However, while possible, we don not believe this was a signficiant factor since the ...... Maclaughlin SM, Walker SK, Roberts CT, Kleemann DO, McMillen IC.
GENE-ENVIRONMENT INTERACTIONS UNDERLYING THE DEVELOPMENTAL ORIGINS OF HEALTH AND DISEASE
By
Brian S. Knight
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology University of Toronto
© Copyright by Brian S. Knight 2008
Gene-Environment Interactions Underlying the Developmental Origins of Health and Disease Brian S. Knight 2008 Doctor of Philosophy Department of Physiology University of Toronto
ABSTRACT: Retrospective epidemiological studies of British cohorts have found an inverse relationship between birth size and rates of mortality from cardiovascular disease and stroke. Subsequently, further studies in humans and in animals have demonstrated that there is an inverse relationship with a combination of suboptimal prenatal and postnatal environments and the development of the metabolic syndrome (insulin resistance, hypertension, obesity and dyslipidemia). However, recently it has been reported that not all individuals exposed to these environments develop these conditions, suggesting that an individual’s genotype may contribute to the eventual outcome. Phylogenetically distinct, murine strains allow the genetic dissection of complex phenotypic traits; however, to date, they have not been utilized to evaluate the geneenvironment interaction underlying these inverse relationships. Thus, A/J and C57BL/6J mice were subjected to prenatal undernutrition, to model an adverse intra-uterine environment, and although prenatal undernutrition resulted in fetal growth restriction of equal magnitude, remarkable strain differences were observed. At the end of gestation C57BL/6J mice showed significant alterations in fetal organ weights (liver, kidneys and placenta) and glucocorticoids (elevation). Postnatally, C57BL/6J offspring demonstrated catch-up growth, obesity, impaired glucose tolerance, insulin resistance, increased blood pressure, liver dysfunction and altered cardiovascular function compared to strain and gender matched controls. The A/J strain was resistant to the development of prenatal and postnatal pathologies, except they also demonstrated alterations in cardiovascular function. Females of both strains displayed a more moderate phenotype than the males. Although feeding undernutrition mice an atherogenic diet postnatally did not exacerbate the phenotype, postnatal dietary supplementation of omega-3 long chain
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unsaturated fatty acids completely reversed the undernutrition induced altered metabolic phenotype in C57BL/6J. Microarray analysis revealed that adult A/J and C57BL/6J mice have distinct gene expression profiles, under control dietary conditions, and that this differential strain expression profile changes in adult offspring in response to prenatal undernutrition. The expression profiles predicted onset of metabolic and liver diseases within the C57BL/6J strain, clearly linking the observed phenotype to alterations in gene expression. These expression differences were also linked to inherent strain differences in the genetic code where a disproportionate number of differential expressed genes had function altering polymorphisms.
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ACKNOWLEDGEMENTS
I would like to thank my supervisor, Dr Stephen Lye, for his guidance, strong encouragement and consistent regard for my well being both in the laboratory and the Research Institute as a whole. I would also like to thank him for the freedom he had given me in the development and the completion of my project, as well as the support for my career aspirations. I would also like to express my appreciation of my advisory committee, Dr S. Lee Adamson, Dr Janet Rossant and Dr Craig Pennell for their criticism and for pushing me in new directions, allowing for a greater learning experience. I would like to thank all the members of the Lye Lab for their support and advice in the lab. In particular, to my editor-in-chief, Julie Wright, who tirelessly read and edited this document. Finally, I would like to thank Dr Craig Pennell, for reason too many to list in entirety.
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CONTRIBUTIONS The following persons and organizations contributed to results presented in this thesis:
Chapter 2 11βHSD2 western blot analysis was performed by Reema Shah (Summer-Student, Lye Lab, Samuel Lunenfeld Research Institute).
Chapter 4 The electrocardiography procedure was performed by Nana Sunn (former postdoctoral fellow, Samuel Lunenfeld Research Institute).
Chapter 5 Alexandra Oldenhof (former technician, Samuel Lunenfeld Research Institute) assisted with some of the animal husbandry, adult tissue dissections and glucose tolerance testing of the mice used in the glucose/insulin trial.
Chapter 6 The Centre for Applied Genomics performed RNA Agilent analysis, RNA reverse transcription, cRNA labeling, microarray hybridization, and microarray scanning and image quantification.
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TABLE OF CONTENTS GENE-ENVIRONMENT INTERACTIONS UNDERLYING THE DEVELOPMENTAL ORIGINS OF HEALTH AND DISEASE ...................................................................... II ACKNOWLEDGEMENTS ........................................................................................... IV CONTRIBUTIONS.......................................................................................................... V LIST OF TABLES ........................................................................................................... X LIST OF FIGURES ........................................................................................................ XI LIST OF APPENDICES ............................................................................................. XIV CHAPTER 1 LITERATURE REVIEW ......................................................................... 1 1.1 ORIGINS OF THE DEVELOPMENTAL ORIGIN OF HEALTH AND DISEASE HYPOTHESIS................................................................................................... 2 1.2
DEVELOPMENT OF CARDIOVASCULAR DISEASE AND HYPERTENSION 4 1.2.1 DOHaD, Cardiovascular Disease and Hypertension................................... 4 1.2.2 Role of Circulatory System in DOHaD....................................................... 8 1.2.3 Relationship between Cardiovascular Disorders and the Metabolic Axis .. 9
1.3
DEVELOPMENT OF DIABETES AND THE METABOLIC SYNDROME .... 10 1.3.1 DOHaD, Diabetes and Metabolic Syndrome ............................................ 11 1.3.2 Induction of the Metabolic Syndrome ....................................................... 13 1.3.3 Role of Organ Developmental Alterations in the Metabolic Syndrome ... 15
1.4
MECHANISMS OF DISEASE ............................................................................ 20 1.4.1 Hypothalamic-Pituitary-Adrenal Axis ...................................................... 20 1.4.2 Prenatal vs. Postnatal................................................................................. 24 1.4.3 Males vs. Females ..................................................................................... 27
1.5
ROLE OF EVOLUTION IN THE PROCESSES UNDERLYING DOHAD ...... 29
1.6
GENETIC BASIS OF DISEASE.......................................................................... 30 1.6.1 Genomic .................................................................................................... 30 vi
1.6.2 Epigenomic................................................................................................ 34 1.7 ADVANTAGES OF USING MICE TO UNDERSTAND GENEENVIRONMENT INTERACTIONS ............................................................................... 36 1.8
RATIONALE ........................................................................................................ 39
1.9
HYPOTHESIS ...................................................................................................... 40
1.10
OBJECTIVES ....................................................................................................... 41
CHAPTER 2 STRAIN DIFFERENCES IN THE IMPACT OF DIETARY RESTRICTION ON FETAL GROWTH AND PREGNANCY IN MICE ............................................. 43 2.1
INTRODUCTION ................................................................................................ 44
2.2
METHODS ........................................................................................................... 46 2.2.1 Animal Protocols ....................................................................................... 46 2.2.2 Tissue Collection ....................................................................................... 47 2.2.3 Hormonal Assays ...................................................................................... 47 2.2.4 Western Blot Analysis ............................................................................... 47 2.2.5 Statistical Analysis .................................................................................... 48
2.3
RESULTS ............................................................................................................. 49
2.4
DISCUSSION ....................................................................................................... 51
CHAPTER 3 THE IMPACT OF MURINE STRAIN AND GENDER ON POSTNATAL DEVELOPMENT AFTER MATERNAL DIETARY RESTRICTION DURING PREGNANCY ................................................................................................................. 62 3.1
INTRODUCTION ................................................................................................ 63
3.2
METHOD ............................................................................................................. 64 3.2.1 Animals ..................................................................................................... 64 3.2.2 Growth and Body Composition................................................................. 65 3.2.3 Glucose Tolerance ..................................................................................... 65 3.2.4 Statistical Analysis .................................................................................... 66
3.3
RESULTS ............................................................................................................. 66
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3.4
DISCUSSION ....................................................................................................... 69
CHAPTER 4 DEVELOPMENTAL REGULATION OF CARDIOVASCULAR FUNCTION IS DEPENDENT ON GENETIC BACKGROUND AND ENVIRONMENT ........................................................................................................................................... 77 4.1
INTRODUCTION ................................................................................................ 78
4.2
METHODS ........................................................................................................... 79
4.3
RESULTS ............................................................................................................. 80
4.4
DISCUSSION ....................................................................................................... 81
CHAPTER 5 INDUCTION OF THE METABOLIC SYNDROME WITH PRENATAL NUTRIENT RESTRICTION AND ITS REVERSAL WITH AN OMEGA-3 FATTY ACID SUPPLEMENTATION IN MICE ................................................................................. 89 5.1
INTRODUCTION ................................................................................................ 90
5.2
METHOD ............................................................................................................. 91 5.2.1 Animal Protocols ....................................................................................... 91 5.2.2 Glucose Handling ...................................................................................... 92 5.2.3 Liver Function and morphology ................................................................ 92 5.2.4 Statistical Analysis .................................................................................... 92
5.3
RESULTS & DISCUSSION ................................................................................ 93 5.3.1 Glucose Insulin Phenotype ........................................................................ 93 5.3.2 Liver Phenotype ........................................................................................ 94 5.3.3 Effects of Omega Fatty-3 Acids in B6 ...................................................... 96 5.3.4 Conclusions ............................................................................................... 98
CHAPTER 6 INHERENT STRAIN DIFFERENCES IN GENETIC BACKGROUND MAY ACCOUNT FOR THE PRENATALLY INDUCED DIFFERENTIAL METABOLIC SYNDROME PHENOTYPE AND GENE EXPRESSION BETWEEN A/J AND C57BL/6J MICE.............................................................................................................................. 108 6.1
INTRODUCTION .............................................................................................. 109
6.2
METHOD ........................................................................................................... 110
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6.2.1 Animal Protocols ..................................................................................... 110 6.2.2 Proportional variability analysis of the phenotype .................................. 110 6.2.3 Microarray analysis for identifying differentially expressed genes ........ 111 6.2.4 In Silico Genome and Pathway Analysis ................................................ 112 6.2.5 Real-time RT-PCR Analysis ................................................................... 113 6.3
RESULTS & DISCUSSION .............................................................................. 113 6.3.1 Proportional variability modeling of gene-environment contributions to murine phenotype................................................................................................ 113 6.3.2 Differential Genome-Wide Expression Profile ....................................... 114 6.3.3 Confirmation of Array Results by real-time RT-PCR ............................ 116 6.3.4 Pathway and Ontology Analysis ............................................................. 117 6.2.5 Inherit Strain Genomic Differences and Relation to Expression ............ 119 6.2.6 Conclusions ............................................................................................. 121
FINAL CONCLUSION ................................................................................................ 154 APPENDIX .................................................................................................................... 159 Fetal alterations in gluconeogensis precede adult onset of the metabolic syndrome. ... 159 REFERENCES.............................................................................................................. 170
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LIST OF TABLES Table 2-1. Comparison of the effect of dietary restriction on fetal growth Table 3-1. Body composition measured by PIXImus densitometry at 77 and 182 days of age Table 4-1. Hemodynamics after postnatal control and high fat diets: Ascending aortic peak blood velocity and aortic diameter Table 5-1. Fatty Acid profile of n-3 diet Table 6-1. Primers used for RT-PCR analyses Table 6-2. R2 analysis of metabolic outcomes Table 6-3. R2 analysis of cardiovascular outcomes Table 6-4. The 239 known genes with strain differences in expression Table 6-5. The 64 known genes that are important in the restriction differential phenotype Table 6-6. Summary of SNPs and predictive functional analysis for key genes involved in the restriction phenotype
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LIST OF FIGURES Figure 1-1. Summary of the developmental origins of health and disease Figure 2-1. Food consumption during pregnancy and maternal weight Figure 2-2. Pregnancy outcomes from spontaneous delivery Figure 2-3. Comparison of the effect of dietary restriction on fetal growth Figure 2-4. Serum estradiol, progesterone, cortisol, and corticosterone levels on day 18.5 of pregnancy Figure 2-5. The relative optical density of 32kD placental 11β-HSD protein expression in day 18.5 mouse placenta Figure 3-1. Growth trajectories of offspring from 2 days postpartum until 182 days of age measured in body weights Figure 3-2. A comparison of the percent change in fat and lean body mass Figure 3-3. Glucose tolerance, represented as area under the curve, measured at 80 and 186 days of age Figure 4-1. Arterial pressure and heart rate at 9 and 25 weeks of age Figure 4-2. The ratio of the peak velocity of E-wave to the peak velocity of A-wave and Fractional Shortening, as measured by an echocardiographic exam at 26 weeks of age Figure 4-3. The Tei Index, calculated from the 26 week echocardiographic exam using ICT, IRT and ET measurements Figure 5-1. Serum glucose and insulin concentrations, post glucose loading in 12 week old mice Figure 5-2. Serum glucose and insulin concentrations, post glucose loading in 26 week old mice Figure 5-3. Serum levels of total protein, albumin, and globulin in 26 week old mice Figure 5-4. Serum ALT and AST levels in 26 week old mice Figure 5-5. HE and Nile blue staining of 26 week old mice livers Figure 5-6. Serum cholesterol and triglyceride levels in 26 week old male and female mice Figure 5-7. Serum glucose and insulin concentrations, post glucose loading in 11 week old males, fed either control or n-3 enriched postnatal diets Figure 5-8. Weight of 11 week old males fed either control or n-3 enriched postnatal diets Figure 6-1. Hierarchical clustering of microarray transcript expression based on all 32 samples
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Figure 6-2. Hierarchical clustering of microarray transcript expression based on the 16 samples in AJ Figure 6-3. Hierarchical clustering of microarray transcript expression based on the 16 samples in B6 Figure 6-4. Venn diagram of the overlap in transcript expression between the four supervised statistical comparisons Figure 6-5. Comparison of array data to RT-PCR data Figure 6-6. Lipid metabolism and small molecule biochemistry IPA pathway from between strain control comparison of gene expression differences Figure 6-7. Cellular development IPA pathway from between strain control comparison of gene expression differences Figure 6-8. Cellular growth and proliferation IPA pathway from between strain control comparison of gene expression differences Figure 6-9. Lipid metabolism and small molecule biochemistry IPA pathway from between strain restricted comparison of gene expression differences Figure 6-10. Organ development and morphology IPA pathway from between strain restricted comparison of gene expression differences Figure 6-11. Cell death IPA pathway from between strain restricted comparison of gene expression differences Figure 6-12. Key genes involved in the restriction phenotype IPA pathway on gene expression Figure 6-13. Key genes involved in the restriction phenotype IPA pathway on cellular growth and proliferation, metabolic disease Appendix Figure 1. Gluconeogenesis and glycolysis pathways Appendix Figure 2. Phosphoenolpyruvate Carboxykinase mRNA and protein expression in the fetal liver Appendix Figure 3. Glucose-6-phosphatase and Pyruvate Carboxylase mRNA expression in the fetal liver Appendix Figure 4. Fetal blood glucose concentration Appendix Figure 5. Insulin Receptor Substrate 1 mRNA and protein expression in the fetal liver Appendix Figure 6. Glucocorticoid Receptor mRNA and protein expression in the fetal liver
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Appendix Figure 7. Peroxisome proliferator-activated receptor-γ mRNA, protein, and its coactivator expression in the fetal liver
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LIST OF APPENDICES Appendix. Fetal alterations in gluconeogensis precede adult onset of the metabolic syndrome.
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CHAPTER 1 LITERATURE REVIEW
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1.1
ORIGINS OF THE DEVELOPMENTAL ORIGIN OF HEALTH AND DISEASE HYPOTHESIS The formation of the Developmental Origins of Health and Disease (DOHaD) hypothesis
has been primarily attributed to the early work of David Barker and colleagues who, during the latter half of 20th century, postulated that events in utero which reduce fetal growth permanently alter both the physiology and structure of the offspring to the extent that their risk of cardiovascular disease and diabetes will be increased later in life. Barker et al. conducted retrospective epidemiological studies of British cohorts and reported an inverse relationship between birth size and rates of mortality from cardiovascular disease and stroke across the normal birthweight range [1, 2]. In these cohorts, which included over 15,000 men and women born between 1911-1930 in Hertfordshire England, there were a disproportionate number of deaths in people who had been born with low birthweight. Later an association with poor weight gain in infancy and coronary heart disease in adult life was also reported [1]. Similar relationships were discovered for other aspects of the metabolic syndrome including hypertension, stroke, insulin resistance, type 2 diabetes and dyslipidemia [3]. Consistent with these early epidemiological observations from Hertfordshire, studies of a cohort of men born between 1924 and 1933 in Helsinki, Finland for whom serial measurements of height and weight from birth to age 12 years were recorded in obstetric, child welfare, and school health records established that death from coronary heart disease was associated with a low ponderal index (birth weight/length3), and to a lesser extent with low birth weight. Following the collection of additional measurements, further analysis of the same cohort demonstrated that low height, weight, and body mass index (weight/height2) at age 1 year and rapid gain in weight and body mass index, after age 1 year, also increased the risk of coronary heart disease in this cohort [4]. In contrast to men in the same Helsinki cohort, women born short (but not thin) who underwent compensatory growth in height during infancy and became thin, exhibited rapid increases in weight and body mass index later in childhood [5]. These findings suggest that women are less vulnerable to undernutrition in utero and are better able to sustain postnatal growth in an adverse environment, as evidence by decreased weight fluctuations [5]. Nevertheless, it appears that even though the growth patterns differ for boys and girls, the end result is the same with alterations in normal growth trajectory increasing the risk of coronary artery disease. One concern with these studies is that its participants were of low socioeconomic
2
status, with poor living conditions; it is thus possible that this introduced some potential bias in the study. Subsequently, numerous epidemiological studies have been performed across different populations, both in the developed and developing world, confirming these relationships [6]. Among the 70,297 women in the American Nurses Study, there was a decline in the relative risk of nonfatal coronary heart disease and stroke across the range of birth weights [7, 8]. Similarly, a study of 22,000 American men, adults who were born weighing less than 5.5 lb had increased relative risks of adult hypertension and type 2 diabetes compared with adults of average birth weight [9]. In 517 men and women in Mysore, South India, low maternal body weight during pregnancy, low birthweight and short length at birth were associated with a raised prevalence of cardiovascular disease [10]. Epidemiological studies are very useful for identifying associations with pathology, however they lack the power to conclusively determine the precise mechanistic relationships that underlie the pathology; questions of this sort are best addressed through experimental studies. Nevertheless, there is an exception to this typical limitation of epidemiology. During World War II, a ban on all food transport to the Netherlands resulted in a famine (1944-1945) where allotted rations had declined from 2000 calories a day to, at a low point, 400 to 800 calories. Since women still conceived and delivered children during this time period, a unique opportunity presented itself for the study of the effects of maternal malnutrition during discreet periods of gestation; indeed today more than 2000 babies born during that time period have reached adulthood. The studies of these survivors suggested that the effects of undernutrition on later adult health depend on its timing during gestation vis-à-vis the organs and systems which develop during the effected window of time [11]. Thus in the Dutch famine, women who ate 65) who have a previous family history (genetic heredity) of the disease [15]. Previously it was believed that adult lifestyle was the primary reason for increased disease risk, with excessive drinking, tobacco smoking and physical inactivity each independently contributing to a doubling in the risk of developing heart disease [15]. However, based on the epidemiological associations supplied by Barker and others, the importance of early life events in the development cardiovascular disease warrants focused consideration and research. 1.2.1 DOHaD, Cardiovascular Disease and Hypertension The early epidemiological studies were critical for establishing the central DOHaD principles, namely that alterations in birthweight and growth early in life is associated with the 4
development of cardiovascular disease in adulthood. A systematic review of the 440,000 men and women from 80 studies by Huxley et al [16] found that both birth weight and head circumference at birth were inversely related to systolic pressure and that accelerated postnatal growth was also associated with raised blood pressure in adulthood. Other studies established another important relationship between fetal growth restriction and placental size, which is inversely related to fetal size [17] and positively related to adult hypertension [18] even though placental size gives only an indirect measure of placental function. Alterations in placental growth, vascular resistance and subsequent nutrient transmission to the fetus have also been associated with the development of cardiovascular disease later in life [19]. Thus, the placental role in regulating nutrient availability to the fetus may deserve careful consideration during nutrient restriction. These observations reveal a robust relationship between the events that alter prenatal and early postnatal growth, and the development of hypertension later in life. Animal models have been successfully used to replicate the epidemiological observations of DOHaD in human studies, but they do so in very different ways. Early examination of the effects of fetal nutrient restriction on postnatal health in animals utilized pregnant guinea pigs in which the ligation of one uterine horn induced severe fetal growth restriction and relative hypertension in the restricted pups [20]. Subsequently, using a rat model, Langley and Jackson demonstrated that feeding rats a low protein diet (60–120g protein/kg diet) during pregnancy also raised systolic blood pressure in the offspring post weaning [21]. The magnitude of the effect of the low-protein diet was greatest when it was consumed during the final week of gestation [22]. These findings parallel those of pregnancies during the Dutch famine where elevated blood pressure was observed in adults who had been exposed to nutrient restriction during late gestation [11]. It has been theorized that the specific nutrient balance in protein undernutrition may be the critical factor that determines the cardiovascular outcome in the offspring. Indeed, glycine supplementation of a low protein diet prevents the induction of increased postnatal systolic blood pressure [23] while methionine supplementation further impairs blood pressure [24]. Thus, specific composition of the maternal diet during pregnancy (and also likely during the early peri-implantation period) can have lasting effects on the health outcome of her offspring. Figure 1-1 summarizes the potential mechanism of how early life alterations lead to the development of cardiovascular disease.
5
The timing and precise nature of nutritional deprivation can also dramatically affect the health status of affected fetuses in adulthood. Nutritional deprivation in the pregnant rat leads to delayed catch-up growth and significantly elevated systolic blood pressure at 30, 48, and 56 weeks of age [25]. However, a balanced reduction (a 50–70% decrease) in maternal nutrient intake produces less consistent effects in both rats and pigs [26]. In sheep, severe undernutrition for either 10 or 20 days from 105 days gestation (term ~147 days gestation), results in elevation of blood pressure, an effect that decreases with increasing birth weight [27]. The offspring born following a period of undernutrition (15% reduction) during the first 70 days of pregnancy also have higher arterial blood pressure in postnatal life [28]. When nutrient restriction (50%) occurs between gestational days 1 to 30, offspring demonstrate increased pulse pressure and a reduced rate pressure product in adult life [29]. Thus animal models have been manipulated to demonstrate that exposure to a period of maternal nutrient restriction during discreet stages of gestation results in the programming of high blood pressure in their offspring later in life. At present, there is mounting evidence that an adverse intrauterine environment can influence the development of the kidney and consequently will have lasting effects on the regulation of blood pressure. The number of nephrons, the filtering unit of the kidney, is determined prior to birth in many species, including rats, humans, and sheep [30]. As the timing of prenatal insults that induce postnatal hypertension correlate to crucial periods in metanephric development, it is not surprising that certain prenatal insults can result in reduced nephron number eventually leading to sodium retention [30]. In rats, bilateral uterine ligation reduced glomeruli number in full-term fetal kidneys [31]. Moreover in piglets, uteroplacental insufficiency has been demonstrated to reduce both nephron number and the glomeruli filtration rate by up to 30% [32]. Similarly, prenatal protein restriction produces deficits in the numbers of nephrons and glomeruli, and in the glomeruli filtration rate (per kidney weight) in early postnatal and adult life [33]. Furthermore, an increase in plasma sodium concentration has been observed after prenatal protein restriction, which may be the consequence of a primary sodium-retaining state resulting from a rightward shift in the pressure-natriuresis curve [34]. Thus in pigs, impaired fetal growth results in decreased renal function. Similarly in sheep where nephrogenesis is completed by 130 days, unilateral nephrectomy at 100 days gestation, produces a compensatory nephrogenesis in utero with reduced glomeruli filtration rates and elevated blood pressure in adult offspring [35]. It appears that some of the maladaptive compensatory changes
6
that arise in response to an adverse intrauterine environment occur intra-renally and compromise both nephrogenesis and blood pressure regulation. The renin-angiotensin system plays an important role in the development of the kidney and is also impacted by prenatal environmental alterations. In rats, a prenatal low-protein diet is associated with a decrease in intrarenal renin-angiotensin system activity including deceased renin and angiotensin II [33]. Furthermore, a high prenatal dose of an angiotensin II receptor antagonist (losartan) resulted in hypertension by 22 weeks of age and decreased nephron number by 42% [36]. In pregnant ewes where the growth and function of the placenta was restricted from the time of conception, renin and angiotensinogen levels were significantly reduced in the fetal kidneys [37]. Thus an inappropriate blockade of the renal renin-angiotensin system or a premature stimulation of the renal renin-angiotensin system in early development produces a nephron deficit and functional abnormalities that are associated with increased blood pressure later in life. A ‘resetting’ of the baroreflex may underlie the adaptations that the fetus makes in response to nutritional deprivation. Nutrient restriction (50%) in sheep not only increased pulse pressure and reduced the rate of pressure product but also produced a leftward shift in the baroreflex curve among the adult offspring [29]. There was also a blunting of baroreflex sensitivity during angiotensin II infusion. In spontaneously hypertensive Dahl salt-sensitive rats, the baroreflex has been found to be altered well in advance of the development and onset of hypertension [38]. Thus, a defective baroreflex function precedes the development of hypertension and may comprise part of the underlying mechanism of the development of the metabolic syndrome. In rats exposed to low protein prenatal diets, hypertension appears to be a consequence of increased peripheral resistance. This finding is supported by studies in rats where low protein prenatal diets effectively lowered the adult offspring pulse rate without inducing cardiac hypertrophy, suggesting that the observed changes are not the result of increased cardiac output [23]. Hypertension is an easy outcome measurement for animal studies, but hypertension is only associated with future cardiac events and thus may not be part of the underlying physiological mechanisms that predispose individuals to heart disease. At present little is known about altered cardiac performance in adult offspring following an adverse intrauterine environment, thus further investigation is required.
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1.2.2 Role of Circulatory System in DOHaD However significant the contribution of hypertension and renal function are in the pathogenesis of cardiovascular disease, alterations in the circulatory system are the principle factors that underlie the onset of cardiovascular disease. Many of the cardiovascular changes that occur throughout coronary heart disease development have early origins in the endothelium [39]. Specifically, endothelial cells of the cardiovascular system play important roles in the physiological response associated with vessel caliber, vascular growth and remodeling, tissue and organ growth, tissue metabolism, immune responses, blood fluidity, platelet and white cell stickiness and vascular permeability [39]. Negative changes in these processes will result in a pathological vessel responses leading to negative cardiovascular outcomes, such as coronary heart disease. Initially, these pathological changes were thought to result from lifestyle factors of individuals in wealthy Western nations, however, the identification of disease incidence in the poorer regions of the world, including parts of the developing world (India) [3], have led many to question these initial assumptions and consider whether cardiovascular disease may have its origins in development. Endothelial dysfunction and loss of arterial elasticity are early markers of atherosclerosis and hypertension [40]. Endothelial dysfunction also plays a role in type 2 diabetes, pre-eclampsia (a placental disorder that can result in intrauterine growth restriction), and the metabolic syndrome characterized by obesity and coronary heart disease [39]. One of the earliest morphological markers of plaque formation and atherosclerosis has been proposed to be intimal thickening [41, 42]. Preliminary evidence to support this hypothesis comes from the observation that the maximum intima-media thickness of the aortic wall was significantly higher in babies with intrauterine growth restriction than those of normal birthweight [41]. An inverse relation between birthweight and aortic intima-media thickness in newborn infants suggests that the predisposition to later cardiovascular risk has prenatal origins [41, 42]. These findings provide clear indications for a fetal contribution to later cardiovascular risk because there were no confounding developmental effects from childhood and adulthood. While some have questioned whether the increased intima-media thickness of the fetal and neonatal aorta actually signals the beginning of a disease process [42], the work of Norman and colleagues has reinforced the endothelial hypothesis through demonstration of a negative correlation between birthweight and
8
carotid artery wall stiffness, in the absence of changes in blood pressure in schoolchildren nine years of age [40]. They were able to measure the onset of impaired endothelial function before the occurrence of the late childhood hypertension that is frequently observed in children programmed by low birthweight. Additionally, they found that when low birthweight was a result of very early preterm birth, the association between endothelial dysfunction and low birthweight is attenuated [43], implying that programming of disease involves some underlying mechanisms, possibility genetic, that are not necessarily related to the factors that cause preterm birth. It is also possible that genetics and the involvement of polymorphisms in the genome create variable susceptibility to the development of disease in the context of the cardiovascular remodeling that has occurred. 1.2.3 Relationship between Cardiovascular Disorders and the Metabolic Axis Perhaps one of the largest contributors to coronary heart disease is the metabolic syndrome, composed of hypertension, abdominal obesity and diabetes mellitus, all of which greatly increase the risk of heart disease [1-3, 6, 14] and are inter-associated risk factors. In general, cardiovascular disease outcomes are associated with five metabolic risk factors: atherogenic dyslipidemia (elevated serum triglycerides, elevated low-density-lipoprotein cholesterol and reduced high-density-lipoprotein cholesterol), elevated blood pressure, insulin resistance, elevated fasting glucose, and a proinflammatory state and prothrombotic state [15]. Consequently, there is much inter-relation between the metabolic axis and cardiovascular disease. It has been theorized that all of these components of the metabolic syndrome which contribute to the development of cardiovascular disease have their origins in perinatal developmental. In support of this, a recent study of a cohort of children aged 9-25 years in Perth, Australia revealed that obesity tracked with increasing age and that the primary predictor of hypertension in men at 25-years was childhood obesity [44]. Furthermore, children with the metabolic syndrome “cluster of high risk features” displayed biomarkers consistent with adverse risk for cardiovascular disease including high C reactive protein and low adiponectin levels [45]. Correspondingly, in a different cohort from the United Kingdom, fetal growth restriction corresponded to high serum concentrations of coronary risk factors such as total cholesterol, LDL cholesterol and apolipoprotein B [6], confirming the relationship between adverse perinatal
9
development and cardiovascular disease indicators across many populations. In addition to predicting obesity in adulthood [44], weight gain patterns during childhood are also powerful predictors of coronary heart disease deaths in adults [4]. Studies of the Raine cohort of Western Australia, which followed children from gestational age 16-18 weeks until 8 years of age, observed a U-shaped relationship between birthweight and the presence of the “cluster of high risk features” of the metabolic syndrome [46]. The U-shaped relationship was confirmed in this contemporary, well-nourished Western population of full-term newborns. The cluster of high risk features which emerged from the study were inappropriate weight, height, BMI, triglycerides, cholesterol, HDL, LDL, resting glucose, systolic blood pressure and diastolic blood pressure. The association between adverse perinatal development and these risk factors provides important insights into the possible mechanisms that may underlie the pathogenesis of metabolic disease.
1.3
DEVELOPMENT OF DIABETES AND THE METABOLIC SYNDROME Diabetes is becoming a global epidemic, with an estimated 246 million people affected
worldwide. The cost of diabetes is also quite high. According to the Canadian Diabetes Association the over 2 million people with diabetes in Canada incur medical costs that are two to three times higher than those without diabetes and have significantly shortened life expectancy (5-15 years shorter). The problem is exacerbated in developing countries where incidence rates are increasing faster than in developed countries. This increase is particularly evident in India where a nutritional transition from undernutrition to increased food availability has resulted in a dramatic 4-fold increase in the rate of diabetes prevalence, and this is predicted to continue to increase over the next decade such that it will surpass rates found in western nations [47]. Diabetes prevalence rates differ for type 1 and type 2, with type 1 accounting for less than 10% of cases. Within the next three decades, the number of people with type 2 diabetes is projected to more than double [48]. Diabetes, particularly type 2, is clearly approaching a pandemic status. The metabolic syndrome is the co-occurrence of metabolic risk factors for type 2 diabetes and/or cardiovascular disease which can include, but are not limited to obesity, hyperglycemia, hyperinsulinaemia, dyslipidemia, and hypertension [49]. According to the American Diabetes Association, the metabolic syndrome is a common phenotype affecting approximately 24% of the US population. Some debate still remains on which factors are more important in the 10
clustering of multiple metabolic abnormalities, as five specific definitions are currently used [50]. However, abdominal obesity and insulin resistance are regarded as the predominant underlying risk factors [49, 50]. Chronic inflammation and physical inactivity have been associated with a clustering of disturbed metabolic symptoms, including insulin resistance and hyperinsulinaemia. Visceral fat accumulation, often caused by over nutrition and physical inactivity, has been linked to pronounced release of free fatty acids [49]. Subsequently, high levels of free fatty acids cause lipotoxic effects and insulin resistance, which lead to the hyperglycemic state [49]. Clearly, the metabolic syndrome is based on a dynamic interplay between contributing factors and recent studies have established that among these are heritable components. Researchers have identified that common genetic variants are associated with the development of this syndrome [50] and that diabetes tends to cluster within families [51]. However, genes alone are not enough; an environmental trigger typically needs to be present in order to manifest the metabolic syndrome or diabetes. 1.3.1 DOHaD, Diabetes and Metabolic Syndrome Over the last several decades, many epidemiological studies have uncovered strong, predictive relationships between adverse development and the incidence of both the metabolic syndrome [52] and diabetes [53] later in life. In the Hertfordshire cohort, low birth weight and low body weight at age 1 year was linked with impaired glucose tolerance and non-insulin dependent diabetes in men at age 64 [53]. The proportion of these individuals with impaired glucose tolerance and diabetes was 26% and 17%, respectively [53], and the association was independent of adult body mass. A follow-up study of 50 year old men and women born in Preston (Lancashire, UK) between 1935 and 1943 demonstrated that among adults, the prevalence of impaired glucose tolerance or diabetes fell from 27% in subjects who weighed 2.50 kg or less at birth to 6% in those who weighed more (p < 0.002 after adjusting for body mass index) [54]. Moreover, plasma glucose and insulin concentration responses to challenge improved with increasing birthweight, decreasing BMI at birth, and decreasing ratios of placental weight to birthweight, [54]. Furthermore, in the same two cohorts, the metabolic syndrome, measured as the combined incidence of type 2 diabetes, hypertension and hyperlipidaemia, was ten times more likely in individuals who had low weight, small head circumference and low ponderal index at birth [55]. These relationships were independent of confounding factors,
11
including duration of gestation, cigarette smoking, alcohol consumption and social class at birth. Disregarding the effects of current adult obesity, the relationship between early life body weight and adult glucose tolerance is far more dramatic: low birth weight males were six times more likely to develop glucose intolerance in adulthood than individuals who were born with normal body weights [51, 54]. Collectively, these findings provide strong support linking physiological indicators in early life with the onset of adult disease and suggest that prenatal undernutrition may play an important role in producing metabolic disease susceptibility in adults. In addition to contributing to the development of diabetes and the metabolic syndrome, prenatal undernutrition has been specifically associated with the development of obesity in children and adults. Evidence demonstrating this relationship can again be derived from individuals conceived during the Dutch famine where maternal malnutrition during early gestation was associated with higher body mass index (BMI) and waist circumference at age 50 [12]. Perturbations of central endocrine regulatory systems established in early gestation may contribute to the development of abdominal obesity in later life. Interestingly, studies demonstrated that as the severity of obesity in children and adolescents increased, so did the prevalence of other metrics of the metabolic syndrome [45]. Specifically, the severity of obesity was linked to increases in C-reactive protein, triglycerides, high-density lipoprotein cholesterol, blood pressure and insulin resistance. Early body mass index appeared to predict body mass index and the development of obesity in the metabolic syndrome later in life. Prenatal undernutrition has also been shown to alter glucose regulation in adults; moreover, the nature of adult glucose impairment has been linked to differences in the type of adversity encountered in the prenatal environment. In the American Nurses Study [8], the relationship between birthweight and glucose tolerance was described as a J-shaped curve with the lowest and highest weights demonstrating the greatest impairment; however, when birthweight was adjusted for adult BMI it demonstrated an inverse relationship. In Dutch Famine, prenatal exposure to undernutrition corresponded to altered adult glucose tolerance and insulin resistance [56], and the greatest impairment was seen in offspring from thin mothers. Outcomes following pregnancies complicated by gestational diabetes also reveal a connection between prenatal undernutrition and impaired adult glucose regulation. In gestational diabetes, the maternal-fetal glucose metabolism is altered and the resulting increased glucose levels lead to increased insulin levels and increased growth in the fetus [57]. The consequences of this insulin
12
imbalance manifest not only in increased birthweight but has been associated with elevated body mass in boys and girls as early 9 years old [57]. Similarly, children of women who were obese during pregnancy or suffered gestational diabetes and who were large-for-gestational-age at birth were at an increased risk of developing the metabolic syndrome by 11 years postnatal age [58]. Specifically, maternal obesity resulted in 2-fold greater risk and gestational diabetes resulted in a 3.6-fold greater risk for developing at least two of the following metabolic syndrome components: obesity, hypertension (systolic or diastolic), glucose intolerance, and dyslipidemia (elevated triglyceride levels or low high density lipids (HDL) levels) [58]. Whether the mother is thin or fat, these findings indicate that intergenerational cycles of obesity and insulin resistance may be perpetuated by the mechanisms that direct early development. Interestingly, the association between thinness in infancy and the presence of impaired glucose tolerance or diabetes in young adulthood is not dependent on the individual experiencing obesity in childhood. A study involving 1492 men and women aged 26 to 32 years in South Delhi, India found impaired glucose tolerance and diabetes in individuals who had had a low body-mass index up to the age of two years, followed by an early adiposity rebound and an accelerated increase in body-mass index until adulthood [47]. Although a sharp deterioration in glucose homeostasis was observed in 15.2 percent of the population at a relatively young age in adult life, it was the altered growth trajectory that best predicted adult impairment [47]. Similarly, among 8760 boys and girls in the Helsinki cohort, childhood obesity was uncommon affecting only 0.4 percent at the age of 12 years [59]. In that study, the 290 children who developed type 2 diabetes in adulthood had below-average body size at birth and low weight at one year of age, followed by an accelerated gain in weight and body-mass index [59]. Thus it is the compensatory growth trajectory, and not specific early life impairments or birthweight per se, that best predict the onset of the metabolic syndrome in adulthood.
1.3.2
Induction of the Metabolic Syndrome The effects of poor fetal nutrition during pregnancy on the metabolic syndrome have
previously been studied using a variety of approaches including uterine artery ligation, which results in a decrease in the maternal-fetal transfer of nutrients [60, 61], a 30–70% decrease in maternal energy intake which results in a decrease in fetal nutrient supply [25, 62] , or an isocaloric low-protein diet (8% vs. 20%) which results in normal fetal glycemia but a decrease in 13
specific amino acid concentrations [63, 64]. In rats, uterine artery ligation in late gestation, inducing fetal growth restriction results in offspring who develop both glucose intolerance and insulin resistance by an early age [60]. Later in adult life these rats develop overt diabetes, characterized by fasting hyperglycemia and hyperinsulinaemia [60]. Global nutrient restriction in gestation, either throughout pregnancy or during late pregnancy alone, results in a decreased nutrient supply to the fetus, a decreased fetal whole body glucose utilization rate, and fetal growth restriction [25, 62, 65]. The offspring subjected to global nutrient restriction have 40% lower insulin content and increased non fasting plasma glucose concentrations by 8 months of age [61, 62]. Offspring from low protein treated mothers have impaired glucose tolerance followed by diabetes [64, 66]. The findings from these inducible animal models of DOHaD parallel the observed phenotypes in humans. Similar, to humans, low birthweight in pigs is associated with poor glucose tolerance [67]. By 12 months of age, pigs that had been born with low birthweight had attained a body weight that was no different than their high birthweight littermates. This equalization was achieved by increased fractional growth rates very early in postnatal life during suckling, and between 3 and 12 months of age. Furthermore, thinness at birth and rapid catch up growth in the first month of life were also associated with increased insulin sensitivity, whereas by 12 months of age, early postnatal catch up growth was associated with insulin resistance [68]. In mice fed high-fat diets after experiencing prenatal undernutrition, weight gain and adiposity is markedly pronounced in comparison with mice who had not been exposed to prenatal nutrient restriction [69]. In rats, maternal food restriction results in offspring with intrauterine growth restriction that exhibit catch-up growth by 9 months of age [70]. When these offspring of nutrient-restricted pregnancies are cross-fostered to control fed mothers, they exhibit rapid catch-up growth by 3 weeks of age; their growth continues to accelerate, resulting in increased weight and percent body fat. In all of these animal models, intrauterine undernutrition results in the development of obesity and may be implicated in detrimental metabolic regulation in adulthood. Thus, intrauterine growth restriction, or just reductions in growth within the normal range, across a variety of species leads to the emergence of the metabolic syndrome through alterations in early growth patterns and the later development of obesity. Maternal hyperglycemia can also induce diabetes in offspring later in life. Experimental administration of streptozotocin in rats results in the induction of gestational diabetes and fetal
14
macrosomia [71-73]. Both male and female macrosomic offspring show accelerated growth during the first 10 weeks of life and by 10 weeks of age, have higher plasma insulin and glucose concentrations post oral glucose challenge [72]. Macrosomic male and female animals are also less sensitive to the actions of insulin, with decreased peripheral insulin sensitivity [71] and decreased responsiveness in the liver [73]. Similar to the offspring of mothers with gestational diabetes in humans, the offspring of diabetic rats develop signs of glucose intolerance during pregnancy with higher glucose and lower insulin concentrations than normal pregnant rats [73]. By inducing permanent changes to the structure and function of the endocrine pancreas, undernutrition during pregnancy has the potential to affect not only maternal glucose metabolism, but to confer metabolic disorder on the offspring and produce lasting generational effects.
1.3.3
Role of Organ Developmental Alterations in the Metabolic Syndrome Compromised prenatal nutrition and its consequences for insulin resistance may directly
influence the development, growth and differentiation of insulin sensitive tissues such as the pancreas, liver, skeletal muscles, and adiposities; in this way, prenatal undernutrition can have broad, far reaching effects on adult physiology. Indeed, alterations in the above mentioned organs have been associated with DOHaD and the onset of the metabolic syndrome [26]. Figure 1-1 summarizes the potential mechanism of how early life alterations lead to the developmental organs changes and the later development of the metabolic syndrome. Animal models in which the metabolic syndrome is induced following compromised fetal growth exhibit alterations in pancreatic function. Moreover, uteroplacental insufficiency results in reduced fetal pancreatic endocrine tissue, β-cell mass, and fetal insulin concentrations [74]. In rats, these effects on β-cell mass and insulin content in the fetal pancreatic islets occur after exposing fetal rats to low-protein diets. [75, 76] and suggest that insufficient protein in the prenatal diet results in decreased proliferation and increased apoptosis in the islet cells [76]. This is in contrast to the mechanism by which global energy restriction alters β-cell levels; here β-cell numbers are decreased by reductions in cellular neogenesis [65]. Nevertheless, despite differences in the mechanistic underpinnings, global nutrient restriction, uteroplacental insufficiency and low-protein availability during fetal development all result in reduced pancreatic cell number, in β-cell mass, islet number, pancreatic insulin content, and circulating 15
fetal insulin concentrations [62, 65]. These alternations in prenatal pancreatic development are carried forward into postnatal life and can have long-term, sustained consequences on the function and health of the entire metabolic system well into adulthood. Thus, alterations in nutrient availability to the fetus impact in utero pancreatic development and this has consequences for the long-term pattern of pancreatic development in postnatal life. Impaired fetal pancreatic development has lasting implications for postnatal pancreatic function. After birth, normal fed rat offspring from protein-restricted dams had small and irregular shaped pancreatic islets and reduced amounts of β-cells [75]. Juvenile offspring that were nutritionally restricted from the fifteenth day of pregnancy and during lactation had significantly reduced β-cell mass, but normal β-cell proliferation and insulin content at 3 weeks of age [65]. However, in the intra-uterine growth retarded offspring, β-cell mass continued to degenerate relative to control treated rats, and their relative β-cell mass was 50% that of normal by 15 weeks of age and less than one-third by 26 wk of age [60]. The reduction in β-cell mass in offspring of global nutrient restricted dams persisted late into adult life [77]. Importantly, all of these postnatal pancreatic changes are associated with altered glucose tolerance and insulin resistance [26]. The persistence of decreased pancreatic function and altered structure in postnatal life suggests that poor maternal nutrition has an adverse effect on pancreatic functional capacity as a consequence of an irreversible developmental deficit. It is possible that during the early stages of obesity, an increase in β-cell mass and function might compensate for peripheral insulin resistance; however, as obesity continues, the β-cell mass may become inadequate and consequently the compensatory adaptation may no longer be sufficient to compensate for the insulin resistance [78]. Obesity is associated with chronically elevated fatty acids and glucose intolerance. This state of hyperlipidemia and hyperglycemia eventually contributes to β-cell dysfunction and a decrease in β-cell mass that characterizes the onset of type 2 diabetes in obese patients [78]. Furthermore, in rats, prenatal undernutrition [65] or maternal low protein diet [61] impairs pancreatic β-cell development, reduces β-cell mass and causes glucose intolerance. Indeed in Hertfordshire cohort it was seen that low birthweight and reduced early growth was related to a raised plasma concentration of 32-33 split proinsulin in adults [53], indicative of potential β-cell dysfunction. Clearly, the pancreas is susceptible to an adverse early environment which alters its structure and impairs its function throughout life.
16
In the rat model, prenatal undernutrition permanently alters the structure and function of the liver by altering hepatic zonation [79]. Protein restricted rat offspring have reduced liver weight [80], liver growth, and hepatocyte proliferation [81]. Hepatic lobular volume was also twice that of control livers, indicating that livers of protein-restricted pups had half the normal number of lobules. Alterations in the prenatal environment produce dramatic structural changes in the liver. Anabolic enzymes, such as glucokinase, located in the perivenous regions were reduced, while catabolic enzymes, such as phosphoenolpyruvate carboxykinase, located in the periportal regions were increased, thus biasing the liver toward production of glucose. Typically, elevated expression of phosphoenolpyruvate carboxykinase (PEPCK) in the liver associates with diabetes [82]. Not surprisingly, offspring from protein restricted dams had impairment in the normal distal perivenous glucose uptake relative to controls [81], leading to increased glucose production from lactate in perfused adult livers. Similarly, following experimental restriction of placental growth, chronically hypoglycemic and hypoinsulinemic fetal sheep displayed a premature increase in hepatic cytosolic PEPCK activity and fetal glucose production [83]. Furthermore, hyperinsulinaemia induced by fetal malnutrition resulted in a dampened suppression of glucose production in the liver [62]. Even though anabolic enzymes were reduced in response to prenatal dietary alterations, glucagon concentrations remained constant [84]. Thus, the switch in liver function, induced during prenatal life, biased the liver towards production instead of storage, and contributes to altered glucose levels later in life. The prenatal switch to glucose production is regulated transcriptionally. The peroxisomeproliferator activated receptor-γ coactivator-1 is a transcriptional coactivator of nuclear receptors that control the hepatic expression of key gluconeogenic enzymes including glucose-6phosphatase, PEPCK, and fructose- 1,6-bisphosphatase [85]. It is increased in the livers of growth restricted rat offspring at birth and later in life and is related to subsequent insulin resistance [86]. Concurrent with the increased peroxisome-proliferator activated receptor-γ coactivator-1 gene expression, there was also an increase in the mRNA levels of each of these enzymes in the liver at birth and on day 21 in the growth-restricted animals, whereas hepatic glucokinase mRNA levels were significantly decreased. These findings suggest that an increase in peroxisome-proliferator activated receptor-γ coactivator-1 expression and subsequent hepatic glucose production contribute to the insulin resistance. Similarly, the offspring of proteinrestricted rat dams display permanent alterations in the activities of these same key hepatic
17
enzymes of glucose metabolism [84]. Thus, the liver plays an essential role in regulation of glucose control and prenatal alterations in nutrition permanently impact its functioning later in life. The metabolic syndrome is also associated with dyslipidemia and alterations in hepatic functions that contribute to the adult phenotype. Uteroplacental insufficiency and subsequent intrauterine growth retardation leads to increased fasting serum triglycerides levels in adults [87]. Increased triglycerides in adults were preceded by altered hepatic fatty acid metabolism in fetal and juvenile livers with decreased expression of acetyl-CoA carboxylase carnitine palmitoyltransferase 1 and the β-oxidation-trifunctional enzymes. Later in adulthood, expression of hepatic malonyl-CoA levels, and acetyl-CoA carboxylase was increased while levels of carnitine palmitoyltransferase 1 and the β-oxidation-trifunctional were decreased [87]. The growth-restricted fetus modifies its hepatic fatty acid metabolism towards increased synthesis and decreased fatty acid oxidation, potentially leading to increased serum levels of hepatic triglycerides in adulthood. These alterations may also impact skeletal muscle insulin sensitivity. Muscle tissue is also susceptible to effects of an adverse environment early in life. In low birthweight lambs, skeletal muscle weight was reduced and the rates of gain in several skeletal muscles, including semitendinosus, were persistently lower [88]. Low birthweight lambs also showed reduced rates of gain in DNA and RNA within skeletal muscles, and these also potentially contributed to impaired growth. The reduced rate of DNA and RNA gain in skeletal muscles indicates an overall reduction in cellular growth and proliferation that will impact the structure of the muscles later in life. Indeed, a decrease in adult lean body mass and musculature is associated with impaired fetal growth in humans [26]. In rats, protein restriction through gestation or immediately after birth resulted in a reduction in skeletal muscle mass [80]. Furthermore, by 15 months of age, there was a decrease in the insulin sensitivity of glucose uptake in skeletal muscle from the group exposed to the low-protein diet in utero [89]. In the rat model of uteroplacental insufficiency, pups developed insulin resistance by a young age and continue to suffer impaired glucose regulation throughout adulthood, leading to diabetes. Muscle from the growth-restricted rats also showed decreased glycogen content and decreased insulinstimulated 2-deoxyglucose uptake [90]. These alterations indicate a potential breakdown of glucose regulation within the muscles and may be associated with a chronic reduction in the supply of ATP available for oxidative phosphorylation and impaired glucose transport. In these
18
studies, oxidative phosphorylation was subsequently shown to be decreased in growth-restricted fetuses, and hepatic cellular energy and redox states were uncoupled resulting in less ATP generated per unit of glucose [90]. Thus, an adverse intrauterine environment leads to a reduction in muscle mass and alterations in muscle glucose metabolism which persist into later life. Development of the metabolic syndrome has also been tied to an increase in total and subcutaneous fat mass. In rats, undernutrition throughout gestation resulted in offspring with an increase in the relative mass of the retroperitoneal fat pad by 100 days [91]. Increased fat to body weight ratios were observed in rats with impaired glucose tolerance after streptozotocin induced fetal macrosomia [1]. The adipocytes of these obese rats also had a higher lipid content and significantly larger cell size than control animals. As subcutaneous fat mass increased, there was an enlargement of subcutaneous adipocytes that were resistant to insulin-mediated glucose uptake and this was associated with the development of overall insulin resistance [92]. Adipocytes isolated from offspring exposed to prenatal low-protein had significantly higher basal and insulin-stimulated glucose uptakes [93]. They also had a threefold increase in insulin receptors [93], possibly as a compensatory mechanism as insulin resistance is increased postnatally. Indeed, hyperglycaemic rats, induced by intraperitoneal insulin antibody injection, showed an increased insulin binding capacity in adipocytes [94]. The increased binding capacity was accompanied by increased insulin sensitivity on lipogenesis in adipocytes [94], which may contribute to the increased dyslipidemia seen in the metabolic syndrome. Similarly, streptozotocin-induced fetal macrosomia also resulted in higher receptor affinities and was associated with the development of hyperinsulinaemia and obesity by 10 weeks postnatal age in the offspring [95]. It is therefore possible that post-receptor deficits in adipocytes may contribute to the abnormal glucose metabolism associated with increased adiposity in DOHaD. DOHaD has also been associated with alterations in the mechanisms that regulate appetite. Offspring subjected to prenatal undernutrition exhibited a premature onset of the neonatal leptin surge compared to offspring with normal intrauterine nutrition; postnally, this was associated with increased adiposity [69]. The premature leptin surge may have the effect of altering hypothalamic regulation of energy and may thus contribute to development of the metabolic syndrome. Typically, circulating leptin levels are increased during adult obesity [26]. Visceral fat accumulation results in dysregulation of adipocytokine secretion, including hyposecretion of adiponectin and hypersecretion of leptin, tumor necrosis factor and interleukin
19
6, each of which has been proposed to mediate many of the changes that culminate in the metabolic syndrome [49]. Neuropeptide Y (NPY) is expressed within the hypothalamus and acts in synchrony with other neuropeptides to regulate energy balance [96]. In rats, maternal protein restriction [97] and uteroplacental insufficiency [98], and, in sheep, maternal undernutrition [99] result in increased fetal NPY expression and low birthweight. Moreover, during postnatal development, these changes are associated with increased appetite and the onset of the metabolic syndrome.
1.4
MECHANISMS OF DISEASE
1.4.1
Hypothalamic-Pituitary-Adrenal Axis The Hypothalamic-Pituitary-Adrenal (HPA) Axis has previously been proposed to be a
major target and mediator of the DOHaD. Linkage of the HPA axis to the fetal programming and the onset of adult disease are supported in many species by different streams of evidence [26, 100, 101]. First, in human neonates umbilical cord blood levels of ACTH and cortisol are increased in association with reduced fetal growth [102]. In independent adult populations, low birth weight has been observed to predict increased plasma cortisol levels [103]. Furthermore, this association between birth weight and glucocorticoid levels is also linked to the development of hypertension and adult onset Type II diabetes [103, 104]. Thus, programming of the HPA axis in utero has been proposed to contribute to the development of cardiovascular disease later life, either directly or indirectly through the effect of the HPA axis on the development of insulin resistance and diabetes (cardiovascular disease risk factors) [105-107]. In particular, the fetal HPA axis is highly vulnerable to changes in the intrauterine environment that can disrupt the delicate balance of fetal HPA axis development and glucocorticoid production. HPA axis hyperactivity has been observed in animal models of DOHaD after prenatal under-nutrition [108], prenatal stress [109] and maternal glucocorticoid administration [110, 111]. In both sheep and humans, exposure of the fetus to synthetic glucocorticoids by repeated maternal administration, in sheep [112] and humans [113], will not only result in fetal and postnatal HPA hyperactivity but it will impair fetal growth. Furthermore, the offspring of pregnant sheep treated with maternal betamethasone (a synthetic glucocorticoid) displayed hyperactivity in HPA axis in early adulthood [114]; however the adrenal glands appeared incapable of sustaining cortisol output later in life as these animals developed relative 20
adrenal insufficiency [115]. These alterations in HPA axis function were most pronounced in offspring that were the most growth restricted at birth [115]. Fetal HPA axis hyperactivity increases the availability of glucocorticoids to the fetus and induces a premature maturation of fetal organs such as the kidneys, liver, lungs and gut [116]. This maturation results when fetal tissues switch from cellular proliferation to differentiation [116]. The consequences of this switch from cell proliferation to differentiation may lead to an inappropriate pattern of growth for the stage of development [116]. Adverse intrauterine environments can therefore alter postnatal HPA axis activity and function, and have the potential to impact the development of disease later in life. It has also been proposed that resetting the endocrine axes, which control growth and development, could be responsible for the developmental programming of later health and well being. There is some evidence that indicates that the HPA axis itself may be programmed, such that that the set point of the axis changes resulting in altered basal and/or stress-induced glucocorticoid responses in postnatal life [26]. An increase in HPA activity throughout life will impact adult health due to altered tissue exposure to endogenous glucocorticoids. Chronically elevated plasma cortisol has been associated with atherosclerosis, immunosuppression, depression and cognitive impairment, as well as elevated cholesterol levels and increased incidence of diabetes [117, 118]. Neuroendocrine programming like this could contribute to the observed association between altered fetal growth and the metabolic syndrome later in life. Evidence for this hypothesis was obtained from epidemiological studies in two populations where adults who had lower birth weight had raised fasting plasma concentrations of cortisol and higher blood pressure [103, 104]. Further support for this hypothesis was derived from studies in rats where maternal administration of synthetic glucocorticoids reduced offspring birthweight, increased plasma corticosterone concentrations, persistently elevated blood pressure and impaired glucose tolerance [119-121]. Similarly, in sheep, even a single dose of synthetic glucocorticoids to the mother during pregnancy increased offspring insulin insensitivity, and glucose intolerance; however, multiple dose were required to induce fetal growth restriction and chronic hypertension [122, 123]. All of these symptoms are factors that are highly related to the metabolic syndrome and the development of cardiovascular disease. Prenatal programming also exists at the level of the central nervous system. Glucocorticoid exposure in utero has widespread acute effects upon neuronal structure and
21
synapse formation [124] and may permanently alter brain structure [106]. Indeed in sheep, prenatal administration of synthetic glucocorticoid reduced the weight of the brain at birth [125] and, in rhesus monkeys, produced a dose-dependent reduction in hippocampal volume and degeneration of hippocampal neurons [111]. The effects of synthetic glucocorticoids on prenatal development are not confined to the brain. Fetal exposure during pregnancy delays neuronal myelination [122, 125, 126] and may affect overall nervous system development. Prenatally programmed increases in HPA function may exacerbate hippocampal deficits (associated with normal aging) by reducing the negative feedback of glucocorticoids, resulting in further increases in HPA axis activity. Michael Meaney’s group have shown in the rat that neonatal handling induces a permanent increase in hippocampal glucocorticoid receptor (GR), involving serotonin-induced methylation of a hippocampal specific GR promoter via alterations in the transcription factor NGFI-A [127]. This alteration provides a mechanism by which the early environment can influence gene expression throughout life. Exposure of the fetal rat to maternal undernutrition during pregnancy produces offspring with reduced adrenal weight, GR and mineralocorticoid receptor (MR) expression in the hippocampus, increased CRH expression in the paraventricular nucleus (PVN), and circulating ACTH [128]. Similarly, Jonathan Seckl’s group has also shown that prenatal exposure to synthetic glucocorticoid in the rat increased CRH mRNA in the PVN, reduced hippocampal GR and MR expression, and increased plasma corticosterone concentrations in offspring [121, 129]. All of these changes may cause a resetting of the feedback mechanism of the HPA axis, potentially explaining differences that are seen in response to stress later in life in offspring that were exposed to prenatal undernutrition [26]. The multi-organ targets of the endocrine axis may explain the degree of the impact following prenatal undernutrition that has been documented across many physiological systems; furthermore, the extensive involvement of the HPA axis in governing development provides a means by which environmental modifications can be translated into changes in biological function. In the rat, prenatal exposure to synthetic glucocorticoids can, in addition to affecting glucocorticoid receptor in the brain, also modify the hepatic expression of the GR and enzymes responsible for glucose regulation [129, 130], thereby contributing directly to changes in metabolic function. Similarly, in genetically obese db/db transgenic mice, PEPCK gene transcription and hyperglycemia were found to be regulated by glucocorticoids [131]. Typically, there is a higher expression of PEPCK in the liver associated with diabetes [82]. Studies in rats
22
and sheep have established that synthetic glucocorticoids also affect the kidney. In these studies, it was observed that maternal administration of synthetic glucocorticoids resulted in reduced nephron number (30%, 40%) in the offspring who develop hypertension later in life in rats [132] and in sheep [30, 133]. Impairments in fetal kidney development will impact overall gene expression and is thought to have lasting effects on the renin-angiotensin system [30] that may contribute to the development of hypertension in other models of DOHaD. Specifically, reninangiotensin system receptor (AT1 & AT2) density and tissue synthesis are affected by antenatal steroid exposure, resulting in a reduced glomerular filtration rate response to angiotensin II [134]. Therefore, it appears that the effects of glucocorticoids are widespread, effecting not only circulation but also basic metabolic processes. The placenta has a major influence on fetal endocrinology and metabolism and may also regulate the long term effects of altered HPA axis activity through glucocorticoids exposure in utero. In the placenta 11β–HSD1 is primarily involved in the conversion of the biologically inactive forms of glucocorticoids to their active counterparts while 11β–HSD2 serves the opposite role [135, 136]; thus together, both regulate glucocorticoid availability to the fetus. In regards to synthetic glucocorticoid models, betamethasone and dexamethasone are poor substrates for 11β–HSD [137] and have the ability to mimic conditions that elevate glucocorticoids, such as prenatal undernutrition. This was demonstrated in rats where a low protein diet not only reduced placental 11β–HSD2 activity but also produced offspring with chronic hypertension [138]. Furthermore, inhibiting placental 11βHSD2 was also shown to lead to hypertension and glucose intolerance in the adult rat offspring by increasing fetal glucocorticoid exposure [139]. Conversely, treating the protein-restricted pregnant rat dams with glucocorticoid synthesis inhibitors protected the offspring from developing hypertension; this protection was reversed when corticosterone was concurrently administered with the glucocorticoids synthesis inhibitors [140]. Several other molecular pathways and axes have been implicated as contributors to the mechanisms of DOHaD. These include: the hypothalamic-pituitary-thyroid axis involved in developing neurotransmitter (serotonin) pathways [127]; the hypothalamic-pituitary-gonadal axis involved in programming sexual behaviour [141]; the growth factor pathway implicated in the pathogenesis of fetal growth restriction [142, 143]; and pathways involved in placental development and trophoblast-maternal interactions [19, 143]. Placental size has an important
23
influence on fetal endocrinology, where fetal serum insulin-like growth factor (IGF) concentrations were reduced with placental restriction [141, 142]. Furthermore, impaired placental development has consequences on nutrient transfer and on maintaining an appropriate partition between the mother and the fetus. Not surprisingly then, alterations in IGF and other growth factors during development have also been correlated to the development of the metabolic syndrome [19].
1.4.2
Prenatal vs. Postnatal Much of the evidence to-date has demonstrated that the environmental factors and
conditions that result in the developmental programming that give rise to altered birth size and compromised cardiovascular/metabolic outcomes exert their effects during early or late pregnancy [3], or even during the peri-conceptual period [144]. However, more recent work has shown that there is an inverse relationship between the pre- and post-natal environment and disease susceptibility later in life. The individuals most affected by adult onset metabolic syndrome were those subjected to the most adverse intrauterine environment and who also underwent rapid weight gain postpartum. These findings, shown in both humans [145, 146] and in animal models [52], demonstrate an interaction between sub-optimal intrauterine and postnatal environments and DOHaD. It is hypothesized that these postnatal alterations are based on the persistence of endocrine, physiological, and metabolic adaptations made by the fetus made when it was undernourished [147]. These adaptations in turn enhance the susceptibility of the offspring to postnatal environmental alterations vis-à-vis the responsiveness of their metabolic and endocrine systems. Programming is not a process confined to the just the extremes in fetal growth (such as intra-uterine growth restriction and macrosomia) but rather one that accompanies the adaptations that every fetus makes to its environment, including subtle variations in growth [3]. Indeed, alterations to the fetus which gives rise to disease susceptibility later in life can occur without producing marked effects on birth weight. Infant size at birth is a frequently cited measurement in DOHaD studies; however, it is just a crude surrogate reflecting the interactions between the fetal environment and the fetal genome. Evidence from both human [3, 12] and animal [52, 148, 149] studies, suggests that the perinatal environment does not necessarily have to produce changes in birth weight in order to affect long term health outcomes. For example, in the Dutch 24
winter famine, women who ate
