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Genetic and Molecular Basis of Obesity
3 Genetic and Molecular Basis of Obesity Adnan Hasan and Tahir Mahmood Department of Obstetrics & Gynaecology, Victoria Hospital, NHSS Fife, Kirkcaldy, UK
Introduction Obesity is recognised as a global epidemic and is rising in children and in both men and women up to the age of 60. In 2008, the WHO reported that among adults, 20 and older, more than 200 million men and nearly 300 million women were obese. The rate of obesity in the United Kingdom has increased by fourfold over the past 30 years, an incidence of 22% in men and 24% in women in 2008/2009 [1]. Similarly, in the United States, comparable figures were 35.5% and 35.8% in 2009/2010 [2]. Obesity (BMI . 30 kg/m2) is a heterogeneous group of disorders that results from the imbalance in energy homeostasis, whereby food energy intake exceeds energy expenditure in genetically susceptible individuals. Such excess energy is stored in the most efficient form as triglycerides in white adipose tissues. Obesity poses serious health problems and reduces longevity from associated co-morbidities such as insulin resistance, type-2 diabetes, hyperlipidaemia, hypertension and various cancers [3,4]. Obesity thus may become the most challenging health problem with huge economic impact on health services. From women’s perspective, obesity further predisposes them to hyperandrogenicity with oligomenorrhoea/amenorrhoea and reduction in fertility in addition to health risks related to pregnancy and childbirth. For most people, the human body maintains its normal weight so that at times of excess nutrients, intake is dissipated through increased physical activity, enhanced basal metabolic rate (BMR) and adaptive thermogenesis in response to daily fluctuation in food intake. The hypothalamus regulates energy homeostasis through integrating neural, hormonal and metabolic signals from peripheral and central nervous system (CNS), endocrine glands, including adipose tissue, and blood metabolites such as glucose and fatty acids. Disruption of the control mechanism whether congenital or acquired results in accumulation of excess energy as lipids. Obesity. DOI: http://dx.doi.org/10.1016/B978-0-12-416045-3.00003-0 © 2013 Elsevier Inc. All rights reserved.
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Hereditary Factors Influencing BMI Studies from twins and adoption that have compared phenotypic correlations for body fat between groups of individuals, varying in genetic relatedness, have yielded interesting observations. It appears that fat mass is genetically influenced among individuals who are more genetically similar such as in the case of monozygotic twins than dizygotic twins [5]. Even for twins reared apart, the estimated heritability was at 65 75% for BMI [6]. Furthermore, results of longitudinal behaviour genetic studies suggest that there are age-specific genetic effects on BMI, such that different obesity-promoting genes may become active at different ages across the lifespan [7]. The ‘Human Obesity Gene Map’ has been annually updated since 1994 [8]. This comprehensive compendium summarises evidences from the four classes of human studies: a. Obesity due to a single gene in digenic mutation. b. Obesity associated with Mendelian disorders such as Prader Willi syndrome or Bardot Biedle syndrome. c. Associated studies that test whether candidate genes are associated with obesity phenotypes among samples of unrelated participants. d. Linkage studies that test for causal association between genomic regions and obesity phenotypes in cohort of families.
This compendium has shown that the number of genes associated with obesityrelated traits has increased dramatically over the past decade, thereby suggesting that the platform of specific genes that might contribute to obesity is large and involves loci throughout the genome.
Genetic Molecular Interaction of Obesity Severe obesity in humans can rarely result from the monogenic mutations of genes involved in hypothalamic appetite control pathways. The genes which encode leptin receptor [9], proopiomelanocortin (POMC) [10], prohormone convertase1 [11], leptin [12], melanocortin 3 receptors (MC3R) [13] and melanocortin 4 receptors (MC4R) [14] are involved. As noted above, human obesity id polygenic in origin [8] and involves a network of genes with several hundred loci located on nearly all the chromosomes including Y chromosome thus affecting obesity-related phenotypes. Monogenic mutations are not involved in the development of common forms of obesity where subtle interaction between numerous related genetic variants and environmental factors, such as diet and exercise, results in the overexpression of molecules that affect energy homeostasis leading to a positive or negative impact. Chronic exposure to environmental factors leads to single nucleotide polymorphism [15] or structural changes in DNA through methylation that promotes gene expression in susceptible individuals [16]. In future, this opens up the possibility of genotyping the individuals in order to identify susceptible individuals to obesogenic stimuli, that may allow for preventive measures to be implemented at individual level.
Genetic and Molecular Basis of Obesity
Regulation of Energy Balance
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Molecular Basis
The main biochemical processes involved in energy regulation are those related to the control of energy intake, energy output (expenditure) and adipogenesis. These inter-related processes are governed at the cellular level by a wide range of inducible molecules and their associated genes, and such cross-regulation may limit the effectiveness of available treatments. This chapter will now focus on the molecules and their related genes responsible for the main biochemical processes that control energy homeostasis.
Regulation of Food Energy Intake Feeding behaviour may be regulated via inter-related short- and long-term control. Short-term control is involved with individual meal intake where hunger and satiety signals reflect the acute energy status of the body. Feeding process is affected by metabolic, neural and endocrine factors and is modified by powerful sensory, emotional and cognitive inputs. Ultimately, all of these factors must be integrated, so that decisions to begin and end periods of feeding will result [17]. Gastrointestinal hormones play an important role in the acute stage of feeding, e.g. plasma levels of ghrelin increase during fasting and infusion of this hormone stimulates appetite, an effect mediated via agoutirelated protein (AgRP) [18]. Likewise, mechanical distension of the stomach and release of gut peptides, such as cholecystokinin, gastrin-releasing peptide 29 (GRP-29) and peptide YY3 36, produce satiety signals [19,20]. Long-term signals that reflect the status of energy stores are mainly that of leptin and insulin, and they provide information to the CNS to regulate feeding behaviour and promote energy homeostasis. As a result of these feedback signals, the energy stores and body weight remain generally stable for most humans over long period of time despite the wide variations in day-to-day food intake. However, chronic excessive food intake combined with reduced energy utilisation (i.e. exercise) limits their effectiveness and leads to increased adiposity that is individually regulated within narrow margins, resulting in a constant set point to which body weight would eventually return after fat loss or gain. In addition, resetting of feedback signals to higher thresholds in hypothalamus and elsewhere results in increased levels of circulating metabolites, e.g. glucose and lipids that reflect resistance to leptin and insulin action.
The Role of Hypothalamus Hypothalamus is critical for the regulation of homeostatic processes such as feeding, thermoregulation and reproduction. It is the primary receptor of peripheral signals that indicate food availability [21]. It receives and integrates mechanical,
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hormonal, metabolic and peripheral neural inputs with those from higher centres: the brainstem, cerebral cortex, olfactory areas and limbic system. Hypothalamus in response produces neurotransmitters and neurohormones resulting in behavioural, autonomic and endocrine responses that control appetite and energy balance. The central role of the hypothalamus in appetite and satiety was established from the studies where lesions in the ventromedial hypothalamus caused obesity, while, conversely, lesions in the lateral hypothalamus resulted in leanness [22]. The arcuate nucleus plays a critical role in the regulation of energy homeostasis. It occupies more than one-half of the hypothalamus and lies around the base of the third ventricle immediately above the median eminence where the capillary endothelium lacks tight junctions forming an incomplete blood brain barrier (BBB) thus allowing larger proteins and hormones to readily access the arcuate nucleus neurons from circulation. The hypothalamic melanocortin signalling system is also heavily regulated by circulating hormones, mainly leptin and insulin, that provide information about body energy status and reserves. Hormonal signals from leptin and insulin inhibit hypothalamic orexigenic neurons [17], while, conversely, the anorexigenic neurons are stimulated by inputs from leptin and insulin [23]. Overall, it is apparent that key neurons in the hypothalamus, most likely in the arcuate nucleus, are primed to integrate a variety of hormonal and metabolic signals in order to interpret the state of energy balance, and, in turn, mediate the necessary metabolic and behavioural responses in order to compensate for deviations from homeostasis.
The Role of Leptin Human leptin (derived from Greek leptos meaning thin) is a protein of 167 amino acids secreted mainly by adipocytes that express the ob gene. The level of circulating leptin is directly proportional to the total amount of body fat [24]. Its levels are higher in women than in men as its production is stimulated by oestrogen and suppressed by testosterone. Leptin levels drop with fasting and increase with food intake, an effect mediated in part by insulin stimulation of adipocytes, and both hormones act on hypothalamus triggering similar responses [25]. Various regulatory elements have been identified within the leptin promoter, e.g. cAMP and glucocorticoid response elements, and CCATT/enhancer binding sites, suggesting a direct regulation of leptin expression through membrane and transcriptional pathways [26]. Leptin is considered the main signal that provides information to the CNS reflecting energy stores in the body. Leptin binds to receptors (Lep R) in the arcuate nucleus and other hypothalamic nuclei resulting in changes in feeding behaviour, with suppression of appetite [27], and an increase in metabolic activity and energy expenditure (thermogenesis) that on the long term regulates energy balance and maintains normal body weight, despite wide variation in daily calorie intake in most individuals.
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The central effects of leptin signals stimulate the anorexigenic neuropeptides, alpha-melonocyte stimulating hormone (α-MSH) and cocaine- and amphetaminerelated transcript (CART), and inhibit the orexigenic neuropeptides, neuropeptides Y (NPY) and agouti-related protein AgRP in the arcuate nucleus [27]. Leptin also inhibits melanin-concentrating hormone (MCH) and orexins in lateral hypothalamus [22]. This in turn stimulates neuroendocrine response that results in the production of thyrotropin-releasing hormone [28] and activates the sympathetic nervous system that results in increased metabolism of tissues mediated via increased metabolic rate. Deficiency of either leptin or its receptors secondary to gene mutation in ob and db (Lep R) loci causes severe obesity, the first of which can be controlled with exogenous leptin. Direct peripheral action of leptin on many tissues, including adipose tissue, stimulates lipolysis and fatty acid oxidation [29]. Local leptin production in the stomach plays a role in short-term energy balance as its level increases under the effect of CCK and gastrin thus mediating satiety signals indicating the filling effect of a meal. Leptin levels in most obese people are high without corresponding gene mutation indicating a disruption in the feedback signals reflecting a state of insensitivity to leptin (hyperleptinaemia) similar to that of insulin. The mechanism involved is related to reduction in leptin transport through BBB where leptin levels in the brain are lower than the plasma levels [30] (Table 3.1).
Melanocortin Signalling Pathway Leptin acts on two distinct subtypes of adjacent neurons in the arcuate nucleus in opposing manner. The first subtype of neurons co-expresses mRNAs encoding anorexigenic peptides, CART and α-MSH (derived from POMC), and leptin induces their expression [22,25,31]. The other subtype co-expresses the orexigenic Table 3.1 Leptin Targets of Neurotransmitters and Peptides that Affect Feeding Process Stimulation of Feeding
Inhibition of Feeding
Neuropeptide Y Agouti-related peptide Melanin-concentrating hormone Orexins Ghrelin Galanin Growth hormone-releasing hormone Opioid peptides Gamma-aminobutyric acid Nitric oxide Noreadrenaline
Alpha-melanocyte stimulating hormone Cocaine- and amphetamine-regulated transcript Corticotropin-releasing hormone Calcitonin gene-related peptide Urocortin Neurotensin Serotonin Glucagon Cholecystokinin Glucagon-like peptide 1 Bombesin/gastrin-releasing peptide
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peptides NPY and AgRP, and leptin reduces their expression [17,22,25,31]. Secreted α-MSH binds to MC4R and to a lesser extent to MR3C to produce tonic inhibition with anorexic effect and increase in thermogenesis. The axonal projections of neuropeptide α-MSH also synapse with second-order neurons in the paraventricular nucleus (PVN) and lateral hypothalamus. The PVN regulates the secretion of pituitary hormones by the release of neuropeptides such as thyrotropin- and corticotrophin-releasing hormone via the projections in the median eminence and regulates autonomic nervous system via the projections to autonomic pregangilionic neurons. Direct projection of these arcuate melanocortinergic neurons (AgRP and α-MSH) onto the neurons within the lateral hypothalamus inhibits the expression of the orexigenic neuropeptides MCH and orexins [32]. Expression of MCH stimulates food intake. Deletion of the MCH gene causes a lean phenotype [33], and transgenic overexpression promotes obesity. Two neuropeptides orexins A and B are involved in the regulation of food intake [34]. Orexins function through G-protein-coupled receptors and their expression in the brain seems to be limited to the neurons of the lateral hypothalamus and nearby region. Central administration of orexins stimulates food intake, and production of orexin increases with fasting [34]. Recent studies indicated that the POMC-derived neuropeptides β-MSH plays a critical role in the hypothalamic control of body weight in humans [35]. Conversely, absence of leptin induced by calorie restriction or fasting activates orexigenic NPY and AgRP [36]. The activation of these orexigenic neurons leads to the inhibition of anorexigenic signalling in two ways. First, AgRP/NPY neurons synapse directly with POMC neurons, providing an inhibitory tone. Second, AgRP itself is an effective antagonist of α-MSH at MC4Rs. Release of AgRP/NPY neuropeptides stimulates food intake and reduces energy expenditure. NPY acts via Y1, Y2 and Y5 G-protein-coupled receptors expressed in the hypothalamic neurons. The Y5 and the Y1 receptors mediate the stimulatory effects of NPY on food intake [37]. The Y2 receptor mediates an inhibitory effect of NPY at low concentrations that could be important for basal control of body weight [38]. The action of NPY on its receptors can be affected by other neurotransmitters, such as glucagon-like peptide 1, which inhibits food intake and diminishes the orexigenic effect of NPY by antagonising NPY receptors Y5 and Y1 [39]. In ob/ob mice model, the role of NPY in full response to leptin deficiency was determined. The results showed that NPY deficiency prevents obesity and other features of ob/ob mice. However, NPY-deficient mice feed normally and gain normal body weight [40]. Adrenal glucocorticoids potentiate the orexigenic effect of NPY by acting as endogenous antagonists of leptin and insulin thus participating in energy regulation [41]. Adrenalectomy mitigates the effect of fasting to increase both appetite and NPY expression and potentiates the anorexigenic and slimming effects of insulin and leptin; administration of glucocorticoids compensates these effects [41]. AgRP antagonises the effect of α-MSH on MC4R. Activation of MC4R by α-MSH reduces food intake, while suppression of MC4R signalling through this receptor by the endogenous antagonist AgRP increases feeding and diminishes
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the hypophagic response to leptin [25,42]. AgRP deficiency leads to a lean phenotype of mice with extended lifespan when the animals received a high-fat diet [43]. Gene mutation at MC4R locus affects 6% of humans with severe obesity, and most affected individuals have a single mutant allele that causes obesity through haploinsufficiency rather than a dominant-negative mechanism [44]. The obesity in several other rare human syndromes also converges on this pathway. Targeted deletion of the MC3R produced obesity in mice [45]. However, obesity from this lesion occurs without the hyperphagia seen in MC4R mutants and may be associated with a loss of lean body mass. Thus, these two melanocortin receptors can cause obesity through distinct physiological mechanisms. Recent studies have strongly implicated that adenosine monophosphate-activated protein kinase (AMPK) pathway plays a central role in hypothalamic control of energy homeostasis by mediating the inputs from multiple hormones, peptides, neurotransmitters and nutrients [46]. Leptin inhibition of AMPK activity in the arcuate nucleus and PVN in hypothalamus is necessary to produce its anorexic effect [47]. The melanocortin system is not the only neuropeptides system involved in weight regulation. Leptin regulates CART expression in arcuate POMC neurons, and CART axons innervate sympathetic preganglionic neurons in the thoracic spinal cord [48]. Among its central effects, leptin further regulates many other neuropeptides, including corticotropin-releasing hormone, growth hormone-releasing hormone and galanin that have been described to participate in energy regulatory pathways. Ghrelin, a peptide expressed in stomach and brain, promotes hyperphagia and obesity, an effect mediated through NPY/AgRP neurons [49]. Noradrenalin, dopamine and serotonin (5-HT) are also known to be involved in central energy balance circuits. Serotonergic neurons within the caudal brainstem project widely within the brain, and elevated levels of 5-HT suppress food intake and reduce body weight [50]. Drugs that increase intrasynaptic 5-HT by blocking the 5-HT uptake have been used for the treatment of obesity. Mice with deletion of the 5-HT2c serotonin receptor subtype developed modest obesity [51]. Leptin increases 5-HT turnover, suggesting that these pathways can converge, but 5-HT2C-deficient mice retained an anorectic response to leptin. In addition to neuropeptides and transmitters, the function of these neural circuits is also influenced by metabolic fuels. Neurons in the hypothalamus that respond to changes in glucose levels may be the same as, or functionally linked to those neurons that respond to leptin and express the peptides discussed earlier. Biosynthesis of long-chain fatty acids and their utilisation in hypothalamus also play an important role in the regulation of energy homeostatic responses [52] (Figure 3.1).
Control of Energy Expenditure The hypothalamic melanocortin pathway regulates the two aspects of energy equation in a coherent way, whereby signals of excess energy result in a reduction of the intake of food energy while activating energy expenditure [21,53]. Human body utilises food
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Obesity Arcuate Nucleus
NPY/AgRP
–
α-MSH/CART +
+
+
–
MC4R/CART receptors
NPY receptors
PVN
–
–
LH
+
+ Thermogenesis
Food intake
Thermogenesis
Adipose tissue
Inhibits
Stimulates Leptin
Figure 3.1 Leptin acts on two subtypes of neurons in the arcuate nucleus. Leptin stimulates the first subtype that produces α-MSH and CART. α-MSH/CART neurons project to PVN and lateral hypothalamus and they inhibit MCH and orexins thus inhibiting food intake and regulate the secretion of pituitary neuropeptides and the sympathetic nervous system to effect energy expenditure. Absence of leptin activates the second subtype of neurons that produce NPY and AgRP. AgRP antagonises α-MSH at MC4R and NPY stimulates Y5 and Y1 receptors in the PVN thus stimulating food intake and reducing energy expenditure.
energy via three forms of thermogenesis: either obligatory related to BMR and part of diet-induced thermogenesis, or facultative which is activated acutely such as through shivering or physical activity when additional heat is required or adaptive when loss of heat energy occurs in response to external stimuli such as exposure to cold, diet and a variety of pathogenic stimuli, including infection, inflammation, cancer, injury and stress [54]. BMR is responsible for cellular metabolism, functioning of organs and maintenance of the body’s vital functions, and normally it accounts for 50 70% of total daily energy expenditure (TEE) [55]. Diet-induced thermogenesis is used to digest and absorb food after a meal. It accounts for about 10% of the TEE and does not vary greatly among individuals [56]. Physical activity accounts for 8 15% of the total energy expenditure where thermogenesis is derived from muscular activity [57]. Adaptive thermogenesis plays a role in energy regulation by heat production without performing actual work through uncoupling phosphorylation in the mitochondrion. Adaptive thermogenesis occurs mainly in the brown adipose tissue and skeletal muscles as a result of hypothalamic activation of sympathetic nervous system and hypothalamic thyroid axis, thus stimulating energy loss in response to various inputs that indicate excess body energy.
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The body produces energy by the oxidation of substrates such as fatty acids and glucose in the inner mitochondrial membrane via the electron transport system of the respiratory chain that results in the production of reducing equivalents nicotinamide adenine dinucleotide and flavin adenine dinucleotide. These are then oxidised to produce protons that are pumped to the outer surface of the inner mitochondrial membrane. Proton transport generates gradient potential that stimulates the oxidative phosphorylation of ADP to ATP via ATP synthase. However, energy coupling to ATP is not 100% efficient due to proton leak into the inner mitochondrial membrane that could contribute to resting metabolic rate by 20 50% [58]. Proton leak increases with the rise in gradient potential across the mitochondrial membrane and is enhanced by binding to uncoupling protein 1 (UCP1) resulting in respiration with heat production not linked to ATP. UCP1 belongs to a group of proteins involved in the transport of anionic fatty acids into the mitochondria that mainly functions in the brown adipose tissue [59]. Fatty acid uncoupling enhances proton binding to the protein thus facilitating entry into the inner mitochondrial membrane resulting in thermogenesis [60]. Other UCPs have been identified in various tissues including UCP2, UCP3, UCP4 and UCP5; however, none of them is involved in thermogenesis. Thermogenesis in brown adipose tissue, which is present in small amounts in human adults, could account for 20% of daily energy expenditure from as little as 50 g [61]. Heat production by brown adipose tissue is triggered and regulated primarily by sympathetic nervous system. Noradrenalin binds to the β3-receptors on the surfaces of brown adipocytes and stimulates lipolysis via cAMP-protein kinase A and activates the expression of PPARγ coactivator 1α (PGC-1α) that controls transcription of the UCP1 genes [62]. Fatty acids produced locally and recruited from the circulation act as substrate for oxidation and activate UCP1 with resultant thermogenesis [63]. The generation of heat is dependent on the role played by peroxisome proliferator-activated receptors (PPARs). In brown adipose tissues, all the three subtypes of PPARs are expressed but PPAR-α is in the highest concentration. PPAR-α activates lipolysis and fatty oxidation required for active thermogenesis. PPARγ is equally expressed in the both white and brown adipose tissues and involved in adipocyte differentiation. Human skeletal muscles compose approximately 40% of the total body mass. They account for 20 30% of the total resting oxygen uptake [64] and contribute to adrenalin-induced thermogenesis by up to 50% [65]. Skeletal muscles can increase heat production in response to cold via mitochondrial uncoupling [66]. However, the mechanism differs from that in brown adipose tissues as skeletal muscles express only UCP3 which is not involved in uncoupling in the muscle [67], and the sarco/endoplasmic reticulum Ca21-ATPase is likely to be the main pathway for adaptive thermogenesis [68]. Activation of hypothalamic thyroid axis is involved indirectly in thermogenesis in brown adipose tissue and skeletal muscle by stimulating lipolysis. Hence, alteration in the uncoupling respiration in brown adipose tissues and skeletal muscles could significantly affect energy balance and influence accumulation of fat stores and thus becomes a target for anti-obesity treatments (Figure 3.2).
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Obesity Noreadrenaline + β-3 receptors + c-AMP + +
Lipolysis
PGC-1 α
+ FFA PPARα
Induces and binds to
Induces
UCP1 H+ Codes for oxidation genes Codes for UCP1 genes
β oxidation
UCP1+ (H+)
Thermogenesis Mitochondria
Figure 3.2 Diet and cold stimulate the sympathetic nervous system. Noreadrenaline acts on β-3 receptors and stimulates cAMP which in turn stimulates lipolysis and PGC-1α. Free fatty acids act as substrate for oxidation, induce UCP1 and form ligands that induce PPARα. PPARα stimulates lipolysis and codes for the expression of genes involved in β oxidation. PGC-1α stimulation leads to the expression of genes that code for UCP1. UCP1 binds to anionic fatty acids and facilitates their transport into mitochondria and this in turn enhances protons produced via the respiratory chain to re-enter across the mitochondrial membrane and produce heat uncoupled to ATP production.
Adipogenesis Adipose tissue is an active endocrine and exocrine organ that responds to and produces hormonal and metabolic signals that play important role in regulation of energy balance and body weight. Thus adipocyte dysfunction could contribute to metabolic diseases in obesity. Moderate obesity results mainly from an increase in the size of the adipocytes due to accumulation of lipids (hypertrophic obesity), while more extreme obesity or obesity in childhood also implies an increase in the number of adipose cells (hyperplasic obesity) [69]. The capacity to make new adipocytes continues throughout life and can be activated by the size, frequency and composition of meals, and by other environmental factors. Almost 10% of adipocytes undergo turnover in the human adipose tissues each year [70]. The adipocyte cell lines are derived from a pluripotent stem cell precursor, which can differentiate into various types of mesodermic cells [71]. Stem cells first
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develop into preadipocytes that exhibit similar morphology to stem cells; however, they are committed to the adipogenic lineage and are no longer able to transform into osteoblasts, myocytes or chondrocytes. Secondly, preadipocytes differentiate to mature fat cells with accumulation of fat. Adipogenesis is tightly regulated at a molecular level by several transcription factors. The activity of these transcription factors is coordinated by extracellular signals regulated by related genes such as WNT signalling pathway. Peptides secreted by the WNT family have been shown to inhibit the early steps of adipogenesis, and suppression of WNT signalling by endogenous inhibitors is essential to generate adipocytes [71]. Two groups of transcription factors seem to be essential for the development of both white and brown adipose tissues CCAAT-enhancer binding proteins (C/EBPs) and PPARs. C/EBPβ and C/EBPδ are the key early regulators of adipogenesis; their expression is induced rapidly by insulin, and both of them are regulated by anti-adipogenic preadipocyte factor 1 [72]. In addition, C/EBPβ is regulated by proadipogenic desumoylating enzyme sentrin-specific peptidase 2 that reduces its degradation by sumoylation [73]. C/EBPβ and C/EBPδ act on the key adipogenic transcription factors C/EBPα, PPARγ and sterol regulatory element-binding protein (SREBP1) [74]. SREBP1, which specifically regulates the aspects of cholesterol and fatty acid metabolism, plays a key role in the generation of the endogenous lipid ligand that activates PPARγ [75]. PPARγ and C/EBPα promote expression of each other via a positive feedback loop. Genowide binding analyses have shown that PPARγ and C/EBPα cooperate on multiple binding sites to regulate a wide range of genes involved in developing and mature adipocytes [76]. They both induce the expression of genes involved in insulin sensitivity, lipogenesis and lypolysis including those encoding glucose transporter type 4, fatty-acid-binding protein 4 also known as adipocyte protein 2, lipoprotein lipase, sn-1-acylglycerol-3-phosphate acyl-transfrase 2, perilipin and the secretion of leptin and adiponectin. This transcription network is regulated by many factors such as C/EBP homologous protein 10 and members of Kruppel-like factor [74,77], in addition to an ever-expanding list of positive and negative regulators including phosphorylation which inhibits the binding of PPARγ to its target promoters [78]. PPARγ is considered a crucial regulator of adipogenesis and of many factors that influence the expansion of white adipose tissues.
Interactions Among Social, Environmental, Genetic and Behavioural Factors in Obesity In a comparative study, 247 Punjabi immigrants in London were noted to have significantly higher BMI values, serum cholesterol and fasting blood glucose against their siblings still living in Punjab, India [79]. Similarly, Ravussin et al. [80] reported a comparative study of Pima Indians living in remote rural regions
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of Mexico with those living in Arizona. Compared to Pima Indians living in rural Mexico, those living in Arizona had significantly higher BMI, higher cholesterol levels and a higher incidence of type-2 diabetes (11% vs 37% in women and 6% vs 54% in men). These two studies suggest that genetic influences on the development of obesity can be mitigated by environmental conditions. Epstein et al. [81] recently reported an association between the ‘reinforcing value of food’ phenotype and ad libitum energy intake moderated by the dopamine transporter gene (SLc6A3) and the dopamine 2 receptor gene (DRD2). In this study of 88 smokers of American European descent, the subjects who lacked the SLc6A4 allele consumed more total energy than the participants with SLC6A3 genotypes. Furthermore, the subjects with AI allele of DRD2 scored high on reinforcing value of food and consumed more total calories compared to participants with any other DRD2 genotype. This unique study has essentially focused on the genetics of food reward as they relate to dopamine pathways. This is an area of future research with considerable potential.
Conclusion Obesity is a complex disorder that results from excessive energy intake without corresponding expenditure leading to accumulation of adipose tissues with serious health consequences. Adiposity is rarely inherited as a monogenic trait, but in the case of 70 80% of obese people, it is a manifestation of interaction between multiple gene loci and environmental factors that induces changes in the biological processes that control energy homeostasis and body weight. Understanding the biochemical and molecular process involved in energy regulation will facilitate the development of innovate preventive/treatment measures in susceptible individuals in a world of abundance of nutrients augmented with reduced physical activity. It must be recognised that the onset of obesity is a developmental process that may be influenced at different genetic or environmental influences at different stages of life. Therefore, prospective studies focusing on the critical growth periods for obesity such as growth in the intra-uterine environment, adiposity in early childhood and adolescence and life experiences specific to those periods would be of immense interest. Furthermore, studies around the genetic environmental interactions could elucidate the nature of environmental influences, reinforcing value of food, delayed satiation, home environments and the effect of cognitive stimulation in childhood and eating in the absence of hunger. All these factors have been linked to obesity status. It will be equally interesting to study the phenotype of individuals who will seek out ‘obesity-promoting environments’ such as fast-food restaurants.
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43. Wortley KE, Anderson KD, Yasenchak J, et al. Agouti-related proteindeficient mice display an age-related lean phenotype. Cell Metab. 2005;2:421 427. 44. Farooqi IS, Yeo GS, Keogh JM, et al. Dominant and recessive inheritance of morbid obesity associated with melanocortin 4 receptor deficiency. J Clin Invest. 2000;106:271 279. 45. Butler AA, Kesterson RA, Khong K, et al. A unique metabolic syndrome causes obesity in the melanocortin-3-receptor-deficient mouse. Endocrinology. 2000;141: 3518 3521. 46. Hardie DG, Hawley SA, Scott JW. AMP-activated protein kinase development of the energy sensor concept. J Physiol. 2006;574:7 15. 47. Minokoshi Y. AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature. 2004;428:569 574. 48. Elias CF. Leptin activates hypothalamic CART neurons projecting to the spinal cord. Neuron. 1998;21:1375 1385. 49. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature. 2000;407:908 913. 50. Leibowitz SF, Alexander JT. Hypothalamic serotonin in control of eating behavior, meal size, and body weight. Biol Psychiatry. 1998;44:851 864. 51. Nonogaki K, Strack AM, Dallman MF, Tecott LH. Leptin-independent hyperphagia and type 2 diabetes in mice with a mutated serotonin 5-HT2C receptor gene. Nat Med. 1998;4:1152 1156. 52. Obici S, Feng Z, Arduini A, Conti R, Rossetti L. Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production. Nat Med. 2003;9:756 761. 53. Cone RD. The central melanocortin system and energy homeostasis. Trends Endocrinol Metab. 1999;10:211 216. 54. Rothwell NJ. CNS regulation of thermogenesis. Crit Rev Neurobiol. 1994;8:1 10. 55. Levine JA. Nonexercise activity thermogenesis liberating the life-force. J Intern Med. 2007;262:273 287. 56. Saito M, Okamatsu-Ogura Y, Matsushita M, Watanabe K, Yoneshiro T, Nio-Kobayasssssshi J. High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity. Diabetes. 2009; 58:1526 1531. 57. Ravussin E, Lillioja S, Anderson TE, Christin L, Bogardus C. Determinants of 24-hour energy expenditure in man. Methods and results using a respiratory chamber. J Clin Invest. 1986;78:1568 1578. 58. Rolfe DF, Brown GC. Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiol Rev. 1997;77:731 758. 59. Villarroya F, Iglesial R, Giralt M. PPARs in the control of uncoupling proteins gene expression. PPAR Res. 2007;2007:74364 [Article ID 74364]. (published on line 2006, November 28, doi: 10.1155/2007/74364). 60. Wojtczak L, Scho¨nfeld P. Effect of fatty acids on energy coupling processes in mitochondria. Biochim Biophys Acta. 1993;1183:41 57. 61. Rothwell NJ, Stock MJ. A role for brown adipose tissue in diet-induced thermogenesis. Nature. 1979;281:31 35. 62. Luquest S, Lopez-Soriano J, Holst D, Gaudel C, Jehl-Pietri C, Fredenrich A. Roles of peroxisome proliferator-activated receptor delta (PPARdelta) in the control of fatty acid catabolism. A new target for the treatment of metabolic syndrome. Biochimie. 2004;86 (11):833 837.
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63. Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev. 2004;84:277 359. 64. Zurlo F, Larson K, Bogardus C, Ravussin E. Skeletal muscle metabolism is a major determinant of resting energy expenditure. J Clin Invest. 1990;86:1423 1427. 65. Astrup A, Bulow J, Madsen J, Christensen NJ. Contribution of BAT and skeletal muscle to thermogenesis induced by ephedrine in man. Am J Physiol. 1985;248:507 515. 66. Sander L, Wijers J, Schrauwen P, Saris WHM, van Marken Lichtenbelt D. Human skeletal muscle mitochondrial uncoupling is associated with cold induced adaptive thermogenesis. PLoS One. 2008;3(3):1777. 67. Azzu V, Jastroch M, Divakaruni AS, Brand MD. The regulation and turnover of mitochondrial uncoupling proteins. Biochim Biophys Acta. 2010;1797:785 791. 68. de Meis L. Role of the sarcoplasmatic reticulum Ca21-ATPase on heat production and thermogenesis. Biosci Rep. 2001;21:113 137. 69. Spiegelman BM, Flier JS. Lipogenesis and obesity: rounding out the picture. Cell. 1996;87:377 389. 70. Spalding KL, Arner E, Westermark PO, et al. Dynamics of fat cell turnover in humans. Nature. 2008;453:783 787. 71. Christodoulides C, Lagathu C, Sethi JK, VidalPuig A. Adipogenesis and WNT signaling. Trends Endocrinol Metab. 2009;20:16 24. 72. Wang Y, Sul HS. Pref-1 regulates mesenchymal cell commitment and differentiation through Sox9. Cell Metab. 2009;9:287 302. 73. Chung SS, Ahn BY, Kim M, et al. Control of adipogenesis by the SUMO specific protease SENP2. Mol Cell Biol. 2010;30:2135 2146. 74. White UA, Stephens JM. Transcriptional factors that promote formation of white adipose tissue. Mol Cell Endocrinol. 2010;318:10 14. 75. Kim JB, Wright HM, Wright M, Spiegelman BM. ADD1/SREBP1 activates PPAR gamma through the production of endogenous ligand. Proc Natl Acad Sci U S A. 1998;95:4333 4337. 76. Lefterova MI, Zhang Y, Steger DJ, et al. PPARgamma and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale. Genes Dev. 2008;22:2941 2952. 77. Rosen ED, MacDougald OA. Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol. 2006;7:885 896. 78. Helenius K, Yang Y, Alasaari J, Makela TP. Mat1 inhibits peroxisome proliferatoractivated receptor gamma-mediated adipocyte differentiation. Mol Cell Biol. 2009;29:315 323. 79. Bhatnagar D, Anand IS, Durrington PN, Patel DJ, et al. Coronary risk factors in people from Indian subcontinent living in West London and their siblings in India. Lancet. 1995;345(8947):405 409. 80. Ravussin E, Valencia ME, Esparza J, Bennett P, Schulz LO. Effects of a traditional lifestyle on obesity in Pima Indians. Diabetes Care. 1994;17(9):1067 1074. 81. Epstein LH, Wright SM, Paluch RA, Leddy JJ, et al. Relation between food reinforcement and deopamine genotypes and its effect on food intake in smokers. Am J Clin Nutr. 2004;80(1):82 88.
Introduction Obesity is recognised as a global epidemic and is rising in children and in both men and women up to the age of 60. In 2008, the WHO reported that among adults, 20 and older, more than 200 million men and nearly 300 million women were obese. The rate of obesity in the United Kingdom has increased by fourfold over the past 30 years, an incidence of 22% in men and 24% in women in 2008/2009 [1]. Similarly, in the United States, comparable figures were 35.5% and 35.8% in 2009/2010 [2]. Obesity (BMI . 30 kg/m2) is a heterogeneous group of disorders that results from the imbalance in energy homeostasis, whereby food energy intake exceeds energy expenditure in genetically susceptible individuals. Such excess energy is stored in the most efficient form as triglycerides in white adipose tissues. Obesity poses serious health problems and reduces longevity from associated co-morbidities such as insulin resistance, type-2 diabetes, hyperlipidaemia, hypertension and various cancers [3,4]. Obesity thus may become the most challenging health problem with huge economic impact on health services. From women’s perspective, obesity further predisposes them to hyperandrogenicity with oligomenorrhoea/amenorrhoea and reduction in fertility in addition to health risks related to pregnancy and childbirth. For most people, the human body maintains its normal weight so that at times of excess nutrients, intake is dissipated through increased physical activity, enhanced basal metabolic rate (BMR) and adaptive thermogenesis in response to daily fluctuation in food intake. The hypothalamus regulates energy homeostasis through integrating neural, hormonal and metabolic signals from peripheral and central nervous system (CNS), endocrine glands, including adipose tissue, and blood metabolites such as glucose and fatty acids. Disruption of the control mechanism whether congenital or acquired results in accumulation of excess energy as lipids. Obesity. DOI: http://dx.doi.org/10.1016/B978-0-12-416045-3.00003-0 © 2013 Elsevier Inc. All rights reserved.
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Hereditary Factors Influencing BMI Studies from twins and adoption that have compared phenotypic correlations for body fat between groups of individuals, varying in genetic relatedness, have yielded interesting observations. It appears that fat mass is genetically influenced among individuals who are more genetically similar such as in the case of monozygotic twins than dizygotic twins [5]. Even for twins reared apart, the estimated heritability was at 65 75% for BMI [6]. Furthermore, results of longitudinal behaviour genetic studies suggest that there are age-specific genetic effects on BMI, such that different obesity-promoting genes may become active at different ages across the lifespan [7]. The ‘Human Obesity Gene Map’ has been annually updated since 1994 [8]. This comprehensive compendium summarises evidences from the four classes of human studies: a. Obesity due to a single gene in digenic mutation. b. Obesity associated with Mendelian disorders such as Prader Willi syndrome or Bardot Biedle syndrome. c. Associated studies that test whether candidate genes are associated with obesity phenotypes among samples of unrelated participants. d. Linkage studies that test for causal association between genomic regions and obesity phenotypes in cohort of families.
This compendium has shown that the number of genes associated with obesityrelated traits has increased dramatically over the past decade, thereby suggesting that the platform of specific genes that might contribute to obesity is large and involves loci throughout the genome.
Genetic Molecular Interaction of Obesity Severe obesity in humans can rarely result from the monogenic mutations of genes involved in hypothalamic appetite control pathways. The genes which encode leptin receptor [9], proopiomelanocortin (POMC) [10], prohormone convertase1 [11], leptin [12], melanocortin 3 receptors (MC3R) [13] and melanocortin 4 receptors (MC4R) [14] are involved. As noted above, human obesity id polygenic in origin [8] and involves a network of genes with several hundred loci located on nearly all the chromosomes including Y chromosome thus affecting obesity-related phenotypes. Monogenic mutations are not involved in the development of common forms of obesity where subtle interaction between numerous related genetic variants and environmental factors, such as diet and exercise, results in the overexpression of molecules that affect energy homeostasis leading to a positive or negative impact. Chronic exposure to environmental factors leads to single nucleotide polymorphism [15] or structural changes in DNA through methylation that promotes gene expression in susceptible individuals [16]. In future, this opens up the possibility of genotyping the individuals in order to identify susceptible individuals to obesogenic stimuli, that may allow for preventive measures to be implemented at individual level.
Genetic and Molecular Basis of Obesity
Regulation of Energy Balance
25
Molecular Basis
The main biochemical processes involved in energy regulation are those related to the control of energy intake, energy output (expenditure) and adipogenesis. These inter-related processes are governed at the cellular level by a wide range of inducible molecules and their associated genes, and such cross-regulation may limit the effectiveness of available treatments. This chapter will now focus on the molecules and their related genes responsible for the main biochemical processes that control energy homeostasis.
Regulation of Food Energy Intake Feeding behaviour may be regulated via inter-related short- and long-term control. Short-term control is involved with individual meal intake where hunger and satiety signals reflect the acute energy status of the body. Feeding process is affected by metabolic, neural and endocrine factors and is modified by powerful sensory, emotional and cognitive inputs. Ultimately, all of these factors must be integrated, so that decisions to begin and end periods of feeding will result [17]. Gastrointestinal hormones play an important role in the acute stage of feeding, e.g. plasma levels of ghrelin increase during fasting and infusion of this hormone stimulates appetite, an effect mediated via agoutirelated protein (AgRP) [18]. Likewise, mechanical distension of the stomach and release of gut peptides, such as cholecystokinin, gastrin-releasing peptide 29 (GRP-29) and peptide YY3 36, produce satiety signals [19,20]. Long-term signals that reflect the status of energy stores are mainly that of leptin and insulin, and they provide information to the CNS to regulate feeding behaviour and promote energy homeostasis. As a result of these feedback signals, the energy stores and body weight remain generally stable for most humans over long period of time despite the wide variations in day-to-day food intake. However, chronic excessive food intake combined with reduced energy utilisation (i.e. exercise) limits their effectiveness and leads to increased adiposity that is individually regulated within narrow margins, resulting in a constant set point to which body weight would eventually return after fat loss or gain. In addition, resetting of feedback signals to higher thresholds in hypothalamus and elsewhere results in increased levels of circulating metabolites, e.g. glucose and lipids that reflect resistance to leptin and insulin action.
The Role of Hypothalamus Hypothalamus is critical for the regulation of homeostatic processes such as feeding, thermoregulation and reproduction. It is the primary receptor of peripheral signals that indicate food availability [21]. It receives and integrates mechanical,
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hormonal, metabolic and peripheral neural inputs with those from higher centres: the brainstem, cerebral cortex, olfactory areas and limbic system. Hypothalamus in response produces neurotransmitters and neurohormones resulting in behavioural, autonomic and endocrine responses that control appetite and energy balance. The central role of the hypothalamus in appetite and satiety was established from the studies where lesions in the ventromedial hypothalamus caused obesity, while, conversely, lesions in the lateral hypothalamus resulted in leanness [22]. The arcuate nucleus plays a critical role in the regulation of energy homeostasis. It occupies more than one-half of the hypothalamus and lies around the base of the third ventricle immediately above the median eminence where the capillary endothelium lacks tight junctions forming an incomplete blood brain barrier (BBB) thus allowing larger proteins and hormones to readily access the arcuate nucleus neurons from circulation. The hypothalamic melanocortin signalling system is also heavily regulated by circulating hormones, mainly leptin and insulin, that provide information about body energy status and reserves. Hormonal signals from leptin and insulin inhibit hypothalamic orexigenic neurons [17], while, conversely, the anorexigenic neurons are stimulated by inputs from leptin and insulin [23]. Overall, it is apparent that key neurons in the hypothalamus, most likely in the arcuate nucleus, are primed to integrate a variety of hormonal and metabolic signals in order to interpret the state of energy balance, and, in turn, mediate the necessary metabolic and behavioural responses in order to compensate for deviations from homeostasis.
The Role of Leptin Human leptin (derived from Greek leptos meaning thin) is a protein of 167 amino acids secreted mainly by adipocytes that express the ob gene. The level of circulating leptin is directly proportional to the total amount of body fat [24]. Its levels are higher in women than in men as its production is stimulated by oestrogen and suppressed by testosterone. Leptin levels drop with fasting and increase with food intake, an effect mediated in part by insulin stimulation of adipocytes, and both hormones act on hypothalamus triggering similar responses [25]. Various regulatory elements have been identified within the leptin promoter, e.g. cAMP and glucocorticoid response elements, and CCATT/enhancer binding sites, suggesting a direct regulation of leptin expression through membrane and transcriptional pathways [26]. Leptin is considered the main signal that provides information to the CNS reflecting energy stores in the body. Leptin binds to receptors (Lep R) in the arcuate nucleus and other hypothalamic nuclei resulting in changes in feeding behaviour, with suppression of appetite [27], and an increase in metabolic activity and energy expenditure (thermogenesis) that on the long term regulates energy balance and maintains normal body weight, despite wide variation in daily calorie intake in most individuals.
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The central effects of leptin signals stimulate the anorexigenic neuropeptides, alpha-melonocyte stimulating hormone (α-MSH) and cocaine- and amphetaminerelated transcript (CART), and inhibit the orexigenic neuropeptides, neuropeptides Y (NPY) and agouti-related protein AgRP in the arcuate nucleus [27]. Leptin also inhibits melanin-concentrating hormone (MCH) and orexins in lateral hypothalamus [22]. This in turn stimulates neuroendocrine response that results in the production of thyrotropin-releasing hormone [28] and activates the sympathetic nervous system that results in increased metabolism of tissues mediated via increased metabolic rate. Deficiency of either leptin or its receptors secondary to gene mutation in ob and db (Lep R) loci causes severe obesity, the first of which can be controlled with exogenous leptin. Direct peripheral action of leptin on many tissues, including adipose tissue, stimulates lipolysis and fatty acid oxidation [29]. Local leptin production in the stomach plays a role in short-term energy balance as its level increases under the effect of CCK and gastrin thus mediating satiety signals indicating the filling effect of a meal. Leptin levels in most obese people are high without corresponding gene mutation indicating a disruption in the feedback signals reflecting a state of insensitivity to leptin (hyperleptinaemia) similar to that of insulin. The mechanism involved is related to reduction in leptin transport through BBB where leptin levels in the brain are lower than the plasma levels [30] (Table 3.1).
Melanocortin Signalling Pathway Leptin acts on two distinct subtypes of adjacent neurons in the arcuate nucleus in opposing manner. The first subtype of neurons co-expresses mRNAs encoding anorexigenic peptides, CART and α-MSH (derived from POMC), and leptin induces their expression [22,25,31]. The other subtype co-expresses the orexigenic Table 3.1 Leptin Targets of Neurotransmitters and Peptides that Affect Feeding Process Stimulation of Feeding
Inhibition of Feeding
Neuropeptide Y Agouti-related peptide Melanin-concentrating hormone Orexins Ghrelin Galanin Growth hormone-releasing hormone Opioid peptides Gamma-aminobutyric acid Nitric oxide Noreadrenaline
Alpha-melanocyte stimulating hormone Cocaine- and amphetamine-regulated transcript Corticotropin-releasing hormone Calcitonin gene-related peptide Urocortin Neurotensin Serotonin Glucagon Cholecystokinin Glucagon-like peptide 1 Bombesin/gastrin-releasing peptide
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peptides NPY and AgRP, and leptin reduces their expression [17,22,25,31]. Secreted α-MSH binds to MC4R and to a lesser extent to MR3C to produce tonic inhibition with anorexic effect and increase in thermogenesis. The axonal projections of neuropeptide α-MSH also synapse with second-order neurons in the paraventricular nucleus (PVN) and lateral hypothalamus. The PVN regulates the secretion of pituitary hormones by the release of neuropeptides such as thyrotropin- and corticotrophin-releasing hormone via the projections in the median eminence and regulates autonomic nervous system via the projections to autonomic pregangilionic neurons. Direct projection of these arcuate melanocortinergic neurons (AgRP and α-MSH) onto the neurons within the lateral hypothalamus inhibits the expression of the orexigenic neuropeptides MCH and orexins [32]. Expression of MCH stimulates food intake. Deletion of the MCH gene causes a lean phenotype [33], and transgenic overexpression promotes obesity. Two neuropeptides orexins A and B are involved in the regulation of food intake [34]. Orexins function through G-protein-coupled receptors and their expression in the brain seems to be limited to the neurons of the lateral hypothalamus and nearby region. Central administration of orexins stimulates food intake, and production of orexin increases with fasting [34]. Recent studies indicated that the POMC-derived neuropeptides β-MSH plays a critical role in the hypothalamic control of body weight in humans [35]. Conversely, absence of leptin induced by calorie restriction or fasting activates orexigenic NPY and AgRP [36]. The activation of these orexigenic neurons leads to the inhibition of anorexigenic signalling in two ways. First, AgRP/NPY neurons synapse directly with POMC neurons, providing an inhibitory tone. Second, AgRP itself is an effective antagonist of α-MSH at MC4Rs. Release of AgRP/NPY neuropeptides stimulates food intake and reduces energy expenditure. NPY acts via Y1, Y2 and Y5 G-protein-coupled receptors expressed in the hypothalamic neurons. The Y5 and the Y1 receptors mediate the stimulatory effects of NPY on food intake [37]. The Y2 receptor mediates an inhibitory effect of NPY at low concentrations that could be important for basal control of body weight [38]. The action of NPY on its receptors can be affected by other neurotransmitters, such as glucagon-like peptide 1, which inhibits food intake and diminishes the orexigenic effect of NPY by antagonising NPY receptors Y5 and Y1 [39]. In ob/ob mice model, the role of NPY in full response to leptin deficiency was determined. The results showed that NPY deficiency prevents obesity and other features of ob/ob mice. However, NPY-deficient mice feed normally and gain normal body weight [40]. Adrenal glucocorticoids potentiate the orexigenic effect of NPY by acting as endogenous antagonists of leptin and insulin thus participating in energy regulation [41]. Adrenalectomy mitigates the effect of fasting to increase both appetite and NPY expression and potentiates the anorexigenic and slimming effects of insulin and leptin; administration of glucocorticoids compensates these effects [41]. AgRP antagonises the effect of α-MSH on MC4R. Activation of MC4R by α-MSH reduces food intake, while suppression of MC4R signalling through this receptor by the endogenous antagonist AgRP increases feeding and diminishes
Genetic and Molecular Basis of Obesity
29
the hypophagic response to leptin [25,42]. AgRP deficiency leads to a lean phenotype of mice with extended lifespan when the animals received a high-fat diet [43]. Gene mutation at MC4R locus affects 6% of humans with severe obesity, and most affected individuals have a single mutant allele that causes obesity through haploinsufficiency rather than a dominant-negative mechanism [44]. The obesity in several other rare human syndromes also converges on this pathway. Targeted deletion of the MC3R produced obesity in mice [45]. However, obesity from this lesion occurs without the hyperphagia seen in MC4R mutants and may be associated with a loss of lean body mass. Thus, these two melanocortin receptors can cause obesity through distinct physiological mechanisms. Recent studies have strongly implicated that adenosine monophosphate-activated protein kinase (AMPK) pathway plays a central role in hypothalamic control of energy homeostasis by mediating the inputs from multiple hormones, peptides, neurotransmitters and nutrients [46]. Leptin inhibition of AMPK activity in the arcuate nucleus and PVN in hypothalamus is necessary to produce its anorexic effect [47]. The melanocortin system is not the only neuropeptides system involved in weight regulation. Leptin regulates CART expression in arcuate POMC neurons, and CART axons innervate sympathetic preganglionic neurons in the thoracic spinal cord [48]. Among its central effects, leptin further regulates many other neuropeptides, including corticotropin-releasing hormone, growth hormone-releasing hormone and galanin that have been described to participate in energy regulatory pathways. Ghrelin, a peptide expressed in stomach and brain, promotes hyperphagia and obesity, an effect mediated through NPY/AgRP neurons [49]. Noradrenalin, dopamine and serotonin (5-HT) are also known to be involved in central energy balance circuits. Serotonergic neurons within the caudal brainstem project widely within the brain, and elevated levels of 5-HT suppress food intake and reduce body weight [50]. Drugs that increase intrasynaptic 5-HT by blocking the 5-HT uptake have been used for the treatment of obesity. Mice with deletion of the 5-HT2c serotonin receptor subtype developed modest obesity [51]. Leptin increases 5-HT turnover, suggesting that these pathways can converge, but 5-HT2C-deficient mice retained an anorectic response to leptin. In addition to neuropeptides and transmitters, the function of these neural circuits is also influenced by metabolic fuels. Neurons in the hypothalamus that respond to changes in glucose levels may be the same as, or functionally linked to those neurons that respond to leptin and express the peptides discussed earlier. Biosynthesis of long-chain fatty acids and their utilisation in hypothalamus also play an important role in the regulation of energy homeostatic responses [52] (Figure 3.1).
Control of Energy Expenditure The hypothalamic melanocortin pathway regulates the two aspects of energy equation in a coherent way, whereby signals of excess energy result in a reduction of the intake of food energy while activating energy expenditure [21,53]. Human body utilises food
30
Obesity Arcuate Nucleus
NPY/AgRP
–
α-MSH/CART +
+
+
–
MC4R/CART receptors
NPY receptors
PVN
–
–
LH
+
+ Thermogenesis
Food intake
Thermogenesis
Adipose tissue
Inhibits
Stimulates Leptin
Figure 3.1 Leptin acts on two subtypes of neurons in the arcuate nucleus. Leptin stimulates the first subtype that produces α-MSH and CART. α-MSH/CART neurons project to PVN and lateral hypothalamus and they inhibit MCH and orexins thus inhibiting food intake and regulate the secretion of pituitary neuropeptides and the sympathetic nervous system to effect energy expenditure. Absence of leptin activates the second subtype of neurons that produce NPY and AgRP. AgRP antagonises α-MSH at MC4R and NPY stimulates Y5 and Y1 receptors in the PVN thus stimulating food intake and reducing energy expenditure.
energy via three forms of thermogenesis: either obligatory related to BMR and part of diet-induced thermogenesis, or facultative which is activated acutely such as through shivering or physical activity when additional heat is required or adaptive when loss of heat energy occurs in response to external stimuli such as exposure to cold, diet and a variety of pathogenic stimuli, including infection, inflammation, cancer, injury and stress [54]. BMR is responsible for cellular metabolism, functioning of organs and maintenance of the body’s vital functions, and normally it accounts for 50 70% of total daily energy expenditure (TEE) [55]. Diet-induced thermogenesis is used to digest and absorb food after a meal. It accounts for about 10% of the TEE and does not vary greatly among individuals [56]. Physical activity accounts for 8 15% of the total energy expenditure where thermogenesis is derived from muscular activity [57]. Adaptive thermogenesis plays a role in energy regulation by heat production without performing actual work through uncoupling phosphorylation in the mitochondrion. Adaptive thermogenesis occurs mainly in the brown adipose tissue and skeletal muscles as a result of hypothalamic activation of sympathetic nervous system and hypothalamic thyroid axis, thus stimulating energy loss in response to various inputs that indicate excess body energy.
Genetic and Molecular Basis of Obesity
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The body produces energy by the oxidation of substrates such as fatty acids and glucose in the inner mitochondrial membrane via the electron transport system of the respiratory chain that results in the production of reducing equivalents nicotinamide adenine dinucleotide and flavin adenine dinucleotide. These are then oxidised to produce protons that are pumped to the outer surface of the inner mitochondrial membrane. Proton transport generates gradient potential that stimulates the oxidative phosphorylation of ADP to ATP via ATP synthase. However, energy coupling to ATP is not 100% efficient due to proton leak into the inner mitochondrial membrane that could contribute to resting metabolic rate by 20 50% [58]. Proton leak increases with the rise in gradient potential across the mitochondrial membrane and is enhanced by binding to uncoupling protein 1 (UCP1) resulting in respiration with heat production not linked to ATP. UCP1 belongs to a group of proteins involved in the transport of anionic fatty acids into the mitochondria that mainly functions in the brown adipose tissue [59]. Fatty acid uncoupling enhances proton binding to the protein thus facilitating entry into the inner mitochondrial membrane resulting in thermogenesis [60]. Other UCPs have been identified in various tissues including UCP2, UCP3, UCP4 and UCP5; however, none of them is involved in thermogenesis. Thermogenesis in brown adipose tissue, which is present in small amounts in human adults, could account for 20% of daily energy expenditure from as little as 50 g [61]. Heat production by brown adipose tissue is triggered and regulated primarily by sympathetic nervous system. Noradrenalin binds to the β3-receptors on the surfaces of brown adipocytes and stimulates lipolysis via cAMP-protein kinase A and activates the expression of PPARγ coactivator 1α (PGC-1α) that controls transcription of the UCP1 genes [62]. Fatty acids produced locally and recruited from the circulation act as substrate for oxidation and activate UCP1 with resultant thermogenesis [63]. The generation of heat is dependent on the role played by peroxisome proliferator-activated receptors (PPARs). In brown adipose tissues, all the three subtypes of PPARs are expressed but PPAR-α is in the highest concentration. PPAR-α activates lipolysis and fatty oxidation required for active thermogenesis. PPARγ is equally expressed in the both white and brown adipose tissues and involved in adipocyte differentiation. Human skeletal muscles compose approximately 40% of the total body mass. They account for 20 30% of the total resting oxygen uptake [64] and contribute to adrenalin-induced thermogenesis by up to 50% [65]. Skeletal muscles can increase heat production in response to cold via mitochondrial uncoupling [66]. However, the mechanism differs from that in brown adipose tissues as skeletal muscles express only UCP3 which is not involved in uncoupling in the muscle [67], and the sarco/endoplasmic reticulum Ca21-ATPase is likely to be the main pathway for adaptive thermogenesis [68]. Activation of hypothalamic thyroid axis is involved indirectly in thermogenesis in brown adipose tissue and skeletal muscle by stimulating lipolysis. Hence, alteration in the uncoupling respiration in brown adipose tissues and skeletal muscles could significantly affect energy balance and influence accumulation of fat stores and thus becomes a target for anti-obesity treatments (Figure 3.2).
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Obesity Noreadrenaline + β-3 receptors + c-AMP + +
Lipolysis
PGC-1 α
+ FFA PPARα
Induces and binds to
Induces
UCP1 H+ Codes for oxidation genes Codes for UCP1 genes
β oxidation
UCP1+ (H+)
Thermogenesis Mitochondria
Figure 3.2 Diet and cold stimulate the sympathetic nervous system. Noreadrenaline acts on β-3 receptors and stimulates cAMP which in turn stimulates lipolysis and PGC-1α. Free fatty acids act as substrate for oxidation, induce UCP1 and form ligands that induce PPARα. PPARα stimulates lipolysis and codes for the expression of genes involved in β oxidation. PGC-1α stimulation leads to the expression of genes that code for UCP1. UCP1 binds to anionic fatty acids and facilitates their transport into mitochondria and this in turn enhances protons produced via the respiratory chain to re-enter across the mitochondrial membrane and produce heat uncoupled to ATP production.
Adipogenesis Adipose tissue is an active endocrine and exocrine organ that responds to and produces hormonal and metabolic signals that play important role in regulation of energy balance and body weight. Thus adipocyte dysfunction could contribute to metabolic diseases in obesity. Moderate obesity results mainly from an increase in the size of the adipocytes due to accumulation of lipids (hypertrophic obesity), while more extreme obesity or obesity in childhood also implies an increase in the number of adipose cells (hyperplasic obesity) [69]. The capacity to make new adipocytes continues throughout life and can be activated by the size, frequency and composition of meals, and by other environmental factors. Almost 10% of adipocytes undergo turnover in the human adipose tissues each year [70]. The adipocyte cell lines are derived from a pluripotent stem cell precursor, which can differentiate into various types of mesodermic cells [71]. Stem cells first
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develop into preadipocytes that exhibit similar morphology to stem cells; however, they are committed to the adipogenic lineage and are no longer able to transform into osteoblasts, myocytes or chondrocytes. Secondly, preadipocytes differentiate to mature fat cells with accumulation of fat. Adipogenesis is tightly regulated at a molecular level by several transcription factors. The activity of these transcription factors is coordinated by extracellular signals regulated by related genes such as WNT signalling pathway. Peptides secreted by the WNT family have been shown to inhibit the early steps of adipogenesis, and suppression of WNT signalling by endogenous inhibitors is essential to generate adipocytes [71]. Two groups of transcription factors seem to be essential for the development of both white and brown adipose tissues CCAAT-enhancer binding proteins (C/EBPs) and PPARs. C/EBPβ and C/EBPδ are the key early regulators of adipogenesis; their expression is induced rapidly by insulin, and both of them are regulated by anti-adipogenic preadipocyte factor 1 [72]. In addition, C/EBPβ is regulated by proadipogenic desumoylating enzyme sentrin-specific peptidase 2 that reduces its degradation by sumoylation [73]. C/EBPβ and C/EBPδ act on the key adipogenic transcription factors C/EBPα, PPARγ and sterol regulatory element-binding protein (SREBP1) [74]. SREBP1, which specifically regulates the aspects of cholesterol and fatty acid metabolism, plays a key role in the generation of the endogenous lipid ligand that activates PPARγ [75]. PPARγ and C/EBPα promote expression of each other via a positive feedback loop. Genowide binding analyses have shown that PPARγ and C/EBPα cooperate on multiple binding sites to regulate a wide range of genes involved in developing and mature adipocytes [76]. They both induce the expression of genes involved in insulin sensitivity, lipogenesis and lypolysis including those encoding glucose transporter type 4, fatty-acid-binding protein 4 also known as adipocyte protein 2, lipoprotein lipase, sn-1-acylglycerol-3-phosphate acyl-transfrase 2, perilipin and the secretion of leptin and adiponectin. This transcription network is regulated by many factors such as C/EBP homologous protein 10 and members of Kruppel-like factor [74,77], in addition to an ever-expanding list of positive and negative regulators including phosphorylation which inhibits the binding of PPARγ to its target promoters [78]. PPARγ is considered a crucial regulator of adipogenesis and of many factors that influence the expansion of white adipose tissues.
Interactions Among Social, Environmental, Genetic and Behavioural Factors in Obesity In a comparative study, 247 Punjabi immigrants in London were noted to have significantly higher BMI values, serum cholesterol and fasting blood glucose against their siblings still living in Punjab, India [79]. Similarly, Ravussin et al. [80] reported a comparative study of Pima Indians living in remote rural regions
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of Mexico with those living in Arizona. Compared to Pima Indians living in rural Mexico, those living in Arizona had significantly higher BMI, higher cholesterol levels and a higher incidence of type-2 diabetes (11% vs 37% in women and 6% vs 54% in men). These two studies suggest that genetic influences on the development of obesity can be mitigated by environmental conditions. Epstein et al. [81] recently reported an association between the ‘reinforcing value of food’ phenotype and ad libitum energy intake moderated by the dopamine transporter gene (SLc6A3) and the dopamine 2 receptor gene (DRD2). In this study of 88 smokers of American European descent, the subjects who lacked the SLc6A4 allele consumed more total energy than the participants with SLC6A3 genotypes. Furthermore, the subjects with AI allele of DRD2 scored high on reinforcing value of food and consumed more total calories compared to participants with any other DRD2 genotype. This unique study has essentially focused on the genetics of food reward as they relate to dopamine pathways. This is an area of future research with considerable potential.
Conclusion Obesity is a complex disorder that results from excessive energy intake without corresponding expenditure leading to accumulation of adipose tissues with serious health consequences. Adiposity is rarely inherited as a monogenic trait, but in the case of 70 80% of obese people, it is a manifestation of interaction between multiple gene loci and environmental factors that induces changes in the biological processes that control energy homeostasis and body weight. Understanding the biochemical and molecular process involved in energy regulation will facilitate the development of innovate preventive/treatment measures in susceptible individuals in a world of abundance of nutrients augmented with reduced physical activity. It must be recognised that the onset of obesity is a developmental process that may be influenced at different genetic or environmental influences at different stages of life. Therefore, prospective studies focusing on the critical growth periods for obesity such as growth in the intra-uterine environment, adiposity in early childhood and adolescence and life experiences specific to those periods would be of immense interest. Furthermore, studies around the genetic environmental interactions could elucidate the nature of environmental influences, reinforcing value of food, delayed satiation, home environments and the effect of cognitive stimulation in childhood and eating in the absence of hunger. All these factors have been linked to obesity status. It will be equally interesting to study the phenotype of individuals who will seek out ‘obesity-promoting environments’ such as fast-food restaurants.
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