specific VEGFB overexpression reduces lipoprotein lipase activity and improves insulin action in rat heart. Am J Physiol Endocrinol Metab. 2021;321:E753-E765. doi: 10.1152/ajpendo.00219.2021 87. Yaribeygi H, Bo S, Ruscica M, Sahebkar A. Ceramides and diabetes mellitus: an update on the potential molecular relationships. Diabet Med. 2020;37:11-19. doi: 10.1111/dme.13943 88. Taskinen MR. Lipoprotein lipase in diabetes. Diabetes Metab Rev. 1987;3:551-570. doi: 10.1002/dmr.5610030208 89. Pulinilkunnil T, Abrahani A, Varghese J, Chan N, Tang I, Ghosh S, Kulpa J, Allard M, Brownsey R, Rodrigues B. Evidence for rapid "metabolic switching" through lipoprotein lipase occupation of endothelial-binding sites. Journal of Molecular and Cellular Cardiology. 2003;35:1093-1103. doi: 10.1016/S0022-2828(03)00205-0 90. Willecke F, Scerbo D, Nagareddy P, Obunike JC, Barrett TJ, Abdillahi ML, Trent CM, Huggins LA, Fisher EA, Drosatos K, et al. Lipolysis, and not hepatic lipogenesis, is the primary modulator of triglyceride levels in streptozotocin-induced diabetic mice. Arterioscler Thromb Vasc Biol. 2015;35:102-110. doi: 10.1161/ATVBAHA.114.304615 91. Levak-Frank S, Hofmann W, Weinstock PH, Radner H, Sattler W, Breslow JL, Zechner R. Induced mutant mouse lines that express lipoprotein lipase in cardiac muscle, but not in skeletal muscle and adipose tissue, have normal plasma triglyceride and high-density lipoprotein-cholesterol levels. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:3165-3170. doi: 10.1073/pnas.96.6.3165 92. Khan RS, Lin Y, Hu Y, Son NH, Bharadwaj KG, Palacios C, Chokshi A, Ji R, Yu S, Homma S, et al. Rescue of heart lipoprotein lipase-knockout mice confirms a role for triglyceride in optimal heart metabolism and function. Am J Physiol Endocrinol Metab. 2013;305:E1339-1347. doi: 10.1152/ajpendo.00349.2013 93. Hinkel R, Howe A, Renner S, Ng J, Lee S, Klett K, Kaczmarek V, Moretti A, Laugwitz KL, Skroblin P, et al. Diabetes Mellitus-Induced Microvascular Destabilization in the Myocardium. J Am Coll Cardiol. 2017;69:131-143. doi: 10.1016/j.jacc.2016.10.058 79 94. Adameova A, Dhalla NS. Role of microangiopathy in diabetic cardiomyopathy. Heart Fail Rev. 2014;19:25-33. doi: 10.1007/s10741-013-9378-7 95. Borradaile NM, Schaffer JE. Lipotoxicity in the heart. Current hypertension reports. 2005;7:412-417. 96. Wilson AJ, Gill EK, Abudalo RA, Edgar KS, Watson CJ, Grieve DJ. Reactive oxygen species signalling in the diabetic heart: emerging prospect for therapeutic targeting. Heart. 2018;104:293-299. doi: 10.1136/heartjnl-2017-311448 97. Volpe CMO, Villar-Delfino PH, Dos Anjos PMF, Nogueira-Machado JA. Cellular death, reactive oxygen species (ROS) and diabetic complications. Cell Death Dis. 2018;9:119. doi: 10.1038/s41419-017-0135-z 98. van de Weijer T, Schrauwen-Hinderling VB, Schrauwen P. Lipotoxicity in type 2 diabetic cardiomyopathy. Cardiovascular research. 2011;92:10-18. doi: 10.1093/cvr/cvr212 99. Wende AR, Symons JD, Abel ED. Mechanisms of lipotoxicity in the cardiovascular system. Current hypertension reports. 2012;14:517-531. doi: 10.1007/s11906-012-0307-2 100. Hagberg CE, Falkevall A, Wang X, Larsson E, Huusko J, Nilsson I, van Meeteren LA, Samen E, Lu L, Vanwildemeersch M, et al. Vascular endothelial growth factor B controls endothelial fatty acid uptake. Nature. 2010;464:917-921. doi: 10.1038/nature08945 101. Falkevall A, Mehlem A, Palombo I, Heller Sahlgren B, Ebarasi L, He L, Ytterberg AJ, Olauson H, Axelsson J, Sundelin B, et al. Reducing VEGF-B Signaling Ameliorates Renal Lipotoxicity and Protects against Diabetic Kidney Disease. Cell Metab. 2017;25:713-726. doi: 10.1016/j.cmet.2017.01.004 102. Hagberg CE, Mehlem A, Falkevall A, Muhl L, Fam BC, Ortsater H, Scotney P, Nyqvist D, Samen E, Lu L, et al. Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes. Nature. 2012;490:426-430. doi: 10.1038/nature11464 103. Mallick R, Yla-Herttuala S. Therapeutic Potential of VEGF-B in Coronary Heart Disease and Heart Failure: Dream or Vision? Cells. 2022;11. doi: 10.3390/cells11244134 104. Holmes DI, Zachary I. The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome Biol. 2005;6:209. doi: 10.1186/gb-2005-6-2-209 105. Olofsson B, Pajusola K, Kaipainen A, von Euler G, Joukov V, Saksela O, Orpana A, Pettersson RF, Alitalo K, Eriksson U. Vascular endothelial growth factor B, a novel growth factor for endothelial cells. Proc Natl Acad Sci U S A. 1996;93:2576-2581. 106. Li X, Aase K, Li H, von Euler G, Eriksson U. Isoform-specific expression of VEGF-B in normal tissues and tumors. Growth Factors. 2001;19:49-59. doi: 10.3109/08977190109001075 107. Aase K, Lymboussaki A, Kaipainen A, Olofsson B, Alitalo K, Eriksson U. Localization of VEGF-B in the mouse embryo suggests a paracrine role of the growth factor in the developing vasculature. Dev Dyn. 1999;215:12-25. doi: 10.1002/(SICI)1097-0177(199905)215:1<12::AID-DVDY3>3.0.CO;2-N 108. Bry M, Kivela R, Leppanen VM, Alitalo K. Vascular endothelial growth factor-B in physiology and disease. Physiological reviews. 2014;94:779-794. doi: 10.1152/physrev.00028.2013 80 109. Makinen T, Olofsson B, Karpanen T, Hellman U, Soker S, Klagsbrun M, Eriksson U, Alitalo K. Differential binding of vascular endothelial growth factor B splice and proteolytic isoforms to neuropilin-1. J Biol Chem. 1999;274:21217-21222. doi: 10.1074/jbc.274.30.21217 110. Lee C, Chen RY, Sun GL, Liu XL, Lin XC, He C, Xing LY, Liu LX, Jensen LD, Kumar A, et al. VEGF-B prevents excessive angiogenesis by inhibiting FGF2/FGFR1 pathway. Signal Transduct Tar. 2023;8. doi: ARTN 305 10.1038/s41392-023-01539-9 111. Li X, Kumar A, Zhang F, Lee C, Tang Z. Complicated life, complicated VEGF-B. Trends Mol Med. 2012;18:119-127. doi: 10.1016/j.molmed.2011.11.006 112. Kivela R, Bry M, Robciuc MR, Rasanen M, Taavitsainen M, Silvola JM, Saraste A, Hulmi JJ, Anisimov A, Mayranpaa MI, et al. VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart. EMBO Mol Med. 2014;6:307-321. doi: 10.1002/emmm.201303147 113. Karpanen T, Bry M, Ollila HM, Seppanen-Laakso T, Liimatta E, Leskinen H, Kivela R, Helkamaa T, Merentie M, Jeltsch M, et al. Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy. Circ Res. 2008;103:1018-1026. doi: 10.1161/CIRCRESAHA.108.178459 114. Arjunan P, Lin X, Tang Z, Du Y, Kumar A, Liu L, Yin X, Huang L, Chen W, Chen Q, et al. VEGF-B is a potent antioxidant. Proceedings of the National Academy of Sciences of the United States of America. 2018;115:10351-10356. doi: 10.1073/pnas.1801379115 115. Li Y, Zhang F, Nagai N, Tang Z, Zhang S, Scotney P, Lennartsson J, Zhu C, Qu Y, Fang C, et al. VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats. J Clin Invest. 2008;118:913-923. doi: 10.1172/JCI33673 116. Shibuya M. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies. Genes & cancer. 2011;2:1097-1105. doi: 10.1177/1947601911423031 117. Sands M, Howell K, Costello CM, McLoughlin P. Placenta growth factor and vascular endothelial growth factor B expression in the hypoxic lung. Respir Res. 2011;12:17. doi: 10.1186/1465-9921-12-17 118. Shibuya M. Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1). Int J Biochem Cell Biol. 2001;33:409-420. 119. Boucher JM, Clark RP, Chong DC, Citrin KM, Wylie LA, Bautch VL. Dynamic alterations in decoy VEGF receptor-1 stability regulate angiogenesis. Nat Commun. 2017;8:15699. doi: 10.1038/ncomms15699 120. Mould AW, Greco SA, Cahill MM, Tonks ID, Bellomo D, Patterson C, Zournazi A, Nash A, Scotney P, Hayward NK, et al. Transgenic overexpression of vascular endothelial growth factor-B isoforms by endothelial cells potentiates postnatal vessel growth in vivo and in vitro. Circ Res. 2005;97:e60-70. doi: 10.1161/01.RES.0000182631.33638.77 121. Zhang F, Tang Z, Hou X, Lennartsson J, Li Y, Koch AW, Scotney P, Lee C, Arjunan P, Dong L, et al. VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis. Proceedings of the 81 National Academy of Sciences of the United States of America. 2009;106:6152-6157. doi: 10.1073/pnas.0813061106 122. Robciuc MR, Kivela R, Williams IM, de Boer JF, van Dijk TH, Elamaa H, Tigistu-Sahle F, Molotkov D, Leppanen VM, Kakela R, et al. VEGFB/VEGFR1-Induced Expansion of Adipose Vasculature Counteracts Obesity and Related Metabolic Complications. Cell Metab. 2016;23:712-724. doi: 10.1016/j.cmet.2016.03.004 123. Kivelä R, Hemanthakumar KA, Vaparanta K, Robciuc M, Izumiya Y, Kidoya H, Takakura N, Peng XY, Sawyer DB, Elenius K, et al. Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2-Mediated Paracrine Signaling. Circulation. 2019;139:2570-2584. doi: 10.1161/Circulationaha.118.036099 124. Bhardwaj S, Roy H, Gruchala M, Viita H, Kholova I, Kokina I, Achen MG, Stacker SA, Hedman M, Alitalo K, et al. Angiogenic responses of vascular endothelial growth factors in periadventitial tissue. Hum Gene Ther. 2003;14:1451-1462. doi: 10.1089/104303403769211664 125. Zhang F, Tang ZS, Hou X, Lennartsson J, Li Y, Koch AW, Scotney P, Lee C, Arjunan P, Dong LJ, et al. VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:6152-6157. doi: 10.1073/pnas.0813061106 126. Lahteenvuo JE, Lahteenvuo MT, Kivela A, Rosenlew C, Falkevall A, Klar J, Heikura T, Rissanen TT, Vahakangas E, Korpisalo P, et al. Vascular endothelial growth factor-B induces myocardium-specific angiogenesis and arteriogenesis via vascular endothelial growth factor receptor-1- and neuropilin receptor-1-dependent mechanisms. Circulation. 2009;119:845-856. doi: 10.1161/CIRCULATIONAHA.108.816454 127. Korpela H, Hätinen OP, Nieminen T, Mallick R, Toivanen P, Airaksinen J, Valli K, Hakulinen M, Poutiainen P, Nurro J, et al. Adenoviral VEGF-B186R127S gene transfer induces angiogenesis and improves perfusion in ischemic heart. Iscience. 2021;24. doi: ARTN 103533 10.1016/j.isci.2021.103533 128. Bry M, Kivela R, Holopainen T, Anisimov A, Tammela T, Soronen J, Silvola J, Saraste A, Jeltsch M, Korpisalo P, et al. Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation. Circulation. 2010;122:1725-1733. doi: 10.1161/CIRCULATIONAHA.110.957332 129. Moessinger C, Nilsson I, Muhl L, Zeitelhofer M, Heller Sahlgren B, Skogsberg J, Eriksson U. VEGF-B signaling impairs endothelial glucose transcytosis by decreasing membrane cholesterol content. EMBO Rep. 2020;21:e49343. doi: 10.15252/embr.201949343 130. Zentilin L, Puligadda U, Lionetti V, Zacchigna S, Collesi C, Pattarini L, Ruozi G, Camporesi S, Sinagra G, Pepe M, et al. Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction. FASEB J. 2010;24:1467-1478. doi: 10.1096/fj.09-143180 131. Lal N, Chiu AP, Wang F, Zhang D, Jia J, Wan A, Vlodavsky I, Hussein B, Rodrigues B. Loss of VEGFB and its signaling in the diabetic heart is associated with increased cell 82 death signaling. American journal of physiology Heart and circulatory physiology. 2017;312:H1163-H1175. doi: 10.1152/ajpheart.00659.2016 132. Huusko J, Lottonen L, Merentie M, Gurzeler E, Anisimov A, Miyanohara A, Alitalo K, Tavi P, Yla-Herttuala S. AAV9-mediated VEGF-B gene transfer improves systolic function in progressive left ventricular hypertrophy. Mol Ther. 2012;20:2212-2221. doi: 10.1038/mt.2012.145 133. Rasanen M, Degerman J, Nissinen TA, Miinalainen I, Kerkela R, Siltanen A, Backman JT, Mervaala E, Hulmi JJ, Kivela R, et al. VEGF-B gene therapy inhibits doxorubicin-induced cardiotoxicity by endothelial protection. Proceedings of the National Academy of Sciences of the United States of America. 2016;113:13144-13149. doi: 10.1073/pnas.1616168113 134. Pepe M, Mamdani M, Zentilin L, Csiszar A, Qanud K, Zacchigna S, Ungvari Z, Puligadda U, Moimas S, Xu X, et al. Intramyocardial VEGF-B167 gene delivery delays the progression towards congestive failure in dogs with pacing-induced dilated cardiomyopathy. Circ Res. 2010;106:1893-1903. doi: 10.1161/CIRCRESAHA.110.220855 135. Bellomo D, Headrick JP, Silins GU, Paterson CA, Thomas PS, Gartside M, Mould A, Cahill MM, Tonks ID, Grimmond SM, et al. Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia. Circ Res. 2000;86:E29-35. doi: 10.1161/01.res.86.2.e29 136. Dijkstra MH, Pirinen E, Huusko J, Kivela R, Schenkwein D, Alitalo K, Yla-Herttuala S. Lack of cardiac and high-fat diet induced metabolic phenotypes in two independent strains of Vegf-b knockout mice. Sci Rep. 2014;4:6238. doi: 10.1038/srep06238 137. Luo X, Li RR, Li YQ, Yu HP, Yu HN, Jiang WG, Li YN. Reducing VEGFB expression regulates the balance of glucose and lipid metabolism in mice via VEGFR1. Mol Med Rep. 2022;26. doi: 10.3892/mmr.2022.12801 138. Fischer C, Mazzone M, Jonckx B, Carmeliet P. FLT1 and its ligands VEGFB and PlGF: drug targets for anti-angiogenic therapy? Nat Rev Cancer. 2008;8:942-956. doi: 10.1038/nrc2524 139. Janes PW, Parslow AC, Cao D, Rigopoulos A, Lee FT, Gong SJ, Cartwright GA, Burvenich IJG, Eriksson U, Johns TG, et al. An Anti-VEGF-B Antibody Reduces Abnormal Tumor Vasculature and Enhances the Effects of Chemotherapy. Cancers (Basel). 2024;16. doi: 10.3390/cancers16101902 140. Albrecht I, Kopfstein L, Strittmatter K, Schomber T, Falkevall A, Hagberg CE, Lorentz P, Jeltsch M, Alitalo K, Eriksson U, et al. Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis. PLoS One. 2010;5:e14109. doi: 10.1371/journal.pone.0014109 141. Zhang S, Tian W, Duan X, Zhang Q, Cao L, Liu C, Li G, Wang Z, Zhang J, Li J, et al. Melatonin attenuates diabetic cardiomyopathy by increasing autophagy of cardiomyocytes via regulation of VEGF-B/GRP78/PERK signaling pathway. Cardiovasc Diabetol. 2024;23:19. doi: 10.1186/s12933-023-02078-x 142. Larsen TS, Aasum E. Metabolic (in)flexibility of the diabetic heart. Cardiovasc Drugs Ther. 2008;22:91-95. doi: 10.1007/s10557-008-6083-1 83 143. Goyal RK, Rodrigues B, McNeill JH. Effect of tri-iodothyronine on cardiac responses to adrenergic-agonists in STZ-induced diabetic rats. Gen Pharmacol. 1987;18:357-362. doi: 10.1016/0306-3623(87)90091-7 144. Shang R, Lee CS, Wang H, Dyer R, Noll C, Carpentier A, Sultan I, Alitalo K, Boushel R, Hussein B, et al. Reduction in Insulin Uncovers a Novel Effect of VEGFB on Cardiac Substrate Utilization. Arterioscler Thromb Vasc Biol. 2024;44:177-191. doi: 10.1161/ATVBAHA.123.319972 145. Vuilleumier R, Lian T, Flibotte S, Khan ZN, Fuchs A, Pyrowolakis G, Allan DW. Retrograde BMP signaling activates neuronal gene expression through widespread deployment of a conserved BMP-responsive cis-regulatory activation element. Nucleic Acids Res. 2019;47:679-699. doi: 10.1093/nar/gky1135 146. Vargas E, Joy NV, Carrillo Sepulveda MA. Biochemistry, Insulin Metabolic Effects. In: StatPearls. Treasure Island (FL); 2024. 147. Ning FCF, Jensen N, Mi JR, Lindström W, Balan M, Muhl L, Eriksson U, Nilsson I, Nyqvist D. VEGF-B ablation in pancreatic β-cells upregulates insulin expression without affecting glucose homeostasis or islet lipid uptake. Sci Rep-Uk. 2020;10. doi: ARTN 923 10.1038/s41598-020-57599-2 148. Wang X, Shen X, Yan Y, Li H. Pyruvate dehydrogenase kinases (PDKs): an overview toward clinical applications. Biosci Rep. 2021;41. doi: 10.1042/BSR20204402 149. Schweiger M, Eichmann TO, Taschler U, Zimmermann R, Zechner R, Lass A. Measurement of lipolysis. Methods Enzymol. 2014;538:171-193. doi: 10.1016/B978-0-12-800280-3.00010-4 150. Ling M, Lai X, Quan L, Li F, Lang L, Fu Y, Feng S, Yi X, Zhu C, Gao P, et al. Knockdown of VEGFB/VEGFR1 Signaling Promotes White Adipose Tissue Browning and Skeletal Muscle Development. Int J Mol Sci. 2022;23. doi: 10.3390/ijms23147524 151. Cao ZL, Zhao H, Fan JJ, Shen YL, Han L, Jing GJ, Zeng X, Jin X, Zhu ZG, Bian Q, et al. Simultaneous blockade of VEGF-B and IL-17A ameliorated diabetic kidney disease by reducing ectopic lipid deposition and alleviating inflammation response. Cell Death Discovery. 2023;9. doi: ARTN 8 10.1038/s41420-023-01304-5 152. Shang R, Rodrigues B. Lipoprotein Lipase and Its Delivery of Fatty Acids to the Heart. Biomolecules. 2021;11. doi: 10.3390/biom11071016 153. Li R, Li Y, Yang X, Hu Y, Yu H, Li Y. Reducing VEGFB accelerates NAFLD and insulin resistance in mice via inhibiting AMPK signaling pathway. J Transl Med. 2022;20:341. doi: 10.1186/s12967-022-03540-2 154. Jin H, Li D, Wang X, Jia J, Chen Y, Yao Y, Zhao C, Lu X, Zhang S, Togo J, et al. VEGF and VEGFB Play Balancing Roles in Adipose Differentiation, Gene Expression, and Function. Endocrinology. 2018;159:2036-2049. doi: 10.1210/en.2017-03246 155. Gomez-Ambrosi J, Catalan V, Rodriguez A, Ramirez B, Silva C, Gil MJ, Salvador J, Fruhbeck G. Involvement of serum vascular endothelial growth factor family members in the development of obesity in mice and humans. J Nutr Biochem. 2010;21:774-780. doi: 10.1016/j.jnutbio.2009.05.004 156. Wu J, Wei H, Qu H, Feng Z, Long J, Ge Q, Deng H. Plasma vascular endothelial growth factor B levels are increased in patients with newly diagnosed type 2 diabetes mellitus 84 and associated with the first phase of glucose-stimulated insulin secretion function of beta-cell. J Endocrinol Invest. 2017;40:1219-1226. doi: 10.1007/s40618-017-0677-z 157. An D, Pulinilkunnil T, Qi D, Ghosh S, Abrahani A, Rodrigues B. The metabolic "switch" AMPK regulates cardiac heparin-releasable lipoprotein lipase. Am J Physiol Endocrinol Metab. 2005;288:E246-253. doi: 10.1152/ajpendo.00211.2004 158. Mowers J, Uhm M, Reilly SM, Simon J, Leto D, Chiang SH, Chang L, Saltiel AR. Inflammation produces catecholamine resistance in obesity via activation of PDE3B by the protein kinases IKKε and TBK1. Elife. 2013;2. doi: ARTN e01119 10.7554/eLife.01119 159. Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F, Nagy JA, Hooper A, Priller J, De Klerck B, et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med. 2002;8:831-840. doi: 10.1038/nm731 160. Kraaijenhof JM, Stroes ESG. Inflammatory Effects of Triglycerides: Relevant or Redundant? JACC Basic Transl Sci. 2023;8:476-478. doi: 10.1016/j.jacbts.2023.04.005 161. Wang Y, Qian Y, Fang Q, Zhong P, Li W, Wang L, Fu W, Zhang Y, Xu Z, Li X, et al. Saturated palmitic acid induces myocardial inflammatory injuries through direct binding to TLR4 accessory protein MD2. Nat Commun. 2017;8:13997. doi: 10.1038/ncomms13997 162. Yamamoto T, Endo J, Kataoka M, Matsuhashi T, Katsumata Y, Shirakawa K, Yoshida N, Isobe S, Moriyama H, Goto S, et al. Sirt1 counteracts decrease in membrane phospholipid unsaturation and diastolic dysfunction during saturated fatty acid overload. J Mol Cell Cardiol. 2019;133:1-11. doi: 10.1016/j.yjmcc.2019.05.019 163. Farvid MS, Ding M, Pan A, Sun Q, Chiuve SE, Steffen LM, Willett WC, Hu FB. Dietary linoleic acid and risk of coronary heart disease: a systematic review and meta-analysis of prospective cohort studies. Circulation. 2014;130:1568-1578. doi: 10.1161/CIRCULATIONAHA.114.010236 164. Hwang EH, Taki J, Yasue S, Fujimoto M, Taniguchi M, Matsunari I, Nakajima K, Shiobara S, Ikeda T, Tonami N. Absent myocardial iodine-123-BMIPP uptake and platelet/monocyte CD36 deficiency. J Nucl Med. 1998;39:1681-1684. 165. He C, Weston TA, Jung RS, Heizer P, Larsson M, Hu X, Allan CM, Tontonoz P, Reue K, Beigneux AP, et al. NanoSIMS Analysis of Intravascular Lipolysis and Lipid Movement across Capillaries and into Cardiomyocytes. Cell Metab. 2018;27:1055-1066 e1053. doi: 10.1016/j.cmet.2018.03.017 166. Chou E, Suzuma I, Way KJ, Opland D, Clermont AC, Naruse K, Suzuma K, Bowling NL, Vlahos CJ, Aiello LP, et al. Decreased cardiac expression of vascular endothelial growth factor and its receptors in insulin-resistant and diabetic States: a possible explanation for impaired collateral formation in cardiac tissue. Circulation. 2002;105:373-379. 167. Litvinukova M, Talavera-Lopez C, Maatz H, Reichart D, Worth CL, Lindberg EL, Kanda M, Polanski K, Heinig M, Lee M, et al. Cells of the adult human heart. Nature. 2020;588:466-472. doi: 10.1038/s41586-020-2797-4
Thesis/Dissertation
eng
Pharmaceutical Sciences
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Rodrigues, Brian
Role of vascular endothelial growth factor B in the cardiovascular complications of diabetes
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