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Role of ghrelin and ghrelin o-acyltransferase in the maintenance of maternal glucose homeostasis in calorie-restricted… Trivedi, Arjun 2013

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ROLE OF GHRELIN AND GHRELIN O-ACYLTRANSFERASE  IN THE MAINTENANCE OF MATERNAL GLUCOSE HOMEOSTASIS IN CALORIE-RESTRICTED MICE  by   Arjun Trivedi B.Sc. (Hons.), Queen?s University, 2009   A THESIS SUBMITTED IN PARTIAL FULFILLMENT  OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE   in  The Faculty of Graduate and Postdoctoral Studies (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)   October 2013   ?Arjun Trivedi, 2013   ii  Abstract  Ghrelin is a hormone that regulates energy homeostasis and is expressed in the stomach, hypothalamus and pituitary of humans and rodents. Ghrelin circulates as acylated (AG) and unacylated (UAG) ghrelin. Acylation is mediated by ghrelin O-acyltransferase (GOAT) and both forms of ghrelin are degraded by plasma proteases. Maintenance of euglycemia is important during pregnancy, where transplacental transfer of glucose is required for optimal fetal outcome. Maternal malnutrition is associated with decreased substrate availability and is a risk for hypoglycemia. AG prevents hypoglycemia in calorie-restricted (CR) mice by stimulating growth hormone (GH) release however its role in pregnancy remains unknown. I hypothesize that CR causes lower blood glucose in pregnant (P) compared to non-pregnant (NP) mice and that the GOAT-ghrelin axis is part of the response to this hypoglycemia. To determine ghrelin?s role in glucose homeostasis during pregnancy, wild-type (WT) and GOAT-KO (KO) mice were time-mated and fed-freely (FF) or CR by 50% for one week beginning at day 10.5 after conception. KO mice showed reduced fertility as conception was rare compared to WT animals. Unexpectedly, KO-CR mice also showed pregnancy termination early after CR was started and failed to survive to day 18 (=sacrifice), suggesting that  AG plays a critical role in maintaining energy homeostasis during pregnancy. To further investigate the effect of ghrelin during pregnancy, several parameters of energy metabolism were analyzed: body composition, blood glucose, plasma AG, UAG, GH, GOAT/ghrelin expression, hepatic glycogen and PCK1 expression. I demonstrate that CR affects glucose metabolism more severely in WT-P and KO-NP mice (which cannot produce AG) compared to WT-NP mice. The additive effect of pregnancy and CR in GOAT-KO mice increases the severity of hypoglycemia. I propose that in WT animals, an increase in AG and UAG levels, whether due to increased production, decreased degradation, or both, serves to mitigate the decrease in blood glucose. The mechanisms of action of AG remain unclear but may involve stimulation of glycogenolysis and/or gluconeogenesis directly or indirectly (via stimulation of GH). My work supports a physiological role for the AG/UAG pathway in the regulation of blood glucose concentrations during pregnancy.              iii  Preface  All of the work presented in this dissertation was conducted in the Diabetes, Nutrition & Metabolism research cluster at the Child & Family Research Institute. All protocols and associated methods were approved by the University of British Columbia Animal Care Committee (A10-0030). C57BL/6 WT and GOAT-KO mice were received in kind donation from Eli Lilly and Company (Indianapolis, IN, USA).  This work was funded by the Canadian Institutes of Health Research (CIHR).  A portion of chapter 2.6.1, a technical study, was published [Trivedi A, Babic S, Chanoine JP. Pitfalls in the determination of human acylated ghrelin plasma concentrations using a double antibody enzyme immunometric assay. Clin Biochem 45(1-2): 178-180, 2012]. I was the lead author, responsible for the design and execution of the experiment as well as writing most of the manuscript. Sandra Babic assisted in processing the samples as well as in the execution of the experiment. Jean-Pierre Chanoine assisted myself in the experimental design, oversaw the study and reviewed the manuscript prior to submission.                                iv  Table of Contents    Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Figures .............................................................................................................................. vii Acknowledgements .................................................................................................................... viii Dedication ..................................................................................................................................... ix  1. Introduction ....................................................................................................................... 1 1.1   Background ................................................................................................................. 1 1.2   Processing of ghrelin................................................................................................... 1 1.3   Acylation of ghrelin by ghrelin O-acyltransferase (GOAT) ....................................... 2 1.4   Deacylation of AG and degradation of AG and UAG ................................................ 2 1.5   Role of UAG ............................................................................................................... 3 1.6   Role of AG .................................................................................................................. 4             1.6.1 Growth-hormone release .................................................................................. 4 1.6.2 Stimulation of food intake ................................................................................ 4 1.6.3 Body composition ............................................................................................. 5 1.6.4 Glucose metabolism ......................................................................................... 6 1.7   Ghrelin during pregnancy ........................................................................................... 7 1.8   Effects of food deprivation and calorie-restriction on the GOAT-ghrelin axis in pregnant and non-pregnant humans and rodents ....................................................... 8 1.9   Project rationale ........................................................................................................ 10 1.10 Relevance to human health ....................................................................................... 10 1.11 Hypotheses and objectives ........................................................................................ 12 1.11.1 Hypotheses .................................................................................................... 12 1.11.2 Overall objective ........................................................................................... 12 1.11.3 Specific objectives ........................................................................................ 12 2. Methods ............................................................................................................................ 13 2.1   Experimental animals................................................................................................ 13 2.1.1 Genetically modified mice ? Wild-type and GOAT knock-out ..................... 13 2.1.2 Diet and priming ............................................................................................. 13 2.2   Experimental procedure ............................................................................................ 14 2.3   Glucose monitoring ................................................................................................... 15 2.4   Body composition analysis ....................................................................................... 15 2.5   Tissue harvest............................................................................................................ 17 2.6   GOAT and ghrelin determination ............................................................................. 18 2.6.1 Plasma AG and UAG ..................................................................................... 18 2.6.2 Tissue specific ghrelin and GOAT mRNA expression................................... 21 2.7   Plasma growth hormone ........................................................................................... 23 2.8   Hepatic glycogen content and gluconeogenesis ....................................................... 24 2.8.1 Hepatic glycogen content determination ........................................................ 25 2.8.2 Hepatic gluconeogenesis determination ......................................................... 27 v  2.9   Statistics .................................................................................................................... 27 3. Results .............................................................................................................................. 30 3.1   Preliminary work ...................................................................................................... 30 3.2   Food intake................................................................................................................ 30 3.3   GOAT-KO mice: Fertility, fetus quantities and response to the 50% CR diet ......... 30 3.4   Body composition ..................................................................................................... 31 3.4.1 Fat mass percent changes in WT mice ........................................................... 32 3.4.2 Fat mass percent changes in GOAT-KO mice ............................................... 33 3.4.3 Comparison of fat mass percent changes between Day -0.5 and Day 17.5 in WT vs. GOAT-KO mice ......................................................................................... 34 3.4.4 Lean mass percent changes in WT mice ........................................................ 35 3.4.5 Lean mass percent changes in GOAT-KO mice ............................................ 37 3.4.6 Comparison of lean mass percent changes between Day -0.5 and Day 17.5 in WT vs. GOAT-KO mice ......................................................................................... 38 3.5   Blood glucose ............................................................................................................ 39 3.5.1 Blood glucose changes in WT mice ............................................................... 40 3.5.2 Blood glucose changes in GOAT-KO mice ................................................... 41 3.5.3 Comparison of blood glucose changes between Day -0.5 and Day 17.5 in WT vs. GOAT-KO mice ................................................................................................. 43 3.6   Plasma AG and UAG concentrations ........................................................................ 43 3.6.1 Plasma AG and UAG concentrations in WT mice ......................................... 44 3.6.2 Plasma UAG concentrations in GOAT-KO mice ........................................... 45 3.6.3 Comparison of plasma UAG concentrations in WT vs. GOAT-KO mice ..... 46 3.7   GOAT and ghrelin mRNA expression in the stomach, hypothalamus and pituitary 46 3.7.1 GOAT mRNA expression in the stomach ...................................................... 47 3.7.2 Ghrelin mRNA expression in the stomach ..................................................... 48 3.7.3 GOAT mRNA expression in the hypothalamus ............................................. 49 3.7.4 Ghrelin mRNA expression in the hypothalamus ............................................ 49 3.7.5 GOAT mRNA expression in the pituitary gland ............................................ 50 3.7.6 Ghrelin mRNA expression in the pituitary gland ........................................... 51 3.8   Plasma growth hormone concentrations ................................................................... 53 3.8.1 Plasma growth hormone concentrations in WT mice ..................................... 53 3.8.2 Plasma growth hormone concentrations in GOAT-KO mice ......................... 54 3.8.3 Comparison of plasma growth hormone concentrations in WT vs. GOAT-KO mice ......................................................................................................................... 55 3.9   Hepatic glycogen content .......................................................................................... 56 3.9.1 Hepatic glycogen content in WT mice ........................................................... 56 3.9.2 Hepatic glycogen content in GOAT-KO mice ............................................... 57 3.9.3 Comparison of hepatic glycogen content in WT vs. GOAT-KO mice .......... 58 3.10  Hepatic PCK1 mRNA expression ............................................................................ 59 3.10.1 Hepatic PCK1 mRNA expression in WT mice ............................................ 59 3.10.2 Hepatic PCK1 mRNA expression in GOAT-KO mice ................................ 59 3.10.3 Comparison of hepatic PCK1 mRNA expression in WT vs. GOAT-KO mice ................................................................................................................................. 59 4. Discussion......................................................................................................................... 61 4.1   Fertility and pregnancy in GOAT-KO mice ............................................................. 63 vi  4.1.1 Fertility in male GOAT-KO mice .................................................................. 63 4.1.2 Fertility in female GOAT-KO mice ............................................................... 63 4.1.3 Hypoglycemia associated with CR in GOAT-KO pregnant mice .................. 64 4.2   Body composition ..................................................................................................... 65 4.3   Blood glucose concentrations ................................................................................... 67 4.4   Association between blood glucose and AG/UAG concentrations .......................... 67 4.5   Tissue specific expression of GOAT and ghrelin mRNA ........................................ 68 4.5.1 Stomach .......................................................................................................... 68 4.5.2 Hypothalamus and pituitary ............................................................................ 68 4.5.3 Degradation of AG/UAG ................................................................................ 70 4.6 Regulatory mechanisms and maintenance of blood glucose ...................................... 70 4.6.1 Glycogenolysis and gluconeogenesis ............................................................. 70 4.6.2 Growth hormone ............................................................................................. 71 4.7 Strengths, limitations and future directions ................................................................ 74  Bibliography ................................................................................................................................ 76                               vii  List of Figures  Figure 2.1     Experimental timeline ............................................................................................. 14 Figure 2.2     Principle of the plasma AG and UAG EIA ............................................................. 21 Figure 2.3     Effects of insulin and glucagon signaling on hepatic glucose metabolism ............. 25 Figure 2.4     Conceptual role of AG in response to starvation-induced hypoglycemia ............... 25 Figure 3.1     Mean percent fat mass changes relative to Day - 0.5 in WT mice .......................... 33 Figure 3.2     Mean percent fat mass changes relative to Day -0.5 in GOAT-KO mice ............... 34 Figure 3.3     Comparison of mean percent fat mass changes at Day 17.5 between WT and GOAT-KO mice ...................................................................................................... 35 Figure 3.4     Mean percent lean mass changes relative to Day -0.5 in WT mice ........................ 36 Figure 3.5     Mean percent lean mass changes relative to Day -0.5 in GOAT-KO mice ............ 38 Figure 3.6     Comparison of mean percent lean mass changes at Day 17.5 between WT and GOAT-KO mice ...................................................................................................... 39 Figure 3.7     Mean blood glucose changes relative to Day -0.5 in WT mice ............................... 41 Figure 3.8     Mean blood glucose changes relative to Day -0.5 in GOAT-KO ........................... 42 Figure 3.9     Comparison of mean blood glucose changes at Day 17.5 between WT and GOAT-KO mice ................................................................................................................... 43 Figure 3.10    Mean plasma AG and UAG concentrations in WT mice ....................................... 44 Figure 3.11    Mean plasma AG and UAG concentrations in GOAT-KO mice ........................... 45 Figure 3.12    Comparison of mean plasma UAG concentrations between WT and GOAT-KO mice ....................................................................................................................... 46 Figure 3.13    Mean relative GOAT mRNA expression in the stomach in WT mice ................... 47 Figure 3.14    Mean relative ghrelin mRNA expression in the stomach in WT and GOAT-KO mice ....................................................................................................................... 48 Figure 3.15    Mean relative GOAT mRNA expression in the hypothalamus in WT mice .......... 49 Figure 3.16    Mean relative ghrelin mRNA expression in the hypothalamus in WT and GOAT-KO mice ................................................................................................................ 50 Figure 3.17    Mean relative ghrelin mRNA expression in the pituitary gland in WT mice ........ 52 Figure 3.18    Mean relative ghrelin mRNA expression in the pituitary gland in GOAT-KO mice............................................................................................................................... 52 Figure 3.19    Comparison of mean relative ghrelin mRNA expression in the pituitary gland between WT and GOAT-KO mice ....................................................................... 53 Figure 3.20    Mean plasma growth hormone concentrations in WT mice ................................... 54 Figure 3.21    Mean plasma growth hormone concentrations in GOAT-KO mice ....................... 55 Figure 3.22    Comparison of mean plasma growth hormone concentrations between WT and GOAT-KO mice .................................................................................................... 56 Figure 3.23    Mean relative hepatic glycogen concentration per gram of liver in WT mice ....... 57 Figure 3.24    Mean relative hepatic glycogen concentration per gram of liver in GOAT-KO mice............................................................................................................................... 58 Figure 3.25    Comparison of mean relative hepatic glycogen concentrations per gram of liver between WT and GOAT-KO mice ....................................................................... 58 Figure 3.26    Mean relative hepatic PCK1 mRNA expression in WT and GOAT-KO mice ...... 60    viii  Acknowledgements  Foremost, I would like to express my sincere gratitude to my supervisor Dr. Jean-Pierre Chanoine for his unconditional and continuous support of my project, for his patience, motivation and immense knowledge. His guidance has not only assisted me in the academic realms alone but has had a peripheral effect on my own personal journey into a lifestyle of hard work, sacrifice and commitment. I could not have imagined having a better mentor during my graduate studies and I would like to thank him for giving me this opportunity to engage in this exciting research in his lab.  In addition to my advisor, I would like to thank members of my defense committee Dr. William Gibson, Dr. Vince Duronio and Dr. Bruce Verchere for their support of my project. Their advice and healthy criticism has helped to refine my thesis and am grateful for their care. I?d like to also thank Dr. William Gibson, in particular, for the use of his laboratory?s EchoMRI system for the body composition analyses portion of my thesis.  I also want to thank our laboratory technician Sandra Babic who without her experimental expertise and technical skill, our findings would not have been possible. She managed the lab for the majority of this project and enhanced my understanding of general laboratory skills.   Last but certainly not the least, I would like to thank my parents, Jay and Daksha, for being the best mom and dad a son could ask for. Their incredible sacrifice, hard work and sense of humility have kept me grounded. My studies would not have been possible if it wasn?t for the temperate but firm motivation by them as well as the time they have invested throughout my life in ensuring I had all the tools to succeed in graduate studies.                      ix  Dedication                ?For my parents, Jay and Daksha, who have given me everything?                  1  1 Introduction  1.1 Background  Ghrelin is a 28 amino acid peptide hormone primarily produced in the gastric oxyntic mucosa of the stomach, specifically the P/D1 cells in humans and X/A-like cells in rodents 1-3. It is also produced, to a lesser extent, by the alpha cells and epsilon cells of the pancreas 4-7. The presence of ghrelin has also been reported in the hypothalamus and pituitary in both humans and rodents 8-10. Its structure and function are highly conserved and rodent ghrelin only differs from human by two amino acids (Arg11-Val12 in humans, Lys11-Ala12 in rodents) 11,12. It results from the processing of a preproghrelin precursor and circulates in two forms, acylated ghrelin (AG) and unacylated ghrelin (UAG) 5,13,14. Acylation occurs by the addition of a medium chain fatty acid to the peptide mediated by an enzyme called ghrelin O-acyltransferase (GOAT) 15. This is a key step as most of the known actions of ghrelin are mediated through the acylated form. Ghrelin has emerged as an important regulator of energy expenditure and as such is attracting increasing interest from the scientific community.  1.2 Processing of ghrelin  The ghrelin gene is located on chromosome 3p25-26 in humans and contains 4 exons and 3 introns where exons 1 and 2 code for the mature ghrelin peptide 16.  In mice, it is located on chromosome 6 8. Translation of the mRNA produces a 117 amino acid precursor, preproghrelin, containing a signal peptide 17.  Upon cleavage of the signal peptide, the proghrelin precursor undergoes an important post-translational modification known as acylation which will be described later. Subsequent enzymatic cleavages take place of this acylated proghrelin precursor takes place in the Golgi apparatus and is mediated by prohormone convertase 1/3 (PC 1/3). This results in a 28 amino acid AG or UAG peptide and 2  in a 23 amino acid putative hormone called obestatin whose physiological role remains controversial 5,16. Similar to ghrelin, PC 1/3 is also expressed in the stomach, hypothalamus and pituitary 18,19. Thus, ghrelin?s processing and activation are mediated by local prohormone convertases expressed in these ghrelin-secreting glands. 1.3 Acylation of ghrelin by ghrelin O-acyltransferase (GOAT)  Acylation of proghrelin by ghrelin O-acyltransferase (GOAT) takes place in the endoplasmic reticulum 15. GOAT transfers a medium chain fatty acid group, octanoate, from an acyl-CoA and attaches it to the serine-3 of the peptide 20. GOAT belongs to a family of 16 hydrophobic membrane-bound O-acyltransferases known as ?MBOATs .? G2$T?s only known role is to acylate ghrelin and conversely GOAT is also the only enzyme known to mediate the acylation of ghrelin 15. Similar to ghrelin, G2$T?s structure and its activity are highly conserved throughout evolution 15,21. GOAT mRNA expression in various tissues has been shown to overlap with those of ghrelin 22.These expression patterns are confined mainly to the stomach and intestine (5-fold greater GOAT mRNA expression) and to a lesser extent the hypothalamus, pituitary gland and pancreas 23,24. In essence, ghrelin and GOAT expression are co-localized in the same tissues and thus changes in expression of each peptide can influence the production of AG and UAG released into the circulation. 1.4 Deacylation of AG and degradation of AG and UAG Once AG and UAG are released into the circulation they elicit different biological responses which will be discussed later. Spontaneous deacylation of AG into UAG is rapid and accounts for the observed short half-life of AG at 9-13 minutes in humans 25. This rapid deacylation likely explains the high UAG to AG ratio observed in the circulation in both mice and humans.  3  The mechanisms underlying the degradation of AG and UAG remain unclear.  In vitro studies suggest that deacylation is mediated in part by the enzymes cholinesterase in humans and carboxylesterase in rodents, although this remains debated 26. However, in studies from our group, inhibition of butyrylcholinesterase and carboxyesterase did not affect AG deacylation, whereas inhibition of serine proteases caused a marked decrease in AG degradation in adult non-pregnant rats 27. These data suggest that while the mechanisms leading to deacylation of AG into UAG remain poorly understood, degradation into fragments occurs predominantly by plasma proteases 27.     2verall, the regulation of $G?s physiological effects seems to depend on the fine tuning of two mechanisms: 1) acylation by GOAT and 2) deacylation and proteasomal degradation in the circulation. Both of these embody a highly regulated, fine-tuning mechanism that ultimately determines the extent of plasma AG concentrations and thus influences its ascribed effects.   1.5 Role of UAG Whether UAG is also an active hormone remains controversial. Since it lacks the octanoate group on its serine-3, it cannot bind to the growth hormone secretagogue receptor type 1a (GHSR) and therefore cannot elicit the ascribed responses of AG (described below). Some studies have shown that U$G and its analogues promote pancreatic ?-cell survival in streptozotocin-treated rats 28. Others have demonstrated UAG to promote fatty acid uptake in the cardiomyocytes, myotubes and adipocytes 29 but the underlying mechanisms remain unclear. The receptor by which UAG exerts some of the mentioned effects remains unknown.   4  1.6 Role of AG  1.6.1 Growth-hormone release GH release has long been known to be stimulated by the hypothalamic release of growth-hormone-releasing hormone (GHRH) into the hypophyseal portal system acting on the GHRH receptor (GHRH-R) on the anterior pituitary gland17 and inhibited by somatostatin. Ghrelin represents a novel pathway by which the hypothalamo-pituitary axis releases GH 30 when injected in humans or rodents. This second pathway is mediated by the growth hormone secretagogue receptor type 1a (GHSR) also expressed in the pituitary gland 31. The GHSR is a G protein-coupled receptor that is widely expressed in different tissues of the body including the hypothalamus and pituitary gland 32. AG is the only known ligand for the GHSR and upon binding in the pituitary results in an increase in intracellular Ca2+ concentrations which in turn induce GH secretion whereas UAG (lacking the acylation) has no affinity for the GHSR and therefore cannot elicit a response 17,33,34. GH is a crucial hormone to sustain growth, cell division and regeneration in the body. Some of the reported effects in GH-deficient patients after intravenous administration of GH include decreased body fat, increased muscle mass, bone density, sexual function and improved immune system functioning 35. Importantly for my studies, GH has also been demonstrated to play an important role in glucose metabolism (discussed later) 36, suggesting that the acylation of UAG by GOAT may be a key step in the maintenance of euglycemia.  1.6.2 Stimulation of food intake Another well-described role of AG is its ability to increase food intake when injected into rodents (orexigenic hormone) 30,37-39. In addition to this, endogenous AG levels in humans (and rodents) have been shown to rise before and decrease after meals and positively 5  correlate with hunger and negatively correlate with satiation 40. UAG has no effect on initiating appetite thus making GOAT an attractive anti-obesity target as it is the only enzyme responsible for generating AG41. AG stimulates appetite by activating neuropeptide Y (NPY) neurons in the arcuate nucleus located in the hypothalamus 30,42. An increase in NPY activity has been shown in rodents to increase appetite whereas administration of NPY inhibitors reduces appetite 43. Moreover, in rodents, GHSR and NPY mRNA have been found to be co-localized in the arcuate nucleus suggesting that the observed increase in appetite may be through the stimulation of the arcuate NPY neurons mediated by AG binding to the GHSR 44. In humans, the effects of gastric bypass on weight loss are thought to be due in part to a reduction in AG (GOAT and ghrelin are highly expressed in the stomach) which would cause a decrease in appetite 45.  Relevant to my study, pregnancy is a period of increased maternal appetite and food consumption in all species, likely attributed to an evolutionary adaptation to provide the fetus and mother with sufficient nutrition 46. This increase in maternal food intake may in part be mediated through $G?s ore[igenic effects. In essence, the acylation of ghrelin by GOAT may play an important role in energy balance by affecting food intake. However, similar to the pharmacological role of AG in GH release, whether AG plays a physiological role in appetite regulation remains unknown. 1.6.3 Body composition Ghrelin has also been shown to affect body composition in both rodents and humans. As mentioned before, one of the earliest reported effects of AG is its ability to act as a potent orexigenic. Therefore, ghrelin acylation could potentially regulate food consumption and adiposity, in particular during pregnancy. An attractive target present in this route is GOAT 6  since it is the only enzyme known to facilitate ghrelin acylation. This has been investigated by Yang et al (2008) who have designed GOAT inhibitors that prevent the formation of AG 47. They determined that GOAT activity could be inhibited by an octanoylated ghrelin pentapeptide. Its potency was enhanced significantly if the octanoylated serine-3 was replaced with an octanoylated diaminopropionic acid. These data offer insights into the potential of GOAT inhibitor based therapies for appetite control in the treatment of obesity. In addition to this, a negative correlation has been shown between ghrelin levels and body mass index (BMI). Shiiya et al. (2002) determined that leaner individuals generally have greater levels of circulating AG and UAG than obese individuals 48. This observation suggests that the regulation of ghrelin secretion is tightly controlled in response to various BMIs. Upregulation (in lean individuals) or downregulation of ghrelin (in obese individuals) may be a tailored response to these different settings of energy balance. Furthermore, Tschop et al (2000) demonstrated exogenous AG to decrease lipid mobilization thereby inducing adiposity in rodents. Mice subcutaneously injected with AG (2.4?mol/kg) daily had a greater fat mass than those inMected a vehicle further suggesting ghrelin?s influence on body composition 49. Thus, exogeneous AG has clearly been shown to also induce adiposity independently from food intake and thus to alter BMI in rodents. However, the role of endogenous AG remains unclear.  1.6.4 Glucose metabolism In addition to $G?s effects on food intake and GH release, it plays an important physiological role in energy balance. Ghrelin?s effects on regulating blood glucose levels were first reported by Broglio, et al. (2001) who determined that exogenous AG administration induced a rapid increase in glucose levels and a decrease in insulin levels 7  compared to placebo administration 50. 2ther studies have confirmed $G?s role in the suppression of insulin in both humans and rodents. Exogenous AG inhibits the release of insulin secretion from isolated pancreatic islets 51. In addition, ghrelin and GOAT mRNA expression have been reported in the pancreatic islet epsilon-cells suggesting a paracrine role of ghrelin on insulin-producing beta-cells 52.  The underlying mechanisms involve induction of IA-2 beta expression by AG as well as an increase in AMPK phosphorylation and UCP2 mRNA expression which in turn attenuates insulin secretion 53,54. Furthermore, exogenous AG has been shown to promote gluconeogenesis in the liver both directly (via the GHSR) and indirectly (via inducing pituitary GH secretion) 50,55,56. Overall, AG has a net effect of raising blood glucose concentrations.  1.7 Ghrelin during pregnancy Before discussing the effects of pregnancy on ghrelin, it is important to understand the major differences in the physiology of the GOAT-ghrelin-GH axis in humans and in rodents during pregnancy. In humans, maternal pituitary GH is gradually replaced by a different type of GH secreted by the placenta and known as placental GH (PGH) 57,58. PGH differs from pituitary GH by 13 amino acids and is not secreted into the fetal circulation. Similar to GH, PGH stimulates insulin-like growth factor-1 (IGF-1) secretion as well as gluconeogenesis and lipolysis in the mother, which could ultimately promote fetal nutrition 59.  PGH is secreted in a non-pulsatile manner (continuously secreted) and is inhibited in vitro and in vitro by glucose 59. With regards to ghrelin, GHSRs have been found in the human placenta suggesting that AG could be involved in the regulation of PGH 57,58. However, we have previously shown that maternal plasma AG concentrations are significantly lower during 8  pregnancy compared to the postpartum period and are low when PGH concentrations are elevated so that the role of AG on maternal GH secretion remains unclear 60. In rodents, in contrast to humans, the placenta does not secrete PGH but pituitary GH secretion increases. During the later stages of rodent pregnancy, both plasma GH concentrations and GH pulse amplitude increase markedly 61. The source of this plasma GH has been shown to be derived from the pituitary instead of the placenta as observed in human pregnancies 61. Changes in ghrelin during rodent pregnancy remain unclear and may depend on the assays and the experimental conditions. For example, plasma AG concentrations and ghrelin mRNA expression in the hypothalamus are significantly lower at days 10 and 15 of pregnancy (delivery ?20) compared to non-pregnant rodents 62. Ghrelin mRNA expression in the stomach however remained unchanged 62. Overall, studies describing changes in ghrelin in ad-libitum pregnant and non-pregnant rodents are limited. Interestingly, studies that have administered a reduced quantity of food, via calorie-restriction or food deprivation, to these mice have adequately stimulated the GOAT-ghrelin system to yield promising results. In the next paragraph, I will review the results of studies that aim at elucidating the role of impaired nutrition on the GOAT-ghrelin axis.   1.8 Effects of food deprivation and calorie-restriction on the GOAT-ghrelin axis in pregnant and non-pregnant humans and rodents Food deprivation and calorie-restriction (CR) are periods of negative energy balance where the body needs to upregulate various pathways in order to maintain euglycemia. Several studies performed in non-pregnant rodents have investigated the role of ghrelin and GOAT on glucose metabolism during these periods of negative energy balance. Short-term food deprivation studies of mice subjected to fasts from 12-36 hours have shown to have 9  increased levels of endogenous plasma AG and UAG however decreased levels of gastric GOAT and ghrelin mRNA expression compared to their freely-fed counterparts 63. In contrast, in long-term food deprivation studies of mice subjected to a 35% calorie-restricted (CR) diet (65% of their normal caloric intake), gastric GOAT and ghrelin mRNA expression is increased compared to their freely-fed counterparts 64. Other studies in mice have shown that the increased levels of endogenous AG resulting from long-term CR correct blood glucose levels 34. In support of this hypothesis, the severe hypoglycemia experienced by GOAT-KO mice (that cannot acylate ghrelin) submitted to a 60% CR diet for seven days was mitigated by administration of AG. This effect of AG was thought to be mediated by GH. Overall, these studies suggest that AG is an essential component of glucose regulation in rodents especially during periods of negative energy balance. To this date, the literature pertaining to exogenous or endogenous role of AG in the regulation of blood glucose homeostasis during a CR pregnancy remains unknown.  Other studies have found total plasma ghrelin concentrations to remain constant throughout pregnancy in rats fed ad libitum with elevated levels reported at days 19 and 21 only in dams subjected to a 70% calorie-restricted diet65.  This rise in plasma ghrelin was correlated with elevated gastric ghrelin mRNA levels at these time points. Whether both AG and UAG are affected in these studies is unclear as the assays measured only total ghrelin concentrations (AG + UAG together). Short term fasting studies in rats (36 hours) have shown a significant increase in maternal plasma AG concentrations at day 21 of pregnancy compared to non-fasted controls 66. This was associated with lower blood glucose and elevated gastric ghrelin mRNA expression in these fasted mice.  The role of ghrelin in GH 10  regulation and in the energy balance during rodent pregnancy remains unclear in the literature but will be clarified by the use of GOAT-KO models in my project. 1.9 Project Rationale Exogenous (= injected) and endogenous (= secreted by the body) AG, through binding to the GHSR receptor, has the potential to play a major physiological role in energy balance. 2ne of $G?s roles that has best been characterized is its effects on glucose metabolism, notably: 1) exogeneous administration of AG causes hyperglycemia 50 and endogenous AG protects against hypoglycemia caused by CR in non-pregnant, GOAT-KO mice 34. There are to our knowledge no data on the role of GOAT and AG on glucose metabolism during pregnancy. Maintenance of euglycemia is especially important during pregnancy, where transplacental transfer of glucose is required for optimal fetal outcome. Maternal malnutrition is associated with decreased substrate availability and is a risk for hypoglycemia. This issue is also relevant to human physiology as described below.  Thus, I wish to clarify the role of the GOAT-ghrelin axis on glucose metabolism during pregnancy in the presence of CR. To understand the role of AG, I will use both WT and GOAT KO mice (in which acylation of ghrelin does not take place).    1.10 Relevance to human health Chronic malnutrition can potentially cause hypoglycemia 67. Poor maternal nutrition causes intrauterine growth restriction (IUGR) and low birth weight (LBW) which contributes to more than 8% of deliveries in Canada 68,69. LBW resulting from inadequate maternal nutrition is much higher in developing countries such as India where LBW accounts for 20% of deliveries 70. LBW can lead to fetal mortality and morbidity including suboptimal growth. 11  It is also a risk factor for the development of chronic diseases later in life 68. Furthermore, if maternal undernutrition persists, especially during the third trimester, it has been reported to increase the risk of preterm delivery 71.  In the mother, pregnancy is known to be a period of increased energy demand to support fetal development through the placenta 72. If nutritional needs are not met, it can put the mother at risk for developing hypoglycemia as adaptive responses prioritize providing nutritional support for the developing fetus over the mother 73. Collectively, these factors suggest an accentuation in the risk for developing hypoglycemia during pregnancy compared to non-pregnant individuals in food scarce environments. These mothers must provide adequate energy for their developing fetus whilst attempting to sustain euglycemia.  Maternal undernutrition is still an epidemic affecting many populations in the world such as sub-Saharan Africa and Southeastern Asia 74. In these countries, the prevalence of undernutrition is more prominent in adult women than in children or men 74. Furthermore, the rate of pregnancies is significantly higher in these developing countries thereby putting child-bearing women at risk for hypoglycemia 67,75. In essence, maternal hypoglycemia, as observed in these environments, can have devastating consequences on both the mother and their offspring. Therefore, understanding the biology behind maternal hypoglycemia and more importantly the pathways that prevent its occurrence could offer insights into potential therapeutic targets in the GOAT-ghrelin pathway that may possess clinical relevance.      12  1.11 Hypotheses and objectives 1.11.1 Hypotheses 1) CR will cause lower blood glucose in pregnant compared to non-pregnant mice  2) Upregulation of the GOAT-ghrelin axis is part of the hormonal response to the hypoglycemia induced by a CR diet in pregnant and non-pregnant mice.   1.11.2 Overall objective To compare the effects of CR on glucose homeostasis and the GOAT/ghrelin axis in WT and GOAT-KO non-pregnant and pregnant mice. 1.11.3 Specific objectives 1) To compare the changes in body composition and blood glucose concentrations associated with CR in pregnant and non-pregnant WT and GOAT-KO mice.  2) To assess the changes associated with CR in pregnant and non-pregnant WT and GOAT-KO mice for a. AG and UAG concentrations in the plasma and  b. GOAT and ghrelin mRNA expression in various ghrelin secreting tissues (stomach, pituitary and hypothalamus). 3) To examine several aspects of the counter regulatory cascade of hypoglycemia in pregnant and non-pregnant WT and GOAT-KO mice submitted to CR: plasma GH concentrations, hepatic glycogen content and gluconeogenesis.    13  2 Methods 2.1 Experimental animals 2.1.1 Genetically Modified Mice ? Wild-type and GOAT Knock-Out  All animal experiments were performed with approval from the University of British Columbia animal care and animal ethics committee (Animal Protocol No. A10-0030, A10-0041). In order to assess the effect of CR on the GOAT-ghrelin system, we used C57BL/6 wild-type (WT) and GOAT Knock-out (GOAT-KO or KO) mice. These mice were received as an in kind donation from Eli Lilly and Company (Indianapolis, IN, USA). GOAT-KO mice are genetically modified mice missing the gene that encodes for GOAT (MBOAT4) thereby possessing an inability to attach the octanoate moiety to the ghrelin molecule thus producing a mouse lacking circulating AG but not UAG. These mice normally express ghrelin mRNA and protein in their tissues.  2.1.2 Diet and priming  Female mice were fed a fat enriched diet (9% fat) chow to assist in attaining pregnancy and switched to the Gibson 5P76 (7% fat) chow after conception for the duration of the experiment. Male cage bottoms were switched with the female cage bottoms to present scent and murine pheromones capable of enhancing mouse attractiveness to one another 2 days prior to introducing the male 76. In addition to this, a small pellet of /ove Mash? (. fat) (product#: S3823P, BioServ, Frenchtown, NJ, USA) was added to the mating cages to support the reproductive performance of our mice. Male mice that were effective in impregnating the female mice were kept as studs for the succeeding cohort.    14  2.2 Experimental procedure Cohorts of 11-12 week old female mice were used for this project. One male mouse was introduced into a cage consisting of two female mice that have been primed as per the above protocol. The male mouse was removed the following day and we designated this experimental time point as Day 0. The presence of a vaginal plug indicated that copulation had occurred but was not a definite guarantee of conception. Pregnancy was confirmed at Day 10.5 by daily weight monitoring as a greater rate of weight gain was apparent in pregnant mice. At day 11 all mice, pregnant and non-pregnant, were subjected to either a 50% CR diet or a freely-fed (FF) diet where they were sacrificed at Day 18 (pregnant mice deliver usually around Day 20). The 50% CR was taken as half of the food quantity a mouse fed ad libitum would normally consume per day. This ad libitum quantity of food was measured by averaging the amount of food consumed by each mouse daily for a week prior to Day 10.5 (=50% CR of FF).  CR mice were fed their ration at 1:30PM, after body composition and blood glucose measurements were taken. This experimental procedure was applied to both WT and GOAT-KO mice (Fig. 2.1).    Figure 2.1: Experimental time line. BG was checked prior to EchoMRI for BC. CR mice were given their dietary ration at 1:30PM which was immediately after BG and BC were taken. 15  2.3 Glucose monitoring Blood glucose levels were measured using a glucometer OneTouch? UltraMini? Blood Glucose Meter with OneTouch? Ultra? Test Strips. A 26 gauge needle was used to lance the mouse on the saphenous vein to draw a small volume of blood (~1-2?L) that was collected by the test strip and read on the glucometer (mmol/L). The test strips are coated with glucose oxidase and other enzymes that oxidize the blood glucose to gluconolactone. During this process, the electrons from the glucose are transferred to an oxidized mediator molecule resulting in its reduction. The mediator then delivers the electrons to an electrode in the glucometer for electrochemical measurement that is translated into a blood glucose concentration in mmol/L 77.  Blood glucose was measured just prior to introducing the male to the female littermates for copulation (Day -0.5), prior to the start of the 50% CR diet or continuation of the FF diet regime (Day 10.5) and prior to sacrifice (Day 17.5). Blood glucose and clinical features were stringently monitored near the closing days of the CR period to ensure an end-point for these mice was attained without enduring the complications associated with severe hypoglycemia. Mice that appeared weak and had blood glucose concentrations under 4.0 mmol/L were immediately euthanized.  2.4 Body composition analysis Body composition was determined by the EchoMRI-00? machine lent for my use courtesy Dr. William Gibson?s laboratory. This machine incorporates a quantitative magnetic resonance (QMR) method which gave a measurement of fat and lean mass in the whole mouse. QMR was selected because of the advantages it offers over other instruments also used to determine body composition (EchoMRI?, http://www.echomri.com/qanda.aspx). It does not require the use of anesthesia or restraints thereby allowing the mice to be scanned 16  alive and awake without unwanted effects of sedation and stress which is duly beneficial for the animal and our data. This allowed for the same mouse to be scanned at our experimental time points so that changes in body composition could be observed within each experimental group. Furthermore, the EchoMRI QMR method is time saving as scanning each mouse is completed in less than a minute which allows for cohorts of mice to be scanned at suitable time points. Finally, QMR was selected as it has been shown to have greater precision in lean and fat mass determination in contrast to DEXA with a coefficient of variation of 0.34-0.71% compared to 3.06-12.60% 78.  The EchoMRI? system relies on taking advantage of the differences in rela[ation times of hydrogen proton spins in various tissues (EchoMRI?, http://www.echomri.com/ qanda.aspx). Radio pulses from the instrument cause proton spins to precess and are received by the EchoMRI? system which then analyzes the amplitude, duration and spatial distribution of these signals. These signals are unique to an individual tissue type and can determine quantitatively the amount of fat and lean tissue present in the animal as a function of mass. Fat tissue is measured as the mass of all the fatty acid molecules in the mouse expressed as an equivalent weight of canola oil. Lean tissue is measured as all types muscle tissue (skeletal, cardiac and smooth muscle) in the mouse. In pregnant mice, fetuses have a higher lean-to-fat mass ratio and may cause a higher lean tissue mass reading on the EchoMRI? system 79. Substances that do not contribute to an NMR signal include minerals, hair, claws and whiskers composition (EchoMRI?, http://www.echomri.com/qanda.aspx).  Our mice were scanned just prior to introducing the male breeder (Day -0.5), at the start of the CR period (Day 10.5) and a half-day before sacrifice CR period (Day 17.5). A mouse was scanned after blood glucose was measured to avoid inducing stress which may affect the 17  observed glucose readings. A mouse was placed in a clear plastic tube supplied by the manufacturers of the EchoMRI? system. This tube containing the non-anesthetized mouse was then placed in a compartment in the system where the scanning would commence at our discretion. Each mouse was scanned in duplicate and then placed back in its original cage with its littermate which would be the sequential mouse to be scanned.    2.5 Tissue harvest On Day 18, each mouse was placed in a chamber containing an oxygen supply infused with isoflurane. Isoflurane is a highly stable and potent inhalational anesthetic that was used for our experiment. Compared to other volatile anesthetic agents, such as halothane and methoxyflurane, isoflurane provides the most rapid induction time for the animal due to its low partition coefficient 80,81. Furthermore, it has been known to cause minimal irritation to the respiratory tract and has had no reports of bodily damage to the mouse 82.  After the mouse has reached an anesthetic depth, it was removed and decapitated. Blood was collected into chilled EDTA tubes.  The top part of the mouse?s cranium was laterally cut and the brain was peeled out from the base of the cavity. Upon removal of the brain, the hypothalamus was removed from its base using surgical tweezers. In the sphenoid of the cranial cavity the pituitary was removed from the hypophyseal fossa of the sella turcica. The pituitary gland, hypothalamus and remainder of the brain were placed in aluminum foil, snap frozen in liquid nitrogen and stored in the -0?& free]er. The chest cavity was opened first by spraying the outside of the mouse with 0 ethanol and cutting the mouse?s fur with larger blade surgical scissors. After the fur was removed, finer surgical scissor blades were used to cut the skin of the mouse?s chest and abdominal areas vertically.  18  The pancreas was removed first quickly, placed in aluminum foil and immediately snap frozen. The pancreas unlike other organs is a hostile environment containing exocrine fluids and variety of enzymes that assist in digestion 83. It was therefore selected to be the first organ to be removed in order to avoid activation of these digestive enzymes that could degrade RNA and proteins of interest that are present in its tissue. Despite our attempt to hastily harvest the pancreas, the isolated RNA was found to be degraded and was not used for our qPCR.  The next tissue removed was the stomach, known to be the major ghrelin secreting gland, which was excised by cutting just above the cardiac and pyloric sphincters proximal to the esophagus and duodenum respectively. The stomach was placed in a Petri dish with cold 10X DEPC treated PBS saline solution. The top quarter portion of the stomach was excised and removed then cut along the lesser curvature to open the stomach?s contents. Remnants of food were carefully removed by flushing the lumen of the stomach using a syringe filled with cold DEPC treated PBS saline solution. The stomach tissue was further cleaned 3 times using fresh batches of the same solution. This section of the stomach was chosen for analysis because GOAT (and ghrelin) expression is more concentrated around the corpus area with marginal expression reported in the forestomach which was discarded 22. The liver was the final organ removed by excising a small portion of it. Both the liver and stomach were snap frozen and stored in the -0?& free]er.  2.6 Ghrelin and GOAT determination 2.6.1 Plasma AG and UAG In order to determine the concentration of AG and UAG in the circulation of the mice we used a double-antibody enzyme immunometric assay (EIA) (#10006307 for AG; #10008953 19  for UAG, Cayman Chemical Company?, Ann Harbor, MI, USA). The principle of this assay, supplied as a kit, is as follows (Rat AG & UAG EIA Kit Booklet, Cayman Chemical Company?): The 96 well plate is pre-coated with a monoclonal antibody specific to the C-terminal part of the ghrelin peptide (Fig. 2.2). This antibody would bind to all ghrelin introduced in the wells (AG, UAG and standards). A secondary monoclonal antibody which is specific to the N-terminal part of AG (or UAG) was then added. This secondary antibody is coupled to an AChE-)ab? conMugate that upon addition of an Ellman?s reagent produced a yellow forming product. This plate is then read by a spectrophotometer at 414nm. The intensity of this colour is directly proportional to the amount of AG or UAG present in the wells. By comparing optical density readings of our samples to a standard curve, the concentration of AG and UAG present in the plasma can be determined.  The major originality of this assay is that it only measures full-length AG or UAG, contrasting with single antibody assays that also detect ghrelin fragments.  Upon collection of the blood, 100mM  p-hydroxymercuribenzoic acid (PHMB) was added to prevent proteolytic degradation. I have previously demonstrated that other protease inhibitors such as phenylmethanesulfonylfluoride (PMSF) and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) interfered with this assay by inhibiting the AChE-)ab? tracer, thereby hindering production of the yellow colour 84. In contrast, PHMB did not interfere with the assay. Samples treated with AEBSF and PMSF would have to be subjected to additional washing prior to adding the secondary antibody in order to remove the protease inhibitor.  PHMB was prepared in a 100 times concentrate solution in potassium phosphate buffer containing 1.2% NaOH 10N (v/v). The blood samples were then 20  centrifuged at 3,500rpm for 5 min at 4?C and the plasma supernatant was treated with 1N hydrochloric acid (HCl) and stored in the -20?C freezer as per protocol.  Upon commencing the assay, plasma samples for AG were diluted 1:5 and UAG in 1:10 in EIA buffer. Samples were chosen from the experimental groups with little to no presence of hemolysis in order to deter further interference as I have previously reported erythrocyte lysate to contain certain peroxides that are known inhibit the AChE-)ab? tracer thus resulting in lower than expected plasma ghrelin concentrations84. Standard curves were produced for AG and UAG with concentrations of 250 pg/mL (S1), 125 pg/mL (S2), 62.5 pg/mL (S3), 31.3 pg/mL (S4), 15.6 pg/mL (S5), 7.81 pg/mL (S6), 3.91 pg/mL (S7) and 1.96pg/mL (S8). All reagents and buffers supplied by the kit were reconstituted as per protocol. A blank was used containing only EIA buffer as well as non-specific binding which contained the EIA buffer as well as the AChE-)ab? tracer. The 9 well plate was then incubated at ?& for 20 hours. This time was chosen in order to optimize assay sensitivity (0.2 pg/mL versus 0.7 pg/mL for shorter reactions). After incubation, the 96 well plate was washed with the supplied wash buffer and the Ellman?s reagent (5,5'-dithiobis-(2-nitrobenzoic acid) which was also supplied by the kit was added. The plate was then incubated in darkness at room temperature for 30 min to develop. 21    2.6.2 Tissue specific ghrelin and GOAT mRNA expression In order to assess gene expression at the transcriptional level (mRNA expression), real-time PCR also known as quantitative PCR (qPCR) was used. qPCR is based on the conventional polymerase chain reaction (PCR) which is a molecular biology technique that is used to simultaneously quantify and amplify a specific DNA molecule. qPCR differs from conventional PCR as it allows for the amplified DNA strand to be observed as the reaction progresses or in ?real time? whereas conventional 3&R allows for detection only at the end 85. As we know the massive size of DNA molecules and the plethora of genes they contain would make it difficult to assess specific genes of interest. In order to determine relative mRNA expressions of a specific gene in a sample, GOAT and ghrelin for instance, the use of certain hydrolysis probes such as TaqMan? are used (Real-Time PCR handbook, Life Technologies?). TaTMan? probes consist of a flurophore reporter on the ? end of the oligonucleotide probe and a Tuencher on the ? end. When this probe is intact, the pro[imity of the reporter and the quencher is too close to elicit a detectable fluorescence signal. During Figure 2.2: Principle of the plasma AG and UAG EIA (modified from Rat AG & UAG EIA Kit Booklet, Cayman Chemical Company?) AG or UAG 22  the annealing/extension step of the amplification process, the TaqMan? probe hybridizes to the target gene. During the synthesis of the complementary strand, the ?-? e[onuclease activity of Taq DNA Polymerase cleaves the reporter thereby separating it from its proximity towards the quencher. With each cycle, the reporter dye molecules are cleaved from their probes resulting in an increase in fluorescence intensity. The intensity of this fluorescence is proportional to the amount of amplicon produced. As the number of cycles increases, the amount fluorescence accumulates and reaches a certain threshold that exceeds the background level. This point is referred to as the cycle threshold (Ct) and is used in the ?comparative &t method? to determine the relative mR1$ e[pression of gene in a sample 86. A smaller Ct value is associated with a greater amount of mRNA expression and a greater Ct value is associated with a lesser amount of mRNA expression of the gene of interest. Once Ct values are determined, they are normalized to a housekeeping gene. In our data, we used ?-actin, a cytoskeletal actin, as it is widely used in qPCR assays and had consistent Ct values in all of our groups thus demonstrating it not to be influenced by our experimental conditions 87,88. The normalized values of these samples (?&t &t of target gene ? &t of ?-actin) are then compared to a calibrator to determine fold-expression of the target gene. For our experimental samples, total RNA was isolated from the stomach, hypothalamus, pituitary (for GOAT and ghrelin mRNA analysis) and liver (for PCK1 mRNA analysis, discussed later) using a mixed isolation method that allows for total protein and total RNA isolation. Total RNA was prepared on ice and the use of RNAse free laboratory equipment to reduce the potential of RNAse activity that could lead to RNA degradation. RNA from these tissues was then subjected to a reverse transcriptase reaction using a High Capacity cDNA Reverse Transcription .it (, $pplied Biosystems?, )oster &ity, &$) that 23  transcribes the single-stranded RNA into a more stable double-stranded cDNA. The cDNA was then used as a template for qPCR amplification. The data was generated as a fold expression for each group relative to a calibrator consisting of pooled RNA from the WT-FF-NP group.  2.7 Plasma growth hormone In order to determine the concentration of GH in the circulation in the mice we used a sandwich ELISA (#EZRMGH-45K, EMD Millipore Corporation?, Billerica, MA, USA). The principle of this assay, supplied as a kit, is as follows (Protocol: Rat/Mouse Growth Hormone ELISA Kit, EMD Millipore Corporation?): The 96-well plate is pre-coated with anti-GH polyclonal antibodies. After addition of the samples and standards, the plate is washed to remove any unbound materials. A secondary biotinylated anti-GH polyclonal detection antibody is then added that binds to the captured GH molecules and the plate is again washed to remove unbound materials. An enzyme solution containing horseradish peroxidase is then added to the immobilized biotinylated antibodies and subjected to another washing step to remove free enzyme conjugates. The substrate for the horseradish pero[idase, ,?,,?-tetramethylbenzidine, is then added that generates a blue colour with an intensity proportional to the amount of GH in the wells. A stop solution is then added and the plate is read at 450nm. By comparing the optical density of our readings to a standard curve, the concentration of GH in the plasma can be determined in each mouse. An aliquot of plasma obtained by the plasma AG and UAG EIA protocol was used for plasma GH determination (PHMB and HCl treated). Samples were similarly chosen from experimental groups that had little to no presence of hemolysis to avoid interference with the assay and stored in the -20?C freezer prior to use. Upon commencing the assay, plasma 24  samples for GH were diluted 1:10 in the assay buffer supplied by the kit. A standard curve was produced with concentrations of 50 ng/mL (S1), 16.7 ng/mL (S2), 5.6 ng/mL (S3), 1.9 ng/mL (S4), 0.62 ng/mL (S5), 0.21 ng/mL (S6) and 0.07 ng/mL (S7). All the buffers and reagents supplied by the kit were prepared as per protocol. After addition of the substrate, the plate was incubated for 15 minutes (as per protocol) and then the stop solution was added to all of the wells.  2.8 Hepatic glycogen content and gluconeogenesis  There are two main mechanisms mammals use to prevent hypoglycemia. One mechanism, referred to as glycogenolysis, causes the breakdown of glycogen to release glucose units into the bloodstream during periods of negative energy balance 89. The second mechanism the body uses to prevent hypoglycemia is a process called gluconeogenesis which produces glucose from non-carbohydrate substrates such as pyruvate, lactate, glycerol and certain amino acids 90. Both of these mechanisms primarily occur in the liver and to a lesser extent the muscles and the kidneys 91,92. This is regulated by the hormone glucagon that is released from the alpha cells of the pancreas in response to low blood glucose levels 89. Insulin does the opposite; it is released in response to high blood glucose levels (from the pancreatic beta cells) to facilitate glucose uptake into the tissues (Fig. 2.3) 93. Exogenous AG administration has been shown to stimulate glucagon release in mice 94. Once glucagon is released into the circulation, it acts on the liver to stimulate glycogenolysis and gluconeogenesis, and antagonistically to inhibit glycolysis 95. These mechanisms have a net effect of raising blood glucose levels to prevent hypoglycemia. Another way to prevent hypoglycemia would be to inhibit insulin secretion as well as raising GH levels, both which have been shown to be stimulated by exogenous AG (Fig. 2.4) 96,97. 25              2.8.1 Hepatic glycogen content determination To determine hepatic glycogen content, we used a Glucose (HK) Assay Kit (#GAHK-20, Sigma-Aldrich?, St. Louis, MO) following the acid-hydrolysis method 98. The Glucose (HK) Assay reagent is used for the quantitative determination of glucose present in a given sample. The assay reagent after reconstitution contains 1.5 mM NAD, 1.0 mM ATP, 1.0 unit/mL of Figure 2.4: Conceptual role of AG in response to starvation-induced hypoglycemia 96,97 Figure 2.3: Effects of insulin and glucagon signaling on hepatic glucose metabolism 93 26  hexokinase and 1.0 unit/mL of glucose-6-phosphate dehydrogenase with sodium benzoate and potassium sorbate as preservatives. The principle of this assay works by measuring the concentration of glucose units per gram of wet liver tissue. The glucose units that result from the hydrolysis of glycogen are phosphorylated by hexokinase. The glucose-6-phosphate produced is oxidized to 6-phosphogluconate in the presence of NAD which is catalyzed by the enzyme glucose-6-phosphate dehydrogenase. During this oxidation reaction, a stoichiometric equimolar amount of NAD is reduced to NADH. The amount of NADH produced is readable at 340 nm using a spectrophotometer. Liver tissues from our experimental groups were removed from the -80?C freezer and each individual liver was minced into two portions (~10-20 mg per portion). The first portion was transferred into an Eppendorf tube containing homogenizing beads and hot 2.0 M HCl. The second portion of the same liver sample was transferred into an Eppendorf tube also containing homogenizing beads but with hot 2.0 M NaOH.  HCl hydrolyzed all the liver glycogen into glucose units and the NaOH treated portions determined the amount of free glucose present in the glycogen. Free glucose was a measurement of the amount of glucose that was not incorporated into the glycogen but rather residual glucose. By subtracting the amount of free glucose present from the amount of hydrolyzed glucose units, the amount of glycogen present in the liver can be determined upon comparison to a glucose standard curve. The weights of the liver tissue were measured using an analytical laboratory scale to determine the concentration of glucose units per gram of liver. The intensity of the colour is proportional to the amount of NADH which is in turn proportional to the amount of glucose present in the wells.   27  2.8.2 Hepatic gluconeogenesis determination  To determine whether the gluconeogenesis pathway was upregulated under our various experimental conditions, we assessed the mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of this pathway. PEPCK is the rate limiting enzyme involved in the first step of gluconeogenesis 99. It is encoded by the gene PCK1 gene in rodents and is upregulated in response to glucagon which is released during periods of negative energy balance 100. In order to assess the transcriptional regulation of PEPCK, we used qPCR with TaqMan probes specific for the PCK1 gene. The principle of this assay was the same as described earlier for G2$T and ghrelin genes. We then compared these ?&t values to a calibrator consisting of pooled RNA from the WT-FF-NP group to generate a relative fold-expression of PEPCK mRNA for each experimental group.  2.9 Statistics Body composition data for fat and lean mass, in grams, measured by EchoMRI were given in duplicate and averaged for each mouse. These data was then entered into Microsoft Excel 2010 and transformed into a percent change relative to Day -0.5 for each group. The blood glucose data measured by the glucometer was also entered into Microsoft Excel 2010. These data were then transformed into a mmol/L change relative to Day -0.5 for each group. All other data produced from samples at Day 18 (plasma AG, UAG, GH, GOAT/ghrelin/PCK1 expression and hepatic glycogen content) were also entered into Microsoft Excel 2010. All data sets (Days -0.5, 10.5, 17.5 and Day 18) were then transferred to SPSS (PASW Statistics 18) for statistical analyses.  An ANOVA with repeated measures test was performed to determine interactions within each group at the experimental time points. An important assumption for the ANOVA with 28  repeated measures test required that the variances of the differences between all combinations of related groups must be equal also known as sphericity. Mauchly?s test of sphericity was applied to the data and, if violated, a Greenhouse-Geisser correction was used in order to alter the degrees of freedom and produce an F-ratio suitable to reduce Type 1 error rate. The ANOVA with repeated measures was applied only to our body composition and blood glucose analysis as data was progressively collected within each group at multiple time points.  A one-way ANOVA was then performed to determine the overall significance between the groups at each of these time points. An important assumption of the one-way ANOVA required that the data be normally distributed and have homogeneity of variances. Therefore, a Shapiro-Wilk test and a /evene?s Test of Homogeneity were applied to all of our data. If our data was not normally distributed and/or had unequal variances, a Kruskal-Wallis test (non-parametric equivalent of a one-way ANOVA) was used to determine the significance between the experimental groups followed by a series of Mann-Whitney U test for the post-hoc analysis between each individual group. If the data did not violate these assumptions, a Bonferroni correction was performed following the one-way ANOVA to determine interactions between each individual group at Day 17.5. The Bonferroni correction was applied by multiplying the significance between each group by a factor of 4 which represented the number of comparative groups of interests within each strain. In order to compare the changes between WT to the GOAT-KO strains, an independent (2-tailed) t-test was performed between them if the data was normally distributed and had homogeneity of variances. If not, a Mann-Whitney U test was used to determine significance between these groups. A p value less than 0.05 was considered statistically significant for all tests. The one-29  way ANOVA (parametric) or the Kruskal-Wallis (non-parametric) tests were applied to body composition and blood glucose data (Day -0.5, Day 10.5, Day 17.5) and plasma AG, UAG, GH, GOAT/ghrelin/PCK1 mRNA and hepatic glycogen content (Day 18).                      30  3 Results 3.1 Preliminary work In order to determine a suitable level of CR to apply to all of my mice, I did a pilot test to assess the effects of various degrees of CR. I first administered a 70% CR diet (30% of normal daily intake). I found this to be too severe for both pregnant and non-pregnant mice as there were signs of severe hypoglycemia just after 2 days and they had to be euthanized. I then administered a 60% CR diet (40% of normal daily intake), similar to what Zhao et al (2010) administered to non-pregnant mice. I found this degree of CR to be acceptable for my non-pregnant mice as they were able to survive on the CR for the 7 day period. Pregnant mice however showed signs of morbidity after only 2-3 days on the 60% CR diet and this degree of restriction was thus deemed too severe to apply. Finally, I tested a 50% CR diet (50% of normal daily intake) that allowed for WT pregnant mice to be viable throughout course of their pregnancy. This degree of calorie restriction was used throughout this project.  3.2 Food intake Ad libitum WT and GOAT-KO mice consumed on average equal amounts of food regardless of strain (~3-5 g/mouse/day). CR mice immediately consumed their ration of food upon addition to the cage at 1:30PM.  3.3 GOAT-KO mice: Fertility, fetus quantities and response to the 50% CR diet WT and GOAT-KO mice had similar numbers of fetuses present (7.8 fetuses in WT and 7.3 fetuses in GOAT-KO) however GOAT-KO mice had difficulties achieving pregnancy compared to WT. Although a presence of a vaginal plug was noted indicating that these mice 31  copulated, conception did not occur as often as in WT mice. In contrast to the information received from the laboratory that donated the mice to us, the fertility rate appeared to be lower in GOAT-KO mice compared to WT mice. Even when a vaginal plug was noted, progression through pregnancy was not as common. While KO-FF-P mice were able to sustain pregnancy once started, KO-CR-P mice could not.  These mice displayed signs of severe hypoglycemia as determined by daily glucose monitoring and physical examination only after 2-3 days of CR and had to be euthanized prior to our experimental time point at Day 17.5. Others during the CR period either absorbed their fetuses or delivered early and ate their newborns. We therefore could not acquire any useful data at our designated time points for the KO-CR-P group of mice. One KO-CR-P mouse made it to Day 17.5 for a final blood glucose check however had to be euthanized immediately afterwards and thus did not proceed to specimen collection at Day 18. Therefore, in this section, figures summarizing the results for GOAT-KO mice do not have a column for KO-CR-P animals. 3.4 Body Composition The results for fat and lean mass percent changes between Day -0.5 (represented as 0%), Day 10.5 (prior to randomization to FF or CR) and Day 17.5 (Day 18=sacrifice) are shown in Figs 3.1-3.6. Mauchly?s Test of Sphericity indicated that the assumption of sphericity had been violated for WT fat (x2(2)=8.371, p=0.015), WT lean (x2(2)=7.780, p=0.020), GOAT-KO fat (x2(2)=7.518, p=0.023) and GOAT-KO lean (x2(2)=10.671, p=0.005), therefore a Greenhouse-Geisser correction was used for our repeated measures test. There were no outliers and the data was normally distributed as assessed by Normal Q-Q plot and a Shapiro-Wilk test for all groups (p>0.05). Homogeneity of variances was not violated as assessed by 32  the /evene?s Test of Homogeneity of 9ariances (p>0.05) for all experimental groups in our body composition analysis. 3.4.1 Fat mass percent changes in WT mice  A repeated measures ANOVA with a Greenhouse-Geisser correction determined that there was a significant difference between the days (F(1.5, 35.0)=7.5, P=0.004). There was also a days *group interaction suggesting that the changes in fat mass between the days varied as a function of the groups (F(4.6, 34.9), P=0.0). Pair-wise comparisons determined the differences within each group at the different time points. There was a 30-60% increase in percent fat mass within each of the pregnant groups from Day -0.5 to Day 10.5. However no change in fat mass was apparent within the non-pregnant groups (p=0.003). From Day 10.5 to 17.5, CR resulted in a decrease in fat mass in both pregnant (58%) and non-pregnant (41%) groups, contrasting with an increase in the FF-P and FF-NP mice (p=0.02).  There was a statistically significant difference between the groups as determined by one-way ANOVA at Day 10.5 (F(3, 32)=9.0, p=0.0001) and Day 17.5 (F(3,31)=36.2, p=0.0) (Fig. 3.1). A post-hoc analysis using the Bonferroni correction revealed that on Day 17.5, FF-P mice had accumulated more fat mass than FF-NP mice (83% vs. 16%; p=0.0005). CR resulted in a loss in fat mass in non-pregnant (-74%; p=0.0006) and pregnant mice (-124%; p=0.0) compared to their FF counterparts.  Overall, these data show that fat mass accumulates in WT-FF mice and that the rate of accumulation is greater during pregnancy. Furthermore, when WT mice are subjected to CR, fat mass decreased in both pregnant and non-pregnant mice.   33       3.4.2 Fat mass percent changes in GOAT-KO mice A repeated measures ANOVA with a Greenhouse-Geisser correction determined that there were no statistically significant changes in fat mass in GOAT-KO mice over time during the experiment. There was a statistically significant difference between the groups only at Day 17.5 as determined by one-way ANOVA (F(2,18)=5.431, p=0.010) (Fig 3.2). A post-hoc analysis using a Bonferroni correction did not identify statistically significant differences between the groups. These data show that in GOAT-KO mice, pregnancy and CR have a modest effect on fat mass.  Figure 3.1: Mean percent fat mass changes relative to Day -0.5 in WT mice. Absolute values at baseline (Day -0.5) of each group are shown in the top-left corner of the figure. SD bars; n=7-20/gr. ANOVA post-hoc for multiple comparisons. *:p<0.001.  34     3.4.3 Comparison of fat mass percent changes between Day -0.5 and Day 17.5 in WT vs. GOAT-KO mice An independent (2-tailed) t-test suggest that WT-FF-P mice accumulate more fat mass than the KO-FF-P mice at least until Day 10.5 of pregnancy, under FF conditions (83% vs. 23%; p<0.05) (Fig.3.3).  The mean fat mass decrease at Day 17.5 was approximately twice as great in the WT-CR-NP as in the KO-CR-NP mice (-41% vs. -23%; p=0.150) but this difference was not statistically significant. There was no significant difference between WT-FF-NP and KO-FF-NP mice. Figure 3.2: Mean percent fat mass changes relative to Day -0.5 in GOAT-KO mice. Absolute values at baseline (Day -0.5) of each group are shown in the top-left corner of the figure. SD bars; n=8-13 for KO; n=2-6 for KO-FF-P. ANOVA post-hoc for multiple comparisons revealed no significance.  35     3.4.4 Lean mass percent changes in WT mice A repeated measures ANOVA with a Greenhouse-Geisser correction determined that there was a significant difference between the days (F(1.5, 46)=148.3, P=0.0). There was also a days*group interaction suggesting that the changes in lean mass between the days varied as a function of the groups (F(4.6, 46)=72.5, P=0.0). Pair-wise comparisons determined the differences within each group at the different time points. There was a 17-29% increase in percent lean mass within each of the pregnant groups from Day -0.5 to Day 10.5. However no change in lean mass was apparent within the non-pregnant groups (p=0.0). From Day 10.5 to 17.5, CR resulted in an 8% decrease in lean mass in non-pregnant mice however CR in pregnant mice results in a 28% increase in lean mass demonstrating lean tissue growth is sustained in the latter group despite CR. In addition, there is an 85% gain Figure 3.3: Comparison of mean percent fat mass changes at Day 17.5 between WT and GOAT-KO mice. SD error bars; n=7-20 for WT; n=8-13 for KO; n=2-6 for KO-FF-P. Independent (2-tailed) t-test. *:p<0.05 36  compared to a 5% gain in lean mass in the FF-P and FF-NP groups respectively (p=0.0) further showing substantial growth of lean mass during pregnancy.   There was a statistically significant difference between the groups as determined by one-way ANOVA at Day 10.5 (F(3,32)=33.7, p=0.0) and Day 17.5 (F(3, 31)=177.3), p=0.0) (Fig.3.4). A post-hoc analysis using the Bonferroni correction revealed that on Day 17.5, FF pregnant mice had accumulated more lean mass than FF non-pregnant mice  (84% vs. 5%; p=0.0). CR resulted in a loss in lean mass in non-pregnant (-13%; p=0.012) and pregnant mice (-56%; p=0.0) compared to their FF counterparts. Although CR resulted in lower lean mass in pregnant mice compared to FF pregnant mice, significant increases in percent lean mass are observed compared to their non-pregnant counterparts (+36%; p=0.0).  Overall, these data show that lean mass accumulates in WT-FF mice and that the rate of accumulation is greater during pregnancy. Furthermore, when WT mice are subjected to CR, lean mass decreased in both non-pregnant and pregnant mice compared to their FF counterparts. The CR-P group however maintained a relative increase in its lean mass when compared to CR-NP.   Figure 3.4: Mean percent lean mass changes relative to Day -0.5 in WT mice. Absolute values at baseline (Day -0.5) of each group are shown in the top-left corner of the figure.  SD bars; n=7-20/gr. ANOVA post-hoc for multiple comparisons. **:p=0.000; *:p<0.05. 37  3.4.5 Lean mass percent changes in GOAT-KO mice A repeated measures ANOVA with a Greenhouse-Geisser correction determined that there was a significant difference between the days (F(1.2, 10.4)=5.9, p=0.031). There was also a days*group interaction suggesting that the changes in lean mass between the days varied as a function of the groups (F(2.3, 10.4)=7.7, p=0.008). Pair-wise comparisons determined the differences within each group at the different time points. There were no apparent changes in percent lean mass within pregnant and non-pregnant groups from Day -0.5 to Day 10.5 (p=0.306). From Day 10.5 to 17.5, CR resulted in a 10% decrease in lean mass in non-pregnant mice. In addition, there is a 32% gain compared to a 1% gain in lean mass in the FF pregnant and non-pregnant groups respectively (p<0.005). These data show that lean mass increases in GOAT-KO mice, similar to WT mice, during pregnancy.  There was a statistically significant difference between the groups as determined by one-way ANOVA at Day 17.5 (F(2, 18)=43.001, p=0.0) (Fig. 3.5) however no difference at Day 10.5. This was also consistent with the repeated measures test that determined no statistically significant changes within the groups at the Day 10.5 time point. A post-hoc analysis using the Bonferroni correction revealed that on Day 17.5, similar to WT mice, FF pregnant mice accumulated more lean mass than FF non-pregnant mice (32%% vs. 1%; p=0.0). Also similar to WT, CR in non-pregnant mice resulted in a loss in lean mass compared to their FF counterparts (-10%; p=0.004). This contrasts with the modest, non-significant decrease in fat mass mentioned earlier in the KO-CR-NP mice (Fig 3.2). 38     3.4.6 Comparison of lean mass percent changes between Day -0.5 and Day 17.5 in WT vs. GOAT-KO mice The results of an independent (2-tailed) t-test suggest WT-FF-P mice accumulate more lean mass than KO-FF-P mice (84% vs. 32%; p=0.0) (Fig.3.6). All other WT and GOAT-KO mice had similar changes in lean mass. Overall, this demonstrates that lean mass changes associated with CR are similar in both WT and GOAT-KO strains in non-pregnant mice. In pregnant mice however, the gain in lean mass associated with pregnancy in the FF groups is greater in WT.  Figure 3.5: Mean percent lean mass changes relative to Day -0.5 in GOAT-KO mice. Absolute values at baseline (Day -0.5) of each group are shown in the top-left corner of the figure.  SD bars; n=8-13/gr; n=2-6 for KO-FF-P. ANOVA post-hoc for multiple comparisons. **:p=0.000; *:p<0.005. 39     3.5 Blood glucose The results for mean blood glucose changes in mmol/L between Day -0.5 (represented as 0 mmol/L), Day 10.5 (prior to randomization to FF or CR) and Day 17.5 (Day 18=sacrifice) are shown in Figs 3.7-3.9. Error bars represent the standard deviation within each e[perimental group and ?n? indicates the number of mice within each of these groups. Mauchly?s Test of Sphericity indicated that the assumption of sphericity had not been violated for all WT (x2(2)=0.1, p=.975) and GOAT-KO groups (x2(2)=0.1, p=.947). There were no outliers and the data was normally distributed as assessed by Normal Q-Q plot and a Shapiro-Wilk test for all groups (p>0.05). Homogeneity of variances was not violated as assessed by the /evene?s Test of Homogeneity of Variances (p>0.05) for all experimental groups in our blood glucose analysis.  Figure 3.6: Comparison of mean percent lean mass changes at Day 17.5 between WT and GOAT-KO mice. SD bars; n=7-20 for WT; n=8-13 for KO; n=2-6 for KO-FF-P. Independent (2-tailed) t-test. *:p<0.05 40  3.5.1 Blood glucose changes in WT mice A repeated measures ANOVA determined that there were no statistically significant changes in blood glucose in WT mice between the days, however, a days*group interaction was observed suggesting that the changes in blood glucose between the days varied as a function of the groups (F(2, 56)=3.0, P=0.003). Pair-wise comparisons determined the differences within each group at the different time points. Blood glucose concentrations remained constant within all groups from Day -0.5 to Day -10.5. From Day 10.5 to Day 17.5, CR resulted in decreased blood glucose concentrations during pregnancy (-3.3 mmol/L; p<0.05) and a modest effect in the non-pregnant group (-0.7 mmol/L; NS).  There was a statistically significant difference between the groups only at Day 17.5 as determined by one-way ANOVA (F(3,43)=10.184, p=0.00003) (Fig. 3.7).  A post-hoc analysis using the Bonferroni correction revealed that on Day 17.5,  CR resulted in lower average blood glucose concentrations in pregnant mice compared to FF counterparts (4.3 mmol/L vs. 7.0 mmol/L; p=0.0008). CR did not affect blood glucose concentrations in non-pregnant mice compared to FF counterparts. Blood glucose concentrations remained at similar levels between the FF-NP and FF-P groups. CR-P mice had lower blood glucose concentrations than CR-NP mice (4.3 mmol/L vs. 6.1 mmol/L; p=0.002). Overall, these data shows that CR in pregnant mice resulted in low blood glucose concentrations whereas CR in non-pregnant mice had no effect on blood glucose concentrations.   41     3.5.2 Blood glucose changes in GOAT-KO mice A repeated measure ANOVA determined that there was a significant difference between the days (F(2,30)=3.7, P<0.05). There was also a days*groups interaction suggesting that the changes in blood glucose between the days varied as a function of the groups (F(4,30)=4.4, P<0.05). Pair-wise comparisons determined the differences within each group at the different time points. Blood glucose concentrations remained constant within all groups from Day -0.5 to Day 10.5. From Day 10.5 to Day 17.5, CR resulted in decreased blood glucose concentrations in non-pregnant mice (-1.5 mmol/L; p<0.05). As mentioned earlier, there was one KO-CR-P mouse that survived to Day 17.5 but displayed severe hypoglycemia (-5.3 mmol/L compared to Day 10.5).  There was a statistically significant difference between the groups only at Day 17.5 as determined by one-way ANOVA (F(2,38)=17.673, p=0.000004) (Fig. 3.8). A post-hoc Figure 3.7: Mean blood glucose changes relative to Day -0.5 in WT mice. Absolute values at baseline (Day -0.5) of each group are shown in the top-left corner of the figure. SD bars; n=8-20/gr. ANOVA post-hoc for multiple comparisons. *:p<0.005. 42  analysis using the Bonferroni correction revealed that on Day 17.5, CR in resulted in lower blood glucose concentrations in non-pregnant mice compared to FF counterparts (5.3 mmol/L vs. 7.1 mmol/L, p=0.000003). This was in contrast to WT mice where CR in non-pregnant resulted in a modest decrease in blood glucose. CR in our one GOAT-KO pregnant mouse that managed to survive to Day 17.5 (as others were euthanized prior to this date) resulted in a very low blood glucose concentration. This mouse also displayed features of severe hypoglycemia compared to its FF counterparts (2.9 mmol/L vs. 6.4 mmol/L). Blood glucose concentrations remained at similar levels between the FF-P and FF-NP groups. Overall, this shows that CR in GOAT-KO mice causes low blood glucose concentrations in non-pregnant mice and is exacerbated in pregnant mice.      Figure 3.8: Mean blood glucose changes relative to Day -0.5 in GOAT-KO mice. Absolute values at baseline (Day -0.5) of each group are shown in the top-left corner of the figure. SD bars; n=6-19/gr; n=1-3 for KO-CR-P. ANOVA post-hoc for multiple comparisons. *:p<0.00001. 43  3.5.3 Comparison of blood glucose changes between Day -0.5 and Day 17.5 in WT vs. GOAT-KO mice An independent (2-tailed) t-test at Day 17.5 determined that CR results in statistically significantly lower blood glucose concentrations in KO-CR-NP mice compared to WT-CR-NP mice (p<0.05) (Fig.3.9). We can also see lower blood glucose concentrations in the one KO-CR-P mouse compared to WT-CR-P mice. FF mice had similar blood glucose levels regardless of WT and KO strains. Overall, these data show that CR resulted in lower blood glucose concentrations in GOAT-KO pregnant and non-pregnant mice compared to WT.     3.6 Plasma AG and UAG concentrations The results for plasma AG and UAG concentrations at Day 18 (sacrifice) are shown in Fig. 3.10-3.12. There were no outliers and the data was not normally distributed as assessed by Normal Q-Q plot and a Shapiro-Wilk test (p<0.05). The data was then analyzed using a Figure 3.9: Comparison of mean blood glucose changes at Day 17.5 between WT and GOAT-KO mice. SD bars; n=8-20/gr for WT; n=6-19/gr for KO; n=1-3 for KO-CR-P. Independent (2-tailed) t-test. *:p<0.05. 44  non-parametric test known as a Kruskal-Wallis test followed by a series of Mann-Whitney U tests for our post-hoc analysis. 3.6.1 Plasma AG and UAG concentrations in WT mice There was a statistically significant difference between the groups as determined by a Kruskal-Wallis test in plasma AG concentrations at Day 18 (H(3)=13.206, p=0.004) with a mean rank of 17.30 for WT-FF-NP, 23.94 for WT-CR-NP, 12.56 for WT-FF-P and 33.43 for WT-CR-P (Fig. 3.10). In contrast, no difference was observed between plasma UAG concentrations (H(3)=2.291, p=0.514). A series of Mann-Whitney U tests revealed CR resulted in a 9-fold higher plasma AG in pregnant mice compared to FF counterparts (905.8 pg/mL vs. 98.3 pg/mL; U=3, p=0.001). In addition, CR-P mice also had a 3-fold higher plasma AG compared to CR-NP mice (905.8 pg/mL vs. 286 pg/mL; U=26, p=0.047). CR did not significantly affect AG in NP mice. Thus, in WT pregnant mice (but not in non-pregnant mice), CR causes an increase in AG concentrations.     Figure 3.10: Mean plasma acylated (AG) & unacylated ghrelin (UAG) concentrations in WT mice. SD bars; n=6-16/gr. Mann-Whitney post-hoc for multiple comparisons. *:p<0.05; **:p<0.005.  45  3.6.2 Plasma UAG concentrations in GOAT-KO mice As expected, plasma AG concentrations were undetectable in GOAT-KO mice, confirming the absence of ghrelin acylation. There was a statistically significant difference between the groups as determined by a Kruskal-Wallis test in plasma UAG concentrations at Day 18 (H=(2)=8.678, p=0.012) with a mean rank of 10.37 for KO-FF-NP, 22.63 for KO-CR-NP and 13.17 for KO-FF-P (Fig. 3.11). A series of Mann-Whitney tests revealed that CR resulted in 4-fold higher plasma UAG concentrations in non-pregnant mice compared to their FF counterparts (4687.8 pg/mL vs. 1001.4 pg/mL; U=3.5, p=0.004). Plasma UAG concentrations remained at similar levels between FF pregnant and non-pregnant groups.  Thus, in GOAT-KO non-pregnant mice, CR causes an increase in plasma UAG concentrations.     Figure 3.11: Mean plasma acylated (AG) & unacylated ghrelin (UAG) concentrations in GOAT-KO mice. SD bars; n=4-16/gr. Mann-Whitney post-hoc for multiple comparisons. *:p<0.005.  46  3.6.3 Comparison of plasma UAG concentrations in WT vs. GOAT-KO mice A Mann-Whitney U test between WT and GOAT-KO groups at Day 18 show KO-CR-NP mice have ~5-fold higher plasma UAG than WT-CR-NP mice (4688 pg/mL vs. 874.6 pg/mL; U=0, p=0.001) (Fig.3.12). All other experimental groups had similar plasma UAG concentrations regardless of WT or GOAT-KO strains. Overall this shows that CR in GOAT-KO mice resulted in higher plasma UAG than in WT mice.    3.7 GOAT and ghrelin mRNA expression in the stomach, hypothalamus and pituitary The results for GOAT and ghrelin mRNA expressions in the stomach, hypothalamus and pituitary at Day 18 (sacrifice) are shown in Figs. 3.13-3.18. Expression levels for each group are shown as a comparative fold-change to a calibrator consisting of a pooled WT-FF-NP RNA samples for each tissue. Ghrelin (and GOAT) expression per nanogram of cDNA was higher in the stomach calibrator compared to the hypothalamus and pituitary calibrator (5035-fold higher compared to pituitary and 6190-fold higher compared to hypothalamus). Figure 3.12: Comparison of mean plasma UAG concentrations between WT and GOAT-KO mice. SD bars; n=6-16/gr for WT; n=4-16/gr for KO. Mann-Whitney test between WT vs. KO. *:p=0.001. 47  There were no outliers and the data was not normally distributed as assed by a Normal Q-Q plot and a Shapiro-Wilk test (p<0.05). The data was then analyzed using a nonparametric test known as a Kruskal-Wallis test, followed by a series of Mann-Whitney U tests for our post-hoc analysis.  3.7.1 GOAT mRNA expression in the stomach GOAT mRNA was undetectable in GOAT-KO mice as expected. There was a statistically significant difference between the groups as determined by a Kruskal-Wallis test at Day 18 (H(3)=22.048, p=0.00006) with a mean rank of 27.33 for WT-FF-NP, 18.50 for WT-CR-NP, 29.90 for WT-FF-P and 8.18 for WT-CR-P (Fig. 3.13). A series of Mann-Whitney U tests revealed CR in resulted in lower levels of relative GOAT mRNA expression in pregnant mice compared to their FF counterparts (0.57-fold vs. 1.1-fold; U=0, p=0.0). Furthermore, CR-P mice had lower GOAT mRNA expression levels compared to CR-NP mice (0.57-fold vs. 0.85-fold; U=12, p=0.002).  CR-NP mice had similar GOAT mRNA expression levels as FF-NP mice. Thus, in WT pregnant mice (but not in non-pregnant mice), CR causes lower GOAT mRNA expression in the stomach.   Figure 3.13: Mean relative GOAT mRNA expression in the stomach in WT mice. SD error bars; n=9-11/gr. Mann-Whitney post-hoc for multiple comparisons . *:p<0.005;**:p=0.000.  48  3.7.2 Ghrelin mRNA expression in the stomach There was a statistically significant difference between the groups as determined by a Kruskal-Wallis test at Day 18 (H(6)=14.997, p=0.02) with a mean rank of 33.78 for WT-FF-NP, 31.30 for WT-CR-NP, 41.80 for WT-FF-P, 18.32 for WT-CR-P, 44.00 for KO-FF-NP, 22.81 for KO-CR-NP and 30.33 for KO-FF-P (Fig. 3.14). A series of Mann-Whitney U tests revealed that WT-CR-NP mice had similar ghrelin mRNA expression levels as WT-FF-NP mice. On the other hand, KO-CR-NP mice had lower levels of ghrelin mRNA expression compared to KO-FF-NP mice (1.36-fold vs. 0.93-fold, U=12, p=0.038). In addition, WT-CR-P mice had lower levels of ghrelin mRNA expression compared to WT-FF-P mice (1.31-fold vs. 0.86-fold; U=15, p=0.004). There were no statistically significant differences in ghrelin mRNA expression between WT and GOAT-KO strains in each experimental group.  Overall, this shows that CR resulted in lowered ghrelin mRNA expression in the stomach in WT and GOAT-KO pregnant and non-pregnant mice.      Figure 3.14: Mean relative ghrelin mRNA expression in the stomach in WT and GOAT-KO mice. SD bars; n=9-11/gr for WT; n=6-8/gr for KO. Mann-Whitney post-hoc for multiple comparisons and Mann-Whitney test between WT vs. KO. *:p<0.05; **:p<0.005. 49  3.7.3 GOAT mRNA expression in the hypothalamus GOAT mRNA was undetectable in GOAT-KO mice as expected. There was a statistically significant difference between the groups as determined by a Kruskal-Wallis at Day 18 (H(3)=12.636, p=0.005) with a mean rank of 12.00 for WT-FF-NP, 15.00 for WT-CR-NP, 16.38 for WT-FF-P and 27.64 for WT-CR-P (Fig. 3.15). A series of Mann-Whitney tests revealed CR resulted in higher levels of GOAT mRNA expression in pregnant mice compared to their FF counterparts (1.55-fold vs. 1.07-fold; U=16, p=0.02). In addition, these CR-P mice had higher levels of GOAT mRNA expression compared to CR-NP mice (1.55-fold vs. 0.99-fold; U=14, p=0.006). CR-NP mice had similar levels of GOAT mRNA expression levels as FF-NP mice. Thus, in WT pregnant mice (but not in non-pregnant mice), CR causes higher GOAT mRNA expression in the hypothalamus.    3.7.4 Ghrelin mRNA expression in the hypothalamus There was a statistically significant difference between the groups as determined by a Kruskal-Wallis test at Day 18 (H(6)=16.7, p=0.011) with a mean rank of 36.67 for WT-FF-NP, 36.22 for WT-CR-NP, 34.13 for WT-FF-P, 39.41 for WT-CR-P, 15.56 for KO-FF-NP, Figure 3.15: Mean relative GOAT mRNA expression in the hypothalamus in WT mice. SD bars; n=8-11/gr. Mann-Whitney post-hoc for multiple comparisons. *:p<0.05;**:p<0.01.  50  21.88 for KO-CR-NP and 18.00 for KO-FF-P (Fig.3.16). A series of Mann-Whitney tests revealed no statistically significant changes between the groups within WT and GOAT-KO mice. In comparing the groups between these strains, FF non-pregnant and pregnant groups had lower levels of ghrelin mRNA expression in the GOAT-KO strain than in the WT (1.17-fold vs. 0.77-fold for non-pregnant; U=7, p=0.04 and 1.01-fold vs. 0.79-fold for pregnant; U=5, p=0.013).  Overall, this shows that CR did not affect ghrelin mRNA expression in the hypothalamus in WT and GOAT-KO mice. WT mice have higher ghrelin mRNA expression in the hypothalamus than GOAT-KO mice.     3.7.5 GOAT mRNA expression in the pituitary gland Pituitary GOAT mRNA expression was not detectable in any of the experimental groups.   Figure 3.16: Mean relative ghrelin mRNA expression in the hypothalamus in WT and GOAT-KO mice. SD bars; n=8-11/gr for WT; n=6-8/gr for KO. Mann-Whitney post-hoc for multiple comparisons and Mann-Whitney test between WT vs. KO. *:p<0.05. 51  3.7.6 Ghrelin mRNA expression in the pituitary gland There was a statistically significant difference between the groups as determined by a Kruskal-Wallis test at Day 18 (H(3)=16.0, p=0.001) with a mean rank of 18.55 for WT-FF-NP, 43.40 for WT-CR-NP, 39.63 for WT-FF-P, 39.88 for WT-CR-P, 10.44 for KO-FF-NP, 27.88 for KO-CR-NP and 18.13 for KO-FF-P (Fig. 3.17-3.19). A series of Mann-Whitney U tests revealed that WT-FF-P mice had higher ghrelin mRNA expression compared to WT-FF-NP mice (2.95-fold vs. 1.33; U=7, p=0.001) (Fig. 3.17). In addition, the WT-CR-NP mice had higher ghrelin mRNA expression compared to WT-FF-NP mice (3.13-fold vs. 1.33-fold; U=2, p=0.00002). Similarly, KO-CR-NP mice also had higher ghrelin mRNA expression levels compared to KO-FF-NP mice (1.78-fold vs. 1.09-fold, U=7, p=0.007) (Fig. 3.18). Furthermore, WT-CR-P mice higher ghrelin mRNA expression compared to WT-FF-P mice although this was not statistically significant. Overall this shows that CR resulted in elevated ghrelin mRNA expression in the pituitary gland in WT and GOAT-KO mice.  Ghrelin mRNA expression in CR-NP mice and FF-P mice are higher in WT compared to the GOAT-KO strain (3.13-fold vs. 1.78-fold; U=11, p=0.009 for CR non-pregnant & 2.95-fold vs. 1.43-fold; U=4, p=0.048 for FF pregnant) (Fig. 3.19). Thus, WT-CR-P and WT-FF-P mice have higher ghrelin mRNA expression in the pituitary compared to GOAT-KO groups.  52      Figure 3.17: Mean relative ghrelin mRNA expression in the pituitary gland in WT mice. SD bars; n=8-11/gr. Mann-Whitney post-hoc for multiple comparisons. *:p<0.005; **:p=0.000. Figure 3.18: Mean relative ghrelin mRNA expression in the pituitary gland in GOAT-KO mice. SD error bars; n=4-8/gr. Mann-Whitney post-hoc for multiple comparisons. *:p<0.01. 53    3.8 Plasma growth hormone concentrations  The results for plasma growth hormone (GH) concentrations at Day 18 (sacrifice) are shown in Figs. 3.20-3.22. There were no outliers and the data was not normally distributed as assessed by a Normal Q-Q plot and a Shapiro-Wilk test of normality (p<0.05). The data was then analyzed using a non-parametric test known as a Kruskal-Wallis test followed by a series of Mann-Whitney U tests for our post-hoc analysis.  3.8.1 Plasma growth hormone concentrations in WT mice There was a statistically significant difference between the groups determined by a Kruskal-Wallis test in plasma GH concentrations at Day 18 (H(3)=34.8, p=0.0) with a mean rank of 9.50 for WT-FF-NP, 22.94 for WT-CR-NP, 41.31 for WT-FF-P and 37.44 for WT-CR-P (Fig.3.20). A series of Mann-Whitney U tests revealed CR resulted in higher plasma GH in non-pregnant mice compared to FF counterparts (75.5 ng/mL vs. 8.0 ng/mL; U= 28, p<0.0001). CR-P mice had higher plasma GH compared to CR-NP mice (291.1 ng/mL vs. Figure 3.19: Comparison of mean relative ghrelin mRNA expression in the pituitary gland between WT and GOAT-KO mice. SD error bars; n=8-11/gr for WT; n=4-8/gr for KO. Mann-Whitney test between WT vs. KO. *:p<0.05; **:p<0.01. 54  75.5 ng/mL; U=12, p<0.0005). Finally, FF-P mice had higher plasma GH compared to FF-NP mice (370.4 ng/mL vs. 8.0 ng/mL; U=0, p=0.0).  Overall, this shows that WT pregnant mice have markedly higher plasma GH concentrations. In addition, in WT-NP mice, CR resulted in elevated plasma GH concentrations relative to FF-NP mice.  3.8.2 Plasma growth hormone concentrations in GOAT-KO mice  There was a statistically significant difference between the groups as determined by a Kruskal-Wallis test in plasma GH concentrations at Day 18 (H(2)=14.2, p=0.001) with a mean rank of 9.93 for KO-FF-NP, 11.83 for KO-CR-NP and 23.50 for KO-FF-P (Fig. 3.21). A series of Mann-Whitney U tests revealed CR resulted in similar levels of plasma GH in non-pregnant mice as their FF counterparts (8.8 ng/mL vs. 8.1 ng/mL). In addition, FF-P mice had higher plasma GH compared to FF-NP mice (276.3 ng/mL vs. 8.1 ng/mL; U=0, p=0.0).  Figure 3.20: Mean plasma GH concentrations in WT mice. SD bars; n=8-18/gr. Mann-Whitney post-hoc for multiple comparisons. *:p<0.0005; **:p<0.0001 ***:p=0.0. 55  Overall, this shows that unlike WT mice, CR does not affect plasma GH concentrations in GOAT-KO non-pregnant mice. GOAT-KO pregnant mice have ~35-fold higher plasma GH concentrations than non-pregnant groups.     3.8.3 Comparison of plasma growth hormone concentrations in WT vs. GOAT-KO mice CR resulted in a ~9-fold higher plasma GH in WT-NP mice than in KO-NP mice as determined by a Mann-Whitney test (75.5 ng/mL vs. 8.8 ng/mL; U=16, p=0.009) (Fig. 3.22). WT and GOAT-KO FF pregnant mice had similar levels of plasma GH concentrations (370.4 ng/mL vs. 276.3 ng/mL).  Overall, this shows that CR resulted in elevated plasma GH concentrations in WT non-pregnant mice whereas GOAT-KO non-pregnant mice had very low levels. Interestingly, GOAT-KO pregnant mice maintained high plasma GH concentrations similar to the WT pregnant group.  Figure 3.21: Mean plasma GH concentrations in GOAT-KO mice. SD bars; n=6-14/gr. Mann-Whitney post-hoc for multiple comparisons. *:p=0.000. 56     3.9 Hepatic glycogen content The results for hepatic glycogen content at Day 18 (sacrifice) are shown in Figs. 3.23-3.25. There were no outliers and the data was normally distributed as assessed by Normal Q-Q plot and a Shapiro-Wilk test for all groups (p>0.05). Homogeneity of variances was not violated as assessed by /evene?s Test of Homogeneity of 9ariances (p>0.05) for all experimental groups in our hepatic glycogen content analysis.  3.9.1 Hepatic glycogen content in WT mice There was a statistically significant difference between the groups as determined by one-way ANOVA at Day 18 (F(3,51)=3.8, p=0.015) (Fig. 3.23). A post-hoc analysis using the Bonferroni correction revealed that on Day 18, CR and FF non-pregnant mice had similar hepatic glycogen content (33.3 mg/mL/g vs. 38.7 mg/mL/g). CR in pregnant mice on the other hand resulted in a 44% lower hepatic glycogen content than in FF pregnant mice (27.1 mg/mL/g vs. 49.4 mg/mL/g; p=0.008). This shows that CR resulted in a greater depletion of Figure 3.22: Comparison of mean plasma GH concentrations between WT and GOAT-KO mice. SD bars; n=8-18/gr for WT; n=6-14/gr for KO. Mann-Whitney test between WT vs. KO. *:p<0.01. 57  hepatic glycogen content in pregnant mice in contrast to non-pregnant mice where depletion was modest.    3.9.2 Hepatic glycogen content in GOAT-KO mice There was no statistically significant difference between the groups as determined by one-way ANOVA at Day 18 (F(2,18)=0.983, p=0.393) (Fig.3.24). This shows that CR had a modest effect on hepatic glycogen content in non-pregnant mice compared to FF counterparts (50.6 mg/mL/g vs. 47.5 mg/mL/g). Figure 3.23: Mean relative hepatic glycogen concentration per gram of liver in WT mice. SD bars; n=10-20/gr. ANOVA post-hoc for multiple comparisons. *:p<0.001. 58    3.9.3 Comparison of hepatic glycogen content in WT vs. GOAT-KO mice An independent (2-tailed) t-test suggest that WT-CR-NP mice have less hepatic glycogen content than the KO-CR-NP mice (p<0.01) (Fig.3.25). All other experimental groups had similar levels of glycogen content regardless of WT or GOAT-KO strains. Overall, this shows that CR resulted in greater glycogen depletion in WT mice than in GOAT-KO mice.   Figure 3.25: Comparison of mean relative hepatic glycogen concentrations per gram of liver between WT and GOAT-KO mice. SD bars; n=10-20/gr for WT; n=5-8/gr for KO. Independent (2-tailed) t-test. *:p<0.001. Figure 3.24: Mean relative hepatic glycogen concentration per gram of liver in GOAT-KO. SD bars; n=5-8/gr. ANOVA post-hoc for multiple comparisons revealed no significance 59  3.10 Hepatic PCK1 mRNA Expression The results for hepatic PCK1 mRNA expression at Day 18 (sacrifice) are shown in Fig. 3.26. There were no outliers and the data was normally distributed as assessed by Normal Q-Q plot and a Shapiro-Wilk test for all groups (p>0.05). Homogeneity of variances was not violated as assessed by /evene?s Test of Homogeneity of 9ariances (p!0.0) for the WT groups but was violated for the GOAT-KO groups. A one-way ANOVA was used on the WT groups and a non-parametric test known as a Kruskal-Wallis test for the GOAT-KO groups. 3.10.1 Hepatic PCK1 mRNA expression in WT mice Differences in hepatic PCK1 mRNA expression on Day 18 between the groups as determined by one-way ANOVA were of borderline significance (F(3, 28)=2.3, p=0.061) (Fig. 3.26). Globally, there was a tendency towards a greater PCK1 mRNA expression in both NP (2.4 fold) and P (2.1-fold) WT mice submitted to CR compared to their FF counterparts.  3.10.2 Hepatic PCK1 mRNA expression in GOAT-KO mice There was no statistically significant difference between the groups as determined by a Kruskal-Wallis test at Day 18 in relative PCK1 mRNA expression (H(2)=4.200, p=0.122) with a  mean rank of 6.00 for KO-FF-NP, 12.00 for KO-CR-NP and 10.20 for KO-FF-P (Fig. 3.26). Although this was not statistically significant, visual examination of Figure 26 suggests that PCK1 mRNA expression is higher in the presence of CR. 3.10.3 Comparison of hepatic PCK1 mRNA expression in WT vs. GOAT-KO mice An independent (2-tailed) t-test assuming unequal variances between WT and GOAT-KO counterparts determined no statistically significant differences between these two groups in relative hepatic PCK1 mRNA expression (Fig. 3.26). Overall, this shows that PCK1 60  mRNA expression remains at similar levels in all groups regardless of WT and GOAT-KO strains               Figure 3.26: Mean relative hepatic PCK1 mRNA expression. SD bars; n=5-10/gr. No post-hoc was performed since ANOVA one-way and Kruskal-Wallis test were not statistically significant.  61  4 Discussion In my thesis, I aimed at understanding the role of ghrelin and ghrelin acylation in glucose metabolism in NP and P mice submitted to global CR. The main findings are: 1. GOAT-KO mice were less fertile than WT mice. These mice had difficulty conceiving or sustaining a pregnancy. The addition of CR on GOAT-KO pregnant mice resulted in an inability to reach the Day 18 (=sacrifice) time point. These mice were severely hypoglycemic, delivered prematurely or absorbed their pregnancy.    2. In FF-P mice, fat mass gain was greater in WT compared to KO animals. The difference was only significant in FF-P animals. The addition of CR on GOAT-KO non-pregnant mice resulted in a modest decrease in fat mass compared to WT-CR-NP mice that had a significant decrease in fat mass. My results are consistent with the adipogenic role of exogenous AG and suggest that endogenous AG (generated through acylation with GOAT) contributes to adiposity in mice during FF pregnancy.  3. CR caused significant decreases in blood glucose in WT-P and KO-NP mice. In addition, CR caused a severe hypoglycemia in the only KO mouse that could sustain pregnancy until day 17.5. All other KO-CR-P mice terminated pregnancy early and several had evidence of low blood glucose. Taken together, these data suggest that acylation of ghrelin is necessary for maintenance of euglycemia during CR.  4. In WT-CR-P mice and in KO-CR-NP mice (I did not have blood samples for KO-CR-P mice), the lower blood glucose caused by CR are associated with an increase in circulating AG (WT only) and UAG (WT and KO) compared to their FF counterparts. These increased concentrations of ghrelin suggest a greater production (in ghrelin secreting glands) or a lower rate of degradation (in the circulation) or both. 62  5. When examining the potential sources of AG and UAG in CR mice, we found decreased GOAT and ghrelin gene expression in the stomach in WT mice but no change in KO. In contrast, we found an increase in ghrelin mRNA expression in the pituitary in WT and KO groups submitted to CR. Hypothalamic GOAT expression was also increased in WT-CR-P mice. These data suggest that central (but not peripheral) production and acylation of ghrelin may be increased in response to a CR severe enough to cause a decrease in blood glucose. We cannot rule out a decrease in peripheral ghrelin deacylation or degradation. 6. We also investigated complementary aspects of glucose metabolism in order to gather a more complete picture of the effect of CR on blood glucose concentrations. In WT-CR groups with low blood glucose, hepatic glycogen content was decreased and PEPCK (PCK1 gene) expression was increased, suggesting an appropriate response of these pathways. However, in KO-CR-NP mice that also had low blood glucose, hepatic glycogen content did not decrease however PCK1 gene expression was increased suggesting an appropriate gluconeogenic response but an impaired glycogenolytic response. GH concentrations remained low in FF-NP mice but were significantly higher in WT-CR-NP compared to KO-CR-NP mice which suggests it as a possible response to maintaining the euglycemia observed in the WT group (KO-CR-NP had low blood glucose). All WT and KO pregnant mice had significantly higher GH compared to NP mice suggesting GH production in KO-P mice is independent of AG. I also discuss a possible local role of the GOAT-ghrelin system in the CNS in facilitating the release of GH.   63  4.1 Fertility and pregnancy in GOAT-KO mice  Preliminary experiments were performed in WT mice to determine the appropriate degree of CR that was sufficient to cause hypoglycemia during pregnancy without affecting pregnancy outcome. I discovered that, in contrast to the information provided by the company that kindly gifted the animals to us, and at least under my experimental conditions, it was more difficult to achieve pregnancy in GOAT-KO mice than in WT mice. When CR was present, we observed preterm delivery and pregnancy absorption. Assuming that GOAT-KO and WT mice only differ by the absence or presence of the GOAT enzyme, my observations could be explained by a direct role of AG on reproduction, an indirect role through the hypoglycemia associated with CR, or both. 4.1.1 Fertility in male GOAT-KO mice The role of AG on puberty and fertility has been investigated in both male and female rodents. In males, ghrelin and GHSR mRNA are expressed in the testosterone-producing Leydig cells of the testis 101-103. Testicular GHSR mRNA expression is undetectable in prepubertal rodents but increases thereafter suggesting a potential role in the development of secondary sexual characteristics 104. Others have shown that luteinizing hormone (LH), which stimulates testosterone production, also induces testicular ghrelin and GHSR expression 101. These data suggests that male GOAT-KO mice may experience reduced fertility.  4.1.2 Fertility in female GOAT-KO mice In female rodents, ghrelin mRNA expression in the ovaries varies throughout the estrous cycle with peak levels occurring during the luteal phase 105. Interestingly, this peak level of ghrelin expression coincides with that of the functional activity of the corpus luteum suggesting a possible role of AG in its regulation and development 105-107. Martin et al. 64  (2011) also demonstrated that maternal ghrelin deficiency in ghrelin heterozygote mice (+/+ male with +/- female) compromised fertility and as well as decreased litter size in their female offspring compared to WT mice (+/+ male with +/+ female) 108. They determined that ghrelin deficiency in utero altered the expression of genes important for endometrial receptivity and implantation.  4.1.3 Hypoglycemia associated with CR in GOAT-KO pregnant mice When GOAT-KO pregnant mice were put on CR, hypoglycemia, preterm delivery and pregnancy absorption were observed. The symptoms of hypoglycemia started as early as after 2-3 days on the CR regime as determined by blood glucose under 4.0mmol/L (contrasting with an average fasting glucose in WT mice of ~6.0mmol/L). These symptoms included hunched backs, shaking and reluctance to move in these mice. Preterm delivery was confirmed by the presence of defined uterine horns which is indicative of a pregnancy and of traces of the aborted pups in the stomach of the CR mother 109. Pregnancy absorption was suspected when KO-CR-P mice, which had experienced a daily weight gain profile similar to that of KO-FF-P mice and had palpable fetuses until Day 10.5 of pregnancy, reached a weight plateau once placed on the CR diet for 2-3 days, suggesting that the pregnancy had not progressed further. At autopsy, they had defined uterine horns but no fetuses were present.  Taking together, this suggests that the absence of ghrelin acylation and CR have an additive effect on the course of the pregnancy. The lack of AG by itself did affect the fertility, but once pregnancy was achieved, delivery of full term pups was the rule. When CR was added, insufficient weight gain during the third trimester of pregnancy, which has been shown to cause hypoglycemia and increase the risk of preterm delivery in humans 110, did 65  also cause preterm delivery and pregnancy absorption. These were not observed in WT-CR-P (with impaired weight gain but normal AG) and in KO-FF P (with absent AG but normal caloric intake).    4.2 Body composition All FF mice regardless of WT or GOAT-KO strain consumed equal amounts of food. This is consistent with other rodent studies that demonstrated that deletion of the ghrelin gene as a whole did not affect appetite 47,111. This suggests that while exogenous administration of AG stimulates food intake, endogenous AG may not be critical for the regulation of food intake. Whether GOAT inhibitors, which are emerging anti-obesity agents, will be effective in inhibiting food intake remains to be demonstrated. In FF-NP and FF-P mice, fat mass gain at Day 17.5 (expressed as a percent change relative to Day -0.5) was greater in WT compared to GOAT-KO mice. The difference was only significant in FF-P animals (83% in WT vs. 23% in GOAT-KO; p<0.05) (Fig. 3.3). Interestingly, it was shown that exogenous AG administration induces adiposity in rodents and that this gain in adiposity is independent of $G?s ore[igenic effects 112,113. My results suggest that, similar to what is observed with exogenous AG administration, endogenous AG (generated through acylation by GOAT) may contribute to adiposity in mice during FF pregnancy.  Interestingly in CR mice there was a different result. We observed that KO-NP mice lost less fat tissue compared to WT-NP counterparts (23% vs. 41% loss; p=0.150) (Fig.3.3). Although it was not statistically significant, the mean fat mass depletion at Day 17.5 was approximately twice as great in the WT-CR-NP compared to the KO-CR-NP mice. These GOAT-KO mice were also characterized by low blood glucose, low GH concentrations and 66  higher hepatic glycogen content compared to WT counterparts. Since adipose tissue is sequentially metabolized after glycogen stores are used during periods of negative energy balance in order to harness energy and restore euglycemia 114, it suggests that AG may, in part, be responsible for facilitating this process. Indeed, exogenous AG administration was shown to induce lipolysis in rodents and humans 115-118. Whether AG-induced lipolysis is direct (independent of GH) or indirect (through stimulation of GH) or mixed remains unclear. Thus endogenous AG appears to play two roles in fat metabolism: 1. Inducing adiposity in FF mice and 2. Inducing fat breakdown (lipolysis?-oxidation) in CR mice.  Another interesting result was how WT fat loss resulting from CR in both P and NP mice was more substantial than lean tissue loss in these mice. This may reflect metabolic adaptations that favour the use of fat stores over the use of protein as a source of energy in response to CR 114.  Looking further into the data, we could see that lean mass losses in CR-NP mice and gains in FF-NP mice were similar regardless of WT or GOAT-KO strains (Fig.3.6). In contrast, we observed a markedly higher lean mass in WT-FF-P mice compared to KO-FF-P mice accounted for this (84% in WT vs. 32%; p<0.0001) (Fig. 3.6).  The EchoMRI machine is known to count lean tissue as a muscle tissue mass of all body parts containing water but excludes fat, bone and other substances (fur, hair, claws, minerals, etc.). We suspect that the fetuses in the pregnant mice may explain this rise in lean mass in both WT and KO mice as neonatal body composition is almost entirely lean (prominently higher lean-to-fat ratio than adult mice) 79. As fetus quantities in both strains were on average the same (7.8 fetuses in WT and 7.3 fetuses in GOAT-KO), the increased lean mass in WT-FF-P vs. KO-FF-P groups may again lie in endogenous $G?s ability to induce lean tissue mass gain during pregnancy 67  similar to the effects of exogenous AG administration reported in NP mice 113. Thus, presence of GOAT and thus AG in pregnant mice induces increases in lean tissue mass which may reflect higher fetal masses. In WT-CR-P mice, a significant gain in lean mass was observed compared to NP counterparts (+36% gain; p=0.000). Although a loss was evident when compared to FF pregnant mice, we could see a marked lean mass gain (and hence fetal growth) by Day 17.5. In contrast to the consequences observed in the KO-CR-P mice, these WT dams maintained their pregnancy and appeared to project a full-term delivery by ~Day 20. Overall, this suggests that GOAT and thus AG may be essential in ensuring a full-term delivery during negative energy balance.  4.3 Blood glucose concentrations We hypothesized that CR would cause a more severe hypoglycemia in P than NP mice. Indeed, in WT animals, CR restriction was associated with lower blood glucose at Day 17.5 only in pregnant animals. In GOAT-KO mice, the decrease in blood glucose was already observed in NP animals and was much more severe in P animals. This suggests that GOAT (and therefore AG) contributes to the maintenance of euglycemia during periods of negative energy balance. Pregnancy may put mice at risk for hypoglycemia in WT and this risk may be accentuated in GOAT-KO. 4.4 Association between blood glucose and AG/UAG concentrations  To clarify this issue, we first measured AG and UAG plasma concentrations. In WT mice, AG concentrations (and to a lesser extent UAG, although the difference was not significant) were significantly higher in CR-P mice (in which blood glucose was lower) compared to FF-P mice. In the GOAT-KO groups, AG was undetectable as expected. UAG 68  concentrations were also higher in the KO-CR-NP animals (in which blood glucose was lower) compared to KO-FF-NP and KO-FF-P groups, suggesting an increase in the production of UAG or a decrease in its degradation (or both). 4.5 Tissue specific expression of ghrelin and GOAT mRNA  4.5.1 Stomach The stomach is classically considered the major source of circulating ghrelin with lower expression levels reported in the hypothalamus, pituitary and pancreas 1,6-10,119,120. Looking at the mRNA data, levels of ghrelin (and GOAT) expression per nanogram of cDNA (reverse transcription from RNA) were much higher in the stomach calibrator compared to the hypothalamus and pituitary calibrator (5035-fold compared to pituitary and 6190-fold compared to hypothalamus). This was consistent with Nass, et al (2004) where they found ghrelin mRNA expression to be 3000-fold higher in the rat stomach than in the pituitary 121. In addition, the size of the stomach is much higher than the size of the other ghrelin-producing tissues. This suggests gastric ghrelin to be the principal source of circulating AG and UAG in the periphery. However, in my study, higher levels of plasma AG and UAG concentrations (WT-CR-P and KO-CR-NP groups in particular) were associated with lower stomach GOAT and ghrelin expression. This suggests that the stomach may not contribute to these higher circulating AG and UAG concentrations. 4.5.2 Hypothalamus and pituitary Hypothalamic GOAT mRNA expression was significantly higher in WT-CR-P mice compared to WT-FF-P and WT-CR-NP mice. Hypothalamic ghrelin mRNA expression was the same for all groups except WT-FF-P and WT-FF-NP mice that had a higher fold 69  expression compared to GOAT-KO counterparts. The hypothalamus plays an important role in appetite regulation and is a target for AG. Although, my data as well as the literature found no differences in food intake between GOAT-KO and WT mice, I hypothesize that hypothalamic ghrelin and GOAT (and thus AG) may be involved in specific feeding behaviours. GOAT-induced ghrelin acylation is responsible for hedonic appetite 122. As the WT-CR-P mice were in a position of negative energy balance and a significantly higher GOAT mRNA expression was associated with this, it could suggest that locally produced GOAT (in the hypothalamus) is responsible for seeking foods richer in calories rather than the overall amount however this yet remains to be determined 122-124.  Pituitary GOAT mRNA expression was undetectable in all of the mice. This was contrary to the demonstration that GOAT mRNA expression is present in rodent pituitary glands 125,126. It is important to mention that these studies were done in rodent pituitary cell cultures or LacZ reporter gene specific for GOAT mRNA in the pituitary where the latter technique yields a qualitative rather a quantitative assessment of its expression. In my study, GOAT and ghrelin mRNA expression was performed using quantitative real-time PCR whereby pituitary GOAT mRNA expression may have been too low (late cycle threshold) for a detectable signal rather than completely non-existent.  The Kruskal-Wallis test determined an overall statistical significance (p<0.005) between the groups in pituitary ghrelin mRNA expression. Pituitary ghrelin mRNA expression was significantly higher in WT-CR-NP and KO-CR-NP mice compared to WT-FF-NP and KO-FF-NP mice respectively (Fig 3.17-3.18). In addition, pituitary ghrelin mRNA expression was higher (NS) in WT-CR-P mice compared to WT-FF-P mice (6.4-fold vs. 2.9-fold) (Fig.3.17). While the hypothalamus and pituitary may contribute less circulating AG and 70  UAG to the periphery compared to the stomach, these observations collectively suggest a local role for the GOAT-ghrelin system in the CNS. 4.5.3 Degradation of AG/UAG Degradation of AG and UAG occurs predominantly by plasma proteases 26,27. I speculate that the increased concentrations of AG/UAG in the periphery observed in CR mice most likely reflect a decreased rate of degradation rather than increased output by these ghrelin secreting glands. The lower expression of GOAT and ghrelin observed in the stomach which reflect a decreased transcriptional output of these genes, may shed light on a fine tuning switch that facilitates this process. 4.6 Regulatory mechanisms and maintenance of blood glucose.  4.6.1 Glycogenolysis and gluconeogenesis To better understand the overall regulation of glucose in my mice, I first looked at glycogenolysis and at gluconeogenesis. Hepatic glycogen content was used as a marker of glycogenolysis and PCK1 expression, a rate-limiting enzyme, was used as a marker of gluconeogenesis. Overexpression of hepatic PCK1 mRNA in mice causes excessive glucose production (and its silencing prevents its diabetogenic effects 127,128. I can see that WT-CR-P mice, that had decrease blood glucose concentrations, had lower hepatic glycogen content and a 2-fold higher mean PCK1 mRNA expression compared to FF counterparts (Fig. 3.23 & 3.26). They also had a decrease in fat and lean mass (Fig 3.1 and 3.4).  Taken together, these data suggest that in WT-CR-P animals, fat and lean mass are likely to provide substrates for gluconeogenesis during CR and that both glycogenolysis and gluconeogenesis are stimulated in order to mitigate the decrease in blood glucose. Looking at the KO-CR-NP mice, which 71  also had significantly lower blood glucose concentrations, there are differences compared to WT-CR-NP: There was no clear decrease in hepatic glycogen content or fat stores but there was a 2-fold increase in PCK1 mRNA expression (Fig. 3.2, 3.24, 3.25 and 3.26). Therefore, one possibility is that the absence of AG in GOAT-KO mice prevents mobilization of fat (lipolysis) and stimulation of glycogenolysis 129,130, thereby contributing to the lower blood glucose.  4.6.2 Growth hormone Zhao et al (2010) investigated the role of GOAT and ghrelin acylation in preventing hypoglycemia associated with CR in GOAT-KO non pregnant mice. They demonstrated that increased GH production by AG mediated this effect.  Looking at my plasma AG and GH concentrations, I saw similar results with a few important differences. WT-CR-NP mice did not have elevated AG (and UAG) but had a markedly high GH compared to FF counterparts. This elevated GH may have been responsible for sustaining euglycemia, in part, by facilitating glycogenolysis, gluconeogenesis and lipolysis 131,132. GH elevation in this group appears to have been independent of a marked rise in AG. A potential reason for this may have been due to the inhibition of AG secretion by the elevated GH 56,133,134. As elevated GH appears to have been associated with sustaining euglycemia in this group, it may have hindered AG production thus preventing further AG mediated GH secretion. In support of this hypothesis, the KO-CR-NP group (similar to the KO-FF-NP group) had undetectable GH further suggesting that the decreased blood glucose concentrations in this group may have resulted from a failed GH response due to complete AG absence. Plasma AG was undetectable in these mice, as expected, but a considerable elevation in UAG was reported (5-fold greater than WT counterpart) suggesting a possible compensatory attempt to raise 72  circulating ghrelin levels. An impairment of the negative feedback mechanism, likely by elevated GH or AG, used to restrict circulating AG may account for this 47,135. Ghrelin production may go unregulated and could explain the substantial increase in circulating UAG.   Although KO-CR-NP mice had low blood glucose and GH compared to WT-CR-NP mice, they did not appear to endure the consequences associated with hypoglycemia reported Zhao et al. (2010). This was also reported by Yi et al. (2012) where GOAT-KO female mice subjected to the same CR regimen for 20 days experienced rare hypoglycemic events suggesting GOAT is not essential for the prevention of prolonged hypoglycemia 136.  Despite the absence of AG, KO-FF-P mice on the other hand had elevated GH (Fig. 3.21). The elevated GH in in these mice resembled that of a WT-FF and WT-CR pregnancy (Fig. 3.22). In humans, during pregnancy pituitary GH is gradually replaced by GH secreted by the placenta known as placental GH. In mice however, the placenta does not produce GH and the pituitary remains the main source of GH 61. Overall this suggests that GH production during pregnancy in GOAT-KO mice is independent of AG. This contrasts with KO-NP mice, where both AG and GH concentrations are barely detectable. Thus, the role of endogenous AG in GH stimulation may differ in P and NP pregnant animals. In essence, my data demonstrate that the GOAT-ghrelin system may be necessary only during substantial energy demand as observed in the CR mice bearing the energy burdening period of pregnancy.  The presence of GOAT and thus AG may help to mitigate these high-risk periods for hypoglycemia such as CR especially during this time.  73  A possible role of AG in the CNS may mirror that of GHRH influencing pituitary GH release. Hypothalamic AG may be released into the hypophyseal portal system directly to the anterior pituitary to stimulate GH release rather than stomach AG via the peripheral circulation. In support of this, AG and UAG have been shown to cross the blood-brain-barrier in both directions (ie. Blood to brain and brain to blood) in mice and humans but the physiological implications of this finding needs to be further characterized 137. Conversely, it has been demonstrated that GHRH released by the hypothalamus has increased pituitary ghrelin mRNA and protein expression and led to GH secretion 138. Furthermore, GHRH and AG synergistically have been shown to potentate pituitary GH release rather than acting alone 138-140. Another relevant study looking at rodent pregnancy showed increased ghrelin mRNA expression in both the hypothalamus and pituitary at Day 18 similar to my mice (Figs. 3.15-3.19) 141. They also reported that GHRH mRNA expression in the hypothalamus increased and GHRH-R mRNA expression decreased at Day 18 in these mice. Interestingly, this was also associated with increased GHSR expression in the pituitary. This suggested that during pregnancy GHRH may not directly influence GH secretion (via. GHRH-R) but by keeping pituitary ghrelin and GHSR mRNA expression elevated 141. In my results I found this to be true in my WT-FF-P, WT-CR-P, WT-CR-NP and KO-CR-NP mice where they had heightened pituitary ghrelin mRNA expression compared to the control WT-FF-NP group (Figs. 3.17-3.19). My data also looked at GOAT expression in the hypothalamus and pituitary (trace expression potentially in the latter) whereby the referenced study did not which may additionally influence the maintenance of elevated ghrelin expression in the pituitary gland during pregnancy or during similar periods of increased energy demand. More studies looking at the regulation of the GOAT-ghrelin system in the hypothalamic-pituitary 74  axis are needed. Altogether,  in contrast to the majority of literature to date that confines their assessment of ghrelin physiology on its regulation by the stomach alone, my study stresses the importance of looking at its regulation in other ghrelin secreting glands, as local stimulation of the GOAT-ghrelin system (in the CNS) rather than in the stomach may accentuate pituitary GH release.  In summary, I demonstrate that CR affects glucose metabolism more severely in WT-P and in KO-NP mice, which cannot produce AG. The additive effect of pregnancy on GOAT-KO mice increases this severity of hypoglycemia when subjected to CR. I propose that in WT animals, an increase in AG and UAG levels, whether due to increased production, decreased degradation, or both, serve to mitigate the decrease in blood glucose. The mechanisms of action of AG remain unclear but may involve GH and stimulation of glycogenolysis and/or gluconeogenesis. My work supports a physiological role for the AG/UAG pathway in the regulation of blood glucose concentrations during pregnancy.  4.7 Strengths, limitations and future directions of the project A major strength of my project was the use of GOAT-KO mice to understand the role of ghrelin acylation in response to CR. With the GOAT gene completely knocked out, it emphasi]es the importance of acylation as being a key step for $G?s prescribed effects. Studies using strictly ghrelin-KO mice would not allow the researcher to assess UAG concentrations. Another strength was the use of full-length EIAs that allow for the determination of AG and UAG concentrations separately. Previous studies have used assays that assessed only total ghrelin (AG + UAG) concentrations. Another strength was the use of the EchoMRI system which allowed for quantitative measurements of fat and lean mass as 75  the mice progressed through the experimental time points. In addition, the use of hepatic glycogen content in addition to PCK1 as markers for glycogenolysis and gluconeogenesis which are key mechanisms for raising glucose during negative energy balance. Finally, My study itself was novel as it provided insights into ghrelin?s role in pregnancy, an area that remains highly uncharacteri]ed. The vast maMority of literature to date focused on ghrelin?s role in non-pregnant animals although changes in ghrelin levels have been widely reported during pregnancy.  A limitation of my project was that the degree of CR (50%) was too severe for the KO-CR-P mice. While it demonstrated the higher rate of severe hypoglycemia, it made it impossible to collect tissues. Another limitation was that the pancreas, which has been reported to be another ghrelin secreting gland, could not be used as RNA was rapidly degraded following sacrifice. Finally, the lack of a Western blot to give an assessment of GOAT and ghrelin expression at the translational level. Although I had antibodies to analyze these proteins, I could not get them to bind effectively.  There are several future directions where I feel would provide further insight into my findings. Firstly, it would be useful to look at other hormones involved in the counter regulation of hypoglycemia such as insulin, corticosterone or glucagon in these mice. In addition, the use of GHSR-KO (ghrelin receptor), GHR-KO (GH receptor), GHRHR-KO and ghrelin-KO mice would help to confirm the endogenous role of AG (and UAG) observed in my mice as acting independently or dependently of GH?s effects. $nother direction would be to analyze the proteases that are responsible for AG and UAG degradation. This would help to confirm our suggestions that a decreased rate of degradation of AG and UAG may be responsible for its elevated concentration in the circulation.  76  Bibliography 1. Date Y, Kojima M, Hosoda H, et al. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology. 2000;141(11):4255-4261. 2. Rindi G, Necchi V, Savio A, et al. Characterisation of gastric ghrelin cells in man and other mammals: Studies in adult and fetal tissues. Histochem Cell Biol. 2002;117(6):511-519. 3. Stengel A, Tache Y. Ghrelin - a pleiotropic hormone secreted from endocrine x/a-like cells of the stomach. Front Neurosci. 2012;6:24. 4. Date Y, Nakazato M, Hashiguchi S, et al. Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion. Diabetes. 2002;51(1):124-129. 5. Walia P, Asadi A, Kieffer TJ, Johnson JD, Chanoine JP. Ontogeny of ghrelin, obestatin, preproghrelin, and prohormone convertases in rat pancreas and stomach. Pediatr Res. 2009;65(1):39-44. 6. Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B, Sussel L. Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci U S A. 2004;101(9):2924-2929. 7. Inui A, Asakawa A, Bowers CY, et al. Ghrelin, appetite, and gastric motility: The emerging role of the stomach as an endocrine organ. FASEB J. 2004;18(3):439-456. 8. Kineman RD, Gahete MD, Luque RM. Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress. J Mol Endocrinol. 2007;38(5):511-521. 9. Korbonits M, Kojima M, Kangawa K, Grossman AB. Presence of ghrelin in normal and adenomatous human pituitary. Endocrine. 2001;14(1):101-104. 10. Mozid AM, Tringali G, Forsling ML, et al. Ghrelin is released from rat hypothalamic explants and stimulates corticotrophin-releasing hormone and arginine-vasopressin. Horm Metab Res. 2003;35(8):455-459. 11. Banks WA, Tschop M, Robinson SM, Heiman ML. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J Pharmacol Exp Ther. 2002;302(2):822-827. 12. Castaneda TR, Tong J, Datta R, Culler M, Tschop MH. Ghrelin in the regulation of body weight and metabolism. Front Neuroendocrinol. 2010;31(1):44-60. 13. Chanoine JP, Wong ACK, Barrios V. Obestatin, acylated and total ghrelin concentrations in the perinatal rat pancreas. Hormone Research in Paediatrics. 2006;66(2):81-88. 14. Zhu X, Cao Y, Voogd K, Steiner DF. On the processing of proghrelin to ghrelin. J Biol Chem. 2006;281(50):38867-38870. 15. Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell. 2008;132(3):387-396. 16. Lim CT, Kola B, Korbonits M. The ghrelin/GOAT/GHS-R system and energy metabolism. Rev Endocr Metab Disord. 2011;12(3):173-186. 77  17. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 1999;402(6762):656-660. 18. Takumi I, Steiner DF, Sanno N, Teramoto A, Osamura RY. Localization of prohormone convertases 1/3 and 2 in the human pituitary gland and pituitary adenomas: Analysis by immunohistochemistry, immunoelectron microscopy, and laser scanning microscopy. Mod Pathol. 1998;11(3):232-238. 19. Espinosa VP, Ferrini M, Shen X, Lutfy K, Nillni EA, Friedman TC. Cellular colocalization and coregulation between hypothalamic pro-TRH and prohormone convertases in hypothyroidism. Am J Physiol Endocrinol Metab. 2007;292(1):E175-86. 20. Barnett BP, Hwang Y, Taylor MS, et al. Glucose and weight control in mice with a designed ghrelin O-acyltransferase inhibitor. Science. 2010;330(6011):1689-1692. 21. Shlimun A, Unniappan S. Ghrelin o-acyl transferase: Bridging ghrelin and energy homeostasis. Int J Pept. 2011;2011:217957. 22. Kang K, Zmuda E, Sleeman MW. Physiological role of ghrelin as revealed by the ghrelin and GOAT knockout mice. Peptides. 2011;32(11):2236-2241. 23. An W, Li Y, Xu G, et al. Modulation of ghrelin O-acyltransferase expression in pancreatic islets. Cell Physiol Biochem. 2010;26(4-5):707-716. 24. Gahete MD, Cordoba-Chacon J, Salvatori R, Castano JP, Kineman RD, Luque RM. Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach. Mol Cell Endocrinol. 2010;317(1-2):154-160. 25. Akamizu T, Takaya K, Irako T, et al. Pharmacokinetics, safety, and endocrine and appetite effects of ghrelin administration in young healthy subjects. Eur J Endocrinol. 2004;150(4):447-455. 26. De Vriese C, Gregoire F, Lema-Kisoka R, Waelbroeck M, Robberecht P, Delporte C. Ghrelin degradation by serum and tissue homogenates: Identification of the cleavage sites. Endocrinology. 2004;145(11):4997-5005. 27. Ni H, Walia P, Chanoine JP. Ontogeny of acylated ghrelin degradation in the rat. Peptides. 2010;31(2):301-306. 28. Granata R, Settanni F, Julien M, et al. Des-acyl ghrelin fragments and analogues promote survival of pancreatic beta-cells and human pancreatic islets and prevent diabetes in streptozotocin-treated rats. J Med Chem. 2012;55(6):2585-2596. 29. Lear PV, Iglesias MJ, Feijoo-Bandin S, et al. Des-acyl ghrelin has specific binding sites and different metabolic effects from ghrelin in cardiomyocytes. Endocrinology. 2010;151(7):3286-3298. 30. Wren A, Small C, Ward H, et al. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology. 2000;141(11):4325-4328. 31. Korbonits M, Jacobs RA, Aylwin SJ, et al. Expression of the growth hormone secretagogue receptor in pituitary adenomas and other neuroendocrine tumors. J Clin Endocrinol Metab. 1998;83(10):3624-3630. 32. Cruz CR, Smith RG. The growth hormone secretagogue receptor. Vitam Horm. 2008;77:47-88. 33. Yamazaki M, Kobayashi H, Tanaka T, Kangawa K, Inoue K, Sakai T. Ghrelin-induced growth hormone release from isolated rat anterior pituitary cells depends on intracellullar and extracellular Ca2+ sources. J Neuroendocrinol. 2004;16(10):825-831. 78  34. Zhao TJ, Liang G, Li RL, et al. Ghrelin O-acyltransferase (GOAT) is essential for growth hormone-mediated survival of calorie-restricted mice. Proc Natl Acad Sci U S A. 2010;107(16):7467-7472. 35. Molitch ME, Clemmons DR, Malozowski S, et al. Evaluation and treatment of adult growth hormone deficiency: An endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2006;91(5):1621-1634. 36. Moller N, Jorgensen JO, Abildgard N, Orskov L, Schmitz O, Christiansen JS. Effects of growth hormone on glucose metabolism. Horm Res. 1991;36 Suppl 1:32-35. 37. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature. 2000;407(6806):908-913. 38. Nakazato M, Murakami N, Date Y, et al. A role for ghrelin in the central regulation of feeding. Nature. 2001;409(6817):194-198. 39. Asakawa A, Inui A, Kaga T, et al. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology. 2001;120(2):337-345. 40. Cummings DE, Weigle DS, Frayo RS, et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med. 2002;346(21):1623-1630. 41. Tong J, Pfluger PT, Tschop MH. Gastric O-acyl transferase activates hunger signal to the brain. Proc Natl Acad Sci U S A. 2008;105(17):6213-6214. 42. Hewson AK, Dickson SL. Systemic administration of ghrelin induces fos and egr-1 proteins in the hypothalamic arcuate nucleus of fasted and fed rats. J Neuroendocrinol. 2000;12(11):1047-1049. 43. King PJ, Williams G, Doods H, Widdowson PS. Effect of a selective neuropeptide Y Y(2) receptor antagonist, BIIE0246 on neuropeptide Y release. Eur J Pharmacol. 2000;396(1):R1-3. 44. Willesen MG, Kristensen P, Romer J. Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Neuroendocrinology. 1999;70(5):306-316. 45. Cummings DE, Shannon MH. Ghrelin and gastric bypass: Is there a hormonal contribution to surgical weight loss? Journal of Clinical Endocrinology & Metabolism. 2003;88(7):2999-3002. 46. Faas MM, Melgert BN, de Vos P. A brief review on how pregnancy and sex hormones interfere with taste and food intake. Chemosens Percept. 2010;3(1):51-56. 47. Yang J, Zhao TJ, Goldstein JL, Brown MS. Inhibition of ghrelin O-acyltransferase (GOAT) by octanoylated pentapeptides. Proc Natl Acad Sci U S A. 2008;105(31):10750-10755. 48. Shiiya T, Nakazato M, Mizuta M, et al. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab. 2002;87(1):240-244. 49. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature. 2000;407(6806):908-913. 50. Broglio F, Arvat E, Benso A, et al. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab. 2001;86(10):5083-5086. 51. Qader SS, Hakanson R, Rehfeld JF, Lundquist I, Salehi A. Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: A 79  study on isolated islets from mouse and rat pancreas. Regul Pept. 2008;146(1-3):230-237. 52. Dezaki K, Sone H, Koizumi M, et al. Blockade of pancreatic islet-derived ghrelin enhances insulin secretion to prevent high-fat diet-induced glucose intolerance. Diabetes. 2006;55(12):3486-3493. 53. Wang Y, Nishi M, Doi A, et al. Ghrelin inhibits insulin secretion through the AMPK-UCP2 pathway in beta cells. FEBS Lett. 2010;584(8):1503-1508. 54. Doi A, Shono T, Nishi M, Furuta H, Sasaki H, Nanjo K. IA-2beta, but not IA-2, is induced by ghrelin and inhibits glucose-stimulated insulin secretion. Proc Natl Acad Sci U S A. 2006;103(4):885-890. 55. Tong J, Prigeon RL, Davis HW, et al. Ghrelin suppresses glucose-stimulated insulin secretion and deteriorates glucose tolerance in healthy humans. Diabetes. 2010;59(9):2145-2151. 56. Nass RM, Gaylinn BD, Rogol AD, Thorner MO. Ghrelin and growth hormone: Story in reverse. Proc Natl Acad Sci U S A. 2010;107(19):8501-8502. 57. Fuglsang J, Skjaerbaek C, Espelund U, et al. Ghrelin and its relationship to growth hormones during normal pregnancy. Clin Endocrinol (Oxf). 2005;62(5):554-559. 58. Lacroix MC, Guibourdenche J, Frendo JL, Muller F, Evain-Brion D. Human placental growth hormone--a review. Placenta. 2002;23 Suppl A:S87-94. 59. Alsat E, Guibourdenche J, Couturier A, Evain-Brion D. Physiological role of human placental growth hormone. Mol Cell Endocrinol. 1998;140(1-2):121-127. 60. Tham E, Liu J, Innis S, et al. Acylated ghrelin concentrations are markedly decreased during pregnancy in mothers with and without gestational diabetes: Relationship with cholinesterase. Am J Physiol Endocrinol Metab. 2009;296(5):E1093-100. 61. Carlsson L, Eden S, Jansson JO. The plasma pattern of growth hormone in conscious rats during late pregnancy. J Endocrinol. 1990;124(2):191-198. 62. Shibata K, Hosoda H, Kojima M, et al. Regulation of ghrelin secretion during pregnancy and lactation in the rat: Possible involvement of hypothalamus. Peptides. 2004;25(2):279-287. 63. Kirchner H, Gutierrez JA, Solenberg PJ, et al. GOAT links dietary lipids with the endocrine control of energy balance. Nat Med. 2009;15(7):741-745. 64. Reimer RA, Maurer AD, Lau DC, Auer RN. Long-term dietary restriction influences plasma ghrelin and GOAT mRNA level in rats. Physiol Behav. 2010;99(5):605-610. 65. Gualillo O, Caminos JE, Nogueiras R, et al. Effect of food restriction on ghrelin in normal-cycling female rats and in pregnancy. Obes Res. 2002;10(7):682-687. 66. Chanoine JP, Wong AC. Ghrelin gene expression is markedly higher in fetal pancreas compared with fetal stomach: Effect of maternal fasting. Endocrinology. 2004;145(8):3813-3820. 67. Arem R. Hypoglycemia associated with renal failure. Endocrinol Metab Clin North Am. 1989;18(1):103-121. 68. Ohlsson A, Shah P, Institute of Health Economics. Determinants and prevention of low birth weight. . 2008; 2009:271. 69. Sheridan C. Intrauterine growth restriction--diagnosis and management. Aust Fam Physician. 2005;34(9):717-723. 70. Bharati P, Pal M, Bandyopadhyay M, Bhakta A, Chakraborty S, Bharati P. Prevalence and causes of low birth weight in india. Malays J Nutr. 2011;17(3):301-313. 80  71. Siega-Riz AM, Adair LS, Hobel CJ. Maternal underweight status and inadequate rate of weight gain during the third trimester of pregnancy increases the risk of preterm delivery. J Nutr. 1996;126(1):146-153. 72. Butte NF, Wong WW, Treuth MS, Ellis KJ, O'Brian Smith E. Energy requirements during pregnancy based on total energy expenditure and energy deposition. Am J Clin Nutr. 2004;79(6):1078-1087. 73. Belkacemi L, Nelson DM, Desai M, Ross MG. Maternal undernutrition influences placental-fetal development. Biol Reprod. 2010;83(3):325-331. 74. Nube M, Van Den Boom GJ. Gender and adult undernutrition in developing countries. Ann Hum Biol. 2003;30(5):520-537. 75. Singh S, Sedgh G, Hussain R. Unintended pregnancy: Worldwide levels, trends, and outcomes. Stud Fam Plann. 2010;41(4):241-250. 76. Zhang JX, Liu YJ, Zhang JH, Sun L. Dual role of preputial gland secretion and its major components in sex recognition of mice. Physiol Behav. 2008;95(3):388-394. 77. Ginsberg BH. Factors affecting blood glucose monitoring: Sources of errors in measurement. J Diabetes Sci Technol. 2009;3(4):903-913. 78. Taicher GZ, Tinsley FC, Reiderman A, Heiman ML. Quantitative magnetic resonance (QMR) method for bone and whole-body-composition analysis. Anal Bioanal Chem. 2003;377(6):990-1002. 79. Krasnow SM, Nguyen ML, Marks DL. Increased maternal fat consumption during pregnancy alters body composition in neonatal mice. Am J Physiol Endocrinol Metab. 2011;301(6):E1243-53. 80. Eger EI,2nd. The pharmacology of isoflurane. Br J Anaesth. 1984;56 Suppl 1:71S-99S. 81. Johnson CB, Taylor PM. Comparison of the effects of halothane, isoflurane and methoxyflurane on the electroencephalogram of the horse. Br J Anaesth. 1998;81(5):748-753. 82. Dohoo SE. Isoflurane as an inhalational anesthetic agent in clinical practice. Can Vet J. 1990;31(12):847-850. 83. Barnard EA. Biological function of pancreatic ribonuclease. Nature. 1969;221(5178):340-344. 84. Trivedi A, Babic S, Chanoine JP. Pitfalls in the determination of human acylated ghrelin plasma concentrations using a double antibody enzyme immunometric assay. Clin Biochem. 2012;45(1-2):178-180. 85. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6(10):986-994. 86. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101-1108. 87. Li Z, Yang L, Wang J, et al. Beta-actin is a useful internal control for tissue-specific gene expression studies using quantitative real-time PCR in the half-smooth tongue sole cynoglossus semilaevis challenged with LPS or vibrio anguillarum. Fish Shellfish Immunol. 2010;29(1):89-93. 88. Mottershead M, Karteris E, Barclay JY, et al. Immunohistochemical and quantitative mRNA assessment of ghrelin expression in gastric and oesophageal adenocarcinoma. J Clin Pathol. 2007;60(4):405-409. 81  89. Wasserman DH, Spalding JA, Lacy DB, Colburn CA, Goldstein RE, Cherrington AD. Glucagon is a primary controller of hepatic glycogenolysis and gluconeogenesis during muscular work. Am J Physiol. 1989;257(1 Pt 1):E108-17. 90. Pilkis SJ, Granner DK. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Physiol. 1992;54:885-909. 91. Holck P, Rasch R. Structure and segmental localization of glycogen in the diabetic rat kidney. Diabetes. 1993;42(6):891-900. 92. Coyle EF, Coggan AR, Hemmert MK, Ivy JL. Muscle glycogen utilization during prolonged strenuous exercise when fed carbohydrate. J Appl Physiol. 1986;61(1):165-172. 93. Le Lay J, Kaestner KH. The fox genes in the liver: From organogenesis to functional integration. Physiol Rev. 2010;90(1):1-22. 94. Chuang JC, Sakata I, Kohno D, et al. Ghrelin directly stimulates glucagon secretion from pancreatic alpha-cells. Mol Endocrinol. 2011;25(9):1600-1611. 95. Bazotte RB, Constantin J, Hell NS, Iwamoto EL, Bracht A. The relation between inhibition of glycolysis and stimulation of oxygen uptake due to glucagon in livers from rats in different metabolic conditions. Cell Biochem Funct. 1988;6(4):225-230. 96. Reimer MK, Pacini G, Ahren B. Dose-dependent inhibition by ghrelin of insulin secretion in the mouse. Endocrinology. 2003;144(3):916-921. 97. Goldstein JL, Zhao TJ, Li RL, Sherbet DP, Liang G, Brown MS. Surviving starvation: Essential role of the ghrelin-growth hormone axis. Cold Spring Harb Symp Quant Biol. 2011;76:121-127. 98. Passonneau JV, Lauderdale VR. A comparison of three methods of glycogen measurement in tissues. Anal Biochem. 1974;60(2):405-412. 99. Holyoak T, Sullivan SM, Nowak T. Structural insights into the mechanism of PEPCK catalysis. Biochemistry. 2006;45(27):8254-8263. 100. Christ B. Inhibition of glucagon-signaling and downstream actions by interleukin 1beta and tumor necrosis factor alpha in cultured primary rat hepatocytes. Horm Metab Res. 2008;40(1):18-23. 101. Barreiro ML, Gaytan F, Caminos JE, et al. Cellular location and hormonal regulation of ghrelin expression in rat testis. Biol Reprod. 2002;67(6):1768-1776. 102. Tanaka M, Hayashida Y, Iguchi T, Nakao N, Nakai N, Nakashima K. Organization of the mouse ghrelin gene and promoter: Occurrence of a short noncoding first exon. Endocrinology. 2001;142(8):3697-3700. 103. Tena-Sempere M, Barreiro ML, Gonzalez LC, et al. Novel expression and functional role of ghrelin in rat testis. Endocrinology. 2002;143(2):717-725. 104. Barreiro ML, Suominen JS, Gaytan F, et al. Developmental, stage-specific, and hormonally regulated expression of growth hormone secretagogue receptor messenger RNA in rat testis. Biol Reprod. 2003;68(5):1631-1640. 105. Caminos JE, Tena-Sempere M, Gaytan F, et al. Expression of ghrelin in the cyclic and pregnant rat ovary. Endocrinology. 2003;144(4):1594-1602. 106. Garcia MC, Lopez M, Alvarez CV, Casanueva F, Tena-Sempere M, Dieguez C. Role of ghrelin in reproduction. Reproduction. 2007;133(3):531-540. 107. Duggal PS, Weitsman SR, Magoffin DA, Norman RJ. Expression of the long (OB-RB) and short (OB-RA) forms of the leptin receptor throughout the oestrous cycle in the mature rat ovary. Reproduction. 2002;123(6):899-905. 82  108. Martin JR, Lieber SB, McGrath J, Shanabrough M, Horvath TL, Taylor HS. Maternal ghrelin deficiency compromises reproduction in female progeny through altered uterine developmental programming. Endocrinology. 2011;152(5):2060-2066. 109. Forbes TR, Taku E. Vein size in intact and hysterectomized mice during the estrous cycle and pregnancy. Anat Rec. 1975;182(1):61-65. 110. Siega-Riz AM, Adair LS, Hobel CJ. Maternal underweight status and inadequate rate of weight gain during the third trimester of pregnancy increases the risk of preterm delivery. J Nutr. 1996;126(1):146-153. 111. Sun Y, Ahmed S, Smith RG. Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol. 2003;23(22):7973-7981. 112. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature. 2000;407(6806):908-913. 113. Perez-Tilve D, Heppner K, Kirchner H, et al. Ghrelin-induced adiposity is independent of orexigenic effects. FASEB J. 2011;25(8):2814-2822. 114. Stryer L, Berg JM, Tymoczko JL, NCBI Bookshelf. Biochemistry. . 2002. 115. Varela L, Vazquez MJ, Cordido F, et al. Ghrelin and lipid metabolism: Key partners in energy balance. J Mol Endocrinol. 2011;46(2):R43-63. 116. Vestergaard ET, Djurhuus CB, Gjedsted J, et al. Acute effects of ghrelin administration on glucose and lipid metabolism. J Clin Endocrinol Metab. 2008;93(2):438-444. 117. Vestergaard ET, Gormsen LC, Jessen N, et al. Ghrelin infusion in humans induces acute insulin resistance and lipolysis independent of growth hormone signaling. Diabetes. 2008;57(12):3205-3210. 118. Thompson NM, Gill DA, Davies R, et al. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology. 2004;145(1):234-242. 119. Ariyasu H, Takaya K, Tagami T, et al. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab. 2001;86(10):4753-4758. 120. Date Y, Nakazato M, Hashiguchi S, et al. Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion. Diabetes. 2002;51(1):124-129. 121. Nass R, Liu J, Hellmann P, et al. Chronic changes in peripheral growth hormone levels do not affect ghrelin stomach mRNA expression and serum ghrelin levels in three transgenic mouse models. J Neuroendocrinol. 2004;16(8):669-675. 122. Davis JF, Perello M, Choi DL, et al. GOAT induced ghrelin acylation regulates hedonic feeding. Horm Behav. 2012;62(5):598-604. 123. Briggs DI, Enriori PJ, Lemus MB, Cowley MA, Andrews ZB. Diet-induced obesity causes ghrelin resistance in arcuate NPY/AgRP neurons. Endocrinology. 2010;151(10):4745-4755. 124. Cowley MA, Smith RG, Diano S, et al. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron. 2003;37(4):649-661. 125. Gahete MD, Cordoba-Chacon J, Salvatori R, Castano JP, Kineman RD, Luque RM. Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach. Mol Cell Endocrinol. 2010;317(1-2):154-160. 126. Kang K, Schmahl J, Lee JM, et al. Mouse ghrelin-O-acyltransferase (GOAT) plays a critical role in bile acid reabsorption. FASEB J. 2012;26(1):259-271. 83  127. Gomez-Valades AG, Mendez-Lucas A, Vidal-Alabro A, et al. Pck1 gene silencing in the liver improves glycemia control, insulin sensitivity, and dyslipidemia in db/db mice. Diabetes. 2008;57(8):2199-2210. 128. Beale EG, Harvey BJ, Forest C. PCK1 and PCK2 as candidate diabetes and obesity genes. Cell Biochem Biophys. 2007;48(2-3):89-95. 129. St-Pierre DH, Benso A, Gramaglia E, et al. The metabolic response to the activation of the beta-adrenergic receptor by salbutamol is amplified by acylated ghrelin. J Endocrinol Invest. 2010;33(6):363-367. 130. Lattuada D, Crotta K, Tonna N, et al. The expression of GHS-R in primary neurons is dependent upon maturation stage and regional localization. PLoS One. 2013;8(6):e64183. 131. Vidal H, Geloen A, Minaire Y, Riou JP. Effect of growth hormone deficiency on hormonal control of hepatic glycogenolysis in hypophysectomized rat. Metabolism. 1993;42(5):631-637. 132. Fielder PJ, Talamantes F. The lipolytic effects of mouse placental lactogen II, mouse prolactin, and mouse growth hormone on adipose tissue from virgin and pregnant mice. Endocrinology. 1987;121(2):493-497. 133. Seoane LM, Al-Massadi O, Barreiro F, Dieguez C, Casanueva FF. Growth hormone and somatostatin directly inhibit gastric ghrelin secretion. an in vitro organ culture system. J Endocrinol Invest. 2007;30(9):RC22-5. 134. Grey CL, Chang JP. Growth hormone-releasing hormone stimulates GH release while inhibiting ghrelin- and sGnRH-induced LH release from goldfish pituitary cells. Gen Comp Endocrinol. 2013;186:150-156. 135. Yin X, Li Y, Xu G, An W, Zhang W. Ghrelin fluctuation, what determines its production? Acta Biochim Biophys Sin (Shanghai). 2009;41(3):188-197. 136. Yi CX, Heppner KM, Kirchner H, et al. The GOAT-ghrelin system is not essential for hypoglycemia prevention during prolonged calorie restriction. PLoS One. 2012;7(2):e32100. 137. Banks WA, Tschop M, Robinson SM, Heiman ML. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J Pharmacol Exp Ther. 2002;302(2):822-827. 138. Kamegai J, Tamura H, Shimizu T, et al. The role of pituitary ghrelin in growth hormone (GH) secretion: GH-releasing hormone-dependent regulation of pituitary ghrelin gene expression and peptide content. Endocrinology. 2004;145(8):3731-3738. 139. Hataya Y, Akamizu T, Takaya K, et al. A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans. J Clin Endocrinol Metab. 2001;86(9):4552. 140. Nass R, Toogood AA, Hellmann P, et al. Intracerebroventricular administration of the rat growth hormone (GH) receptor antagonist G118R stimulates GH secretion: Evidence for the existence of short loop negative feedback of GH. J Neuroendocrinol. 2000;12(12):1194-1199. 141. Szczepankiewicz D, Skrzypski M, Pruszynska-Oszmalek E, et al. Importance of ghrelin in hypothalamus-pituitary axis on growth hormone release during normal pregnancy in the rat. J Physiol Pharmacol. 2010;61(4):443-449.  

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