PMC:4195273 / 77746-92977 JSONTXT

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of CART in human\nAs introduced earlier, evolutionary conservation has been demonstrated for CART in the neuroendocrine system across various mammalian species in the contexts of isoform structure, expression distribution pattern as well as functional implications, including a role of CART in the regulation of energy balance in human (Hager et al., 1998; Challis et al., 2000; del Giudice et al., 2001; Yamada et al., 2002; Dominguez et al., 2004a; Guerardel et al., 2005; Yanik et al., 2006; Rigoli et al., 2010). First, a genome-wide scan for human obesity-susceptibility loci in obese French Caucasian families (Hager et al., 1998) revealed a clear linkage to the chromosomal locus of 5q13.2 where the human CART gene is encoded (Table 3). Respectively, the expression of CART transcripts and peptides has been characterized in various hypothalamic areas involved in appetite control (Charnay et al., 1999; Elias et al., 2001; Menyhért et al., 2007), as well as in the subcutaneous and visceral white adipose tissues central to the moderation of lipid homeostasis (Vasseur et al., 2007; Banke et al., 2013). Intriguingly, the aforementioned anatomical-functional implications provided by the expression patterns of CART in the human infundibular nucleus, which demonstrated colocalization with the orexigenic NPY/AgRP and segregation from the anorexigenic POMC neurons, had rendered a primary anorectic role of CART appealable (Menyhért et al., 2007).\nTable 3 Examples of human studies demonstrating the association between genetic variations in the CART gene and the development of obesity.\nPublication Genetic association study Ethnicity (sample size) Body weight (sample size) Genetic variation/susceptibility locus Occurrence Feeding behavior and body weight alterations Biochemical alteration Energy and glucose homeostasis\nHager et al., 1998 Genome-wide scan for human obesity-susceptibility loci using model-free multipoint linkage analysis French Caucasian (514) Overweight (72), obese (107), morbidly obese (196), and non-obese controls (139) Chromosomal locus 5q13.2 (CART gene) Higher allele frequencies in overweight and obese sibpairs N/A Linkage with ↑ serum leptin levels ↑ Fasting glucose and insulin levels\nChallis et al., 2000 Mutational analysis and population genetics British Caucasian (902) Morbidly obese (91) and non-obese (811) 1475A\u003eG SNP (3′-UTR of exon 3) NSD in allele frequency between obese and control subjects Potential link to early-onset obesity; ↓ waist-to-hip ratio in male heterozygotes Potential interference with fat distribution and contribution to dyslipidaemia ↓ Fasting plasma insulin and fasting triglycerides in male heterozygotes\ndel Giudice et al., 2001 Single-strand conformation polymorphism and automatic sequencing Italian (230) Obese (130) and non-obese controls (100) Leu34Phe missense mutation in pro-CART (729G\u003eC in exon 2) A large family of obese subjects across three generations Hyperphagia and severe early-onset obesity even when heterozygous for allele Altered post-translational processing; intracellular missorting of proCART; bioactive CART deficiency in the serum; ↑ serum leptin levels ↓ Resting metabolic rates; linked to type II diabetes\nYamada et al., 2002 Single-strand conformation polymorphism and direct sequencing Japanese (558) Overweight and obese (528), non-obese controls (30) 6 polymorphic sites at 5′-flanking region, e.g., −156A\u003eG [corresponds to −175A\u003eG (Guerardel et al., 2005)], −929G\u003eC Higher allele frequencies in obese subjects than controls ↑ Genetic predisposition to obesity when in linkage disequliibrium N/A Potential association with type II diabetes\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French Caucasian (660) Morbidly obese (292) and non-obese controls (368) 1475A\u003eG SNP (3′-UTR of exon 3) Higher allele frequencies in morbidly obese subjects than controls N/A N/A N/A\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French Caucasian (989) Morbidly obese (621) and non-obese controls (368) 5′ SNPs: −3608T\u003eC, −3607C\u003eT, −1702C\u003eT, −175A\u003eG; 3′UTR SNP: ΔA1457 Higher allele frequencies in morbidly obese subjects than controls; association enhanced with the SNP haplotype structure 3608T\u003eC (or 175A\u003eG) and −1702C\u003eT, combined to ΔA1457 N/A N/A N/A\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French (2340) and Swiss (385) Caucasian Moderately obese (619), morbidly obese (1006) and non-obese controls (1100) −3608T\u003eC SNP (promoter region) Higher allele frequencies ↑ Genetic predisposition to obesity Potential modulation of nuclear protein binding affinity N/A\nVasseur et al., 2007 Sequence variability screen and haplotype analysis French Caucasian (840) General population sample 5′ SNPs: −3608T\u003eC, −1702C\u003eT, −175A\u003eG (promoter region) NSD in allele frequency between subjects with different BMI; strong linkage disequilibrium between the SNPs, haplotypic effect attributed to −3608T\u003eC N/A ↓ Plasma LDL-cholesterol level and LDL/HDL ratio; potential protection against atherogenesis Potential association with lipid metabolism and atherogenicity\nRigoli et al., 2010 Family-based association methods Italian (320) Overweight (103), obese (30) and non-obese controls (187) 1475A\u003eG SNP (3′-UTR of exon 3) Higher allele frequencies in overweight (0.07) and obese (0.08) children compared to non-obese unrelated controls (children and/or adults) (0.02); preferential transmission of 1475G allele from heterozygous parents to overweight and obese offspring Early-onset obesity N/A N/A\nWith slight variation between different studies, body weight is categorized according to the body mass index (BMI): non-obese (\u003c25 kg/m2), overweight (25–30 kg/m2), moderately obese (30–40 kg/m2), morbidly obese (\u003e40 kg/m2). LDL, low-density lipoprotein; HDL, high-density lipoprotein; NSD, no significant difference; SNP, single nucleotide polymorphism; UTR, untranslated region; Δ, deletion. In human, alterations in CART have been associated with reduced metabolic rate, hyperphagia, obesity and elevated incidence of type II diabetes (Banke et al., 2013) (Table 3). For example, a Leu34Phe missense mutation in human proCART was discovered in obese members of an Italian family across three generations to affect post-translational processing, which coincided with CART peptide deficiency in the sera and reduced resting energy expenditure, ultimately leading to hyperphagia and severe early-onset obesity (del Giudice et al., 2001; Dominguez et al., 2004a; Yanik et al., 2006). In brief, the mutation was identified to neighbor a cluster of basic amino acids, hence presupposed to influence the specific processing of the proCART (1–89) in generating the biologically active CART I (42–89) and CART II (49–89) peptides (Kuhar and Yoho, 1999; Thim et al., 1999; del Giudice et al., 2001; Dominguez et al., 2004a). To simulate cellular effects of the mutation, subsequent investigations transfected human CART cDNA constructs representing either the wild-type or the mutant into corticotropic AtT-20 cells, a mouse pituitary-derived cell line often used for studying peptide processing and, more importantly, is known to express and process CART peptides (Dominguez et al., 2004a). Notably, in addition to reduced CART peptide levels in cells transfected with the mutated cDNA compared with controls, expression of the mutated proCART was also described to be mis-sorted, poorly processed and secreted, thus disarranging the cellular distribution of CART as a whole (Dominguez et al., 2004a). Other than discoveries that addressed the potentially crucial role of protein biosynthesis in the development of obesity, the majority of the studies directed at the association between CART and obesity focused on polymorphisms in CART within populations.\nPolymorphism studies in the CART gene conducted worldwide have established substantial linkage between a few specific single nucleotide polymorphisms (SNPs) to obesity (del Giudice et al., 2001; Yamada et al., 2002; Guerardel et al., 2005; Rigoli et al., 2010) (Table 3). For instance, a family-based association study of 133 Italian trios has identified significantly higher allele frequencies of the 1475A\u003eG SNP in the CART gene in overweight and obese children compared to non-obese unrelated controls consisting of both adults and children, while preferential transmission of the allele to overweight or obese children from heterozygous parents was predicted (Rigoli et al., 2010). In another Caucasian population, 292 French morbidly obese subjects were recruited for sequence variability screen in the CART gene, where a few SNPs residing in the promoter region, with SNP-3608T\u003eC in particular, were suggested by haplotype analysis to prominently contribute to the genetic risk for obesity (Guerardel et al., 2005). The proposed association was further strengthened by the high prevalence of the specific allele in an expanded genotyping study, with additional populations of European Caucasian origin comprising 619 moderately obese French subjects and 385 morbidly obese Swiss subjects (Guerardel et al., 2005). Extended on the genetic studies, plausible functional effects of the SNP were also investigated by electrophoretic mobility shift assays in cellular system, where modulation of nuclear protein binding affinity was demonstrated to potentially correlate with the obesity phenotype (Guerardel et al., 2005). Besides Caucasians, a sequencing study in 528 Japanese subjects revealed a high level of polymorphisms in the 5′-flanking region of the CART gene housing the putative promoter region, wherein specific polymorphic sites or variants in linkage disequilibrium with each other were identified to associate with genetic predisposition to obesity (Yamada et al., 2002).\nAs mentioned, CART has recently been defined as a component of adipocytes involved in lipid substrate utilization in both human and rodents (Banke et al., 2013). Investigation in a large Caucasian population of approximately 1000 subjects in the United Kingdom identified two common polymorphisms in the 3′-untranslated region of CART, that were implicated to interfere with fat distribution and contribute to dyslipidaemia (Challis et al., 2000; Rogge et al., 2008) (Table 3). Despite a lack of correlation or consistent association with obesity through systematic mutational analysis (Lambert et al., 1998; Challis et al., 2000; Okumura et al., 2000; Rogge et al., 2008), a particular genetic variant 1475A\u003eG among the polymorphisms was illustrated to significantly affect waist-to-hip ratio as well as the levels of plasma insulin and triglycerides (Challis et al., 2000), suggesting a putative pivotal role of CART in glucose- and lipid-homeostasis. Consolidation of the proposition has been illustrated in subsequent haplotypic study in a general population of 840 subjects from northern France (Vasseur et al., 2007), a continuum from the former French project on CART promoter SNPs (Guerardel et al., 2005), where three of the previously identified SNPs were described to affect plasma low-density lipoprotein-cholesterol level and consequently associated with cholesterol metabolism and atherogenicity (Guerardel et al., 2005; Vasseur et al., 2007). Specifically, the functional SNP-3808C\u003eT was of particular interest, as plasma lipid profile traits protective against atherogenesis were displayed in cases bearing the allele, exemplifying the clinical potentials of CART in lipid metabolism and atherogenesis (Vasseur et al., 2007; Banke et al., 2013).\nTaken together, human studies based principally on genetic polymorphisms have provided evidence promoting a role of CART in body weight regulation in humans. Altered CART expression has generally been associated with an elevated genetic predisposition to overweight and obesity, indirectly substantiating the anorexigenic nature of the peptide, although results from the literature show both anorexigenic and orexigenic properties of CART in animal studies. It is also noteworthy to address the plausible challenges imposed on the translatability of results obtained from animal models to the human system, considering the discernible difference in the anatomy of central CART-containing neurons between the two, as discussed above. Furthermore, although overall support has been gained for the hypothesis that inherited variations in CART could influence the development of obesity, such genetic linkage was absent for some other sequence variants detected within the gene, where the polymorphisms have been speculated as insufficient to disturb the peptide structure or create topological and conformational changes in the protein that would ultimately affect the functional activity of the peptide (Echwald et al., 1999; Walder et al., 2000; Rogge et al., 2008). Indeed, recent studies conducting an alanine scan for assessing the importance of the structure-activity relationship of CART demonstrated the dependence of anorexigenic potency on individual disulfide bridges in the peptide (Maixnerova et al., 2007; Maletinska et al., 2007; Blechova et al., 2013). To elucidate the contribution of specific disulfide bridges to maintaining the stability and biological function of CART, analogs with only one or two among the three disulfide bridges in the intact peptide were synthesized, with which binding activities as well as metabolic effects were measured in both cell and animal systems (Maixnerova et al., 2007). Intriguingly, results from binding experiments in PC12 rat pheochromocytoma cells (Maixnerova et al., 2007; Maletinska et al., 2007; Lin et al., 2011) indicated that the preservation of two particular disulfide bridges as well as the full-length peptide was imperative for biological activity, where high affinity of the analog to PC12 cells in both states of native phenotype and differentiated into neurons was measured (Blechova et al., 2013). In mice subjected to i.c.v. administration of the same analog, strong and long-lasting anorexigenic potency was exhibited during food consumption and behavioral tests, further purporting that one particular disulfide bridge could be omitted without a loss of bioactive function (Blechova et al., 2013).\nIn summary, the familial nature of obesity is well-established to be interrelated with a prominent genetic component. The CART system has been evident to constitute a dominant player in feeding control, body weight regulation and energy metabolism, hence a promising candidate for the development of anti-obesity therapeutics. Respectively, population genetics have revealed the potential contributions of polymorphisms in the CART gene to abnormalities in feeding and body weight control, where effects on interactions between the transcription factors and regulatory elements binding to the polymorphic sites may exert phenotypic influence. However, elucidating the mechanisms of CART action as well as investigating and replicating the fine genetic mapping in further populations will be essential for unraveling the authentic role of CART in energy homeostasis and understanding obesity."}

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},{"id":"25352770-17766010-357921","span":{"begin":13583,"end":13587},"obj":"17766010"},{"id":"25352770-17766010-357922","span":{"begin":13692,"end":13696},"obj":"17766010"},{"id":"25352770-17292884-357923","span":{"begin":13717,"end":13721},"obj":"17292884"},{"id":"25352770-21855138-357924","span":{"begin":13735,"end":13739},"obj":"21855138"},{"id":"25352770-23174349-357925","span":{"begin":14030,"end":14034},"obj":"23174349"},{"id":"25352770-23174349-357926","span":{"begin":14333,"end":14337},"obj":"23174349"}],"text":"Roles of CART in human\nAs introduced earlier, evolutionary conservation has been demonstrated for CART in the neuroendocrine system across various mammalian species in the contexts of isoform structure, expression distribution pattern as well as functional implications, including a role of CART in the regulation of energy balance in human (Hager et al., 1998; Challis et al., 2000; del Giudice et al., 2001; Yamada et al., 2002; Dominguez et al., 2004a; Guerardel et al., 2005; Yanik et al., 2006; Rigoli et al., 2010). First, a genome-wide scan for human obesity-susceptibility loci in obese French Caucasian families (Hager et al., 1998) revealed a clear linkage to the chromosomal locus of 5q13.2 where the human CART gene is encoded (Table 3). Respectively, the expression of CART transcripts and peptides has been characterized in various hypothalamic areas involved in appetite control (Charnay et al., 1999; Elias et al., 2001; Menyhért et al., 2007), as well as in the subcutaneous and visceral white adipose tissues central to the moderation of lipid homeostasis (Vasseur et al., 2007; Banke et al., 2013). Intriguingly, the aforementioned anatomical-functional implications provided by the expression patterns of CART in the human infundibular nucleus, which demonstrated colocalization with the orexigenic NPY/AgRP and segregation from the anorexigenic POMC neurons, had rendered a primary anorectic role of CART appealable (Menyhért et al., 2007).\nTable 3 Examples of human studies demonstrating the association between genetic variations in the CART gene and the development of obesity.\nPublication Genetic association study Ethnicity (sample size) Body weight (sample size) Genetic variation/susceptibility locus Occurrence Feeding behavior and body weight alterations Biochemical alteration Energy and glucose homeostasis\nHager et al., 1998 Genome-wide scan for human obesity-susceptibility loci using model-free multipoint linkage analysis French Caucasian (514) Overweight (72), obese (107), morbidly obese (196), and non-obese controls (139) Chromosomal locus 5q13.2 (CART gene) Higher allele frequencies in overweight and obese sibpairs N/A Linkage with ↑ serum leptin levels ↑ Fasting glucose and insulin levels\nChallis et al., 2000 Mutational analysis and population genetics British Caucasian (902) Morbidly obese (91) and non-obese (811) 1475A\u003eG SNP (3′-UTR of exon 3) NSD in allele frequency between obese and control subjects Potential link to early-onset obesity; ↓ waist-to-hip ratio in male heterozygotes Potential interference with fat distribution and contribution to dyslipidaemia ↓ Fasting plasma insulin and fasting triglycerides in male heterozygotes\ndel Giudice et al., 2001 Single-strand conformation polymorphism and automatic sequencing Italian (230) Obese (130) and non-obese controls (100) Leu34Phe missense mutation in pro-CART (729G\u003eC in exon 2) A large family of obese subjects across three generations Hyperphagia and severe early-onset obesity even when heterozygous for allele Altered post-translational processing; intracellular missorting of proCART; bioactive CART deficiency in the serum; ↑ serum leptin levels ↓ Resting metabolic rates; linked to type II diabetes\nYamada et al., 2002 Single-strand conformation polymorphism and direct sequencing Japanese (558) Overweight and obese (528), non-obese controls (30) 6 polymorphic sites at 5′-flanking region, e.g., −156A\u003eG [corresponds to −175A\u003eG (Guerardel et al., 2005)], −929G\u003eC Higher allele frequencies in obese subjects than controls ↑ Genetic predisposition to obesity when in linkage disequliibrium N/A Potential association with type II diabetes\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French Caucasian (660) Morbidly obese (292) and non-obese controls (368) 1475A\u003eG SNP (3′-UTR of exon 3) Higher allele frequencies in morbidly obese subjects than controls N/A N/A N/A\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French Caucasian (989) Morbidly obese (621) and non-obese controls (368) 5′ SNPs: −3608T\u003eC, −3607C\u003eT, −1702C\u003eT, −175A\u003eG; 3′UTR SNP: ΔA1457 Higher allele frequencies in morbidly obese subjects than controls; association enhanced with the SNP haplotype structure 3608T\u003eC (or 175A\u003eG) and −1702C\u003eT, combined to ΔA1457 N/A N/A N/A\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French (2340) and Swiss (385) Caucasian Moderately obese (619), morbidly obese (1006) and non-obese controls (1100) −3608T\u003eC SNP (promoter region) Higher allele frequencies ↑ Genetic predisposition to obesity Potential modulation of nuclear protein binding affinity N/A\nVasseur et al., 2007 Sequence variability screen and haplotype analysis French Caucasian (840) General population sample 5′ SNPs: −3608T\u003eC, −1702C\u003eT, −175A\u003eG (promoter region) NSD in allele frequency between subjects with different BMI; strong linkage disequilibrium between the SNPs, haplotypic effect attributed to −3608T\u003eC N/A ↓ Plasma LDL-cholesterol level and LDL/HDL ratio; potential protection against atherogenesis Potential association with lipid metabolism and atherogenicity\nRigoli et al., 2010 Family-based association methods Italian (320) Overweight (103), obese (30) and non-obese controls (187) 1475A\u003eG SNP (3′-UTR of exon 3) Higher allele frequencies in overweight (0.07) and obese (0.08) children compared to non-obese unrelated controls (children and/or adults) (0.02); preferential transmission of 1475G allele from heterozygous parents to overweight and obese offspring Early-onset obesity N/A N/A\nWith slight variation between different studies, body weight is categorized according to the body mass index (BMI): non-obese (\u003c25 kg/m2), overweight (25–30 kg/m2), moderately obese (30–40 kg/m2), morbidly obese (\u003e40 kg/m2). LDL, low-density lipoprotein; HDL, high-density lipoprotein; NSD, no significant difference; SNP, single nucleotide polymorphism; UTR, untranslated region; Δ, deletion. In human, alterations in CART have been associated with reduced metabolic rate, hyperphagia, obesity and elevated incidence of type II diabetes (Banke et al., 2013) (Table 3). For example, a Leu34Phe missense mutation in human proCART was discovered in obese members of an Italian family across three generations to affect post-translational processing, which coincided with CART peptide deficiency in the sera and reduced resting energy expenditure, ultimately leading to hyperphagia and severe early-onset obesity (del Giudice et al., 2001; Dominguez et al., 2004a; Yanik et al., 2006). In brief, the mutation was identified to neighbor a cluster of basic amino acids, hence presupposed to influence the specific processing of the proCART (1–89) in generating the biologically active CART I (42–89) and CART II (49–89) peptides (Kuhar and Yoho, 1999; Thim et al., 1999; del Giudice et al., 2001; Dominguez et al., 2004a). To simulate cellular effects of the mutation, subsequent investigations transfected human CART cDNA constructs representing either the wild-type or the mutant into corticotropic AtT-20 cells, a mouse pituitary-derived cell line often used for studying peptide processing and, more importantly, is known to express and process CART peptides (Dominguez et al., 2004a). Notably, in addition to reduced CART peptide levels in cells transfected with the mutated cDNA compared with controls, expression of the mutated proCART was also described to be mis-sorted, poorly processed and secreted, thus disarranging the cellular distribution of CART as a whole (Dominguez et al., 2004a). Other than discoveries that addressed the potentially crucial role of protein biosynthesis in the development of obesity, the majority of the studies directed at the association between CART and obesity focused on polymorphisms in CART within populations.\nPolymorphism studies in the CART gene conducted worldwide have established substantial linkage between a few specific single nucleotide polymorphisms (SNPs) to obesity (del Giudice et al., 2001; Yamada et al., 2002; Guerardel et al., 2005; Rigoli et al., 2010) (Table 3). For instance, a family-based association study of 133 Italian trios has identified significantly higher allele frequencies of the 1475A\u003eG SNP in the CART gene in overweight and obese children compared to non-obese unrelated controls consisting of both adults and children, while preferential transmission of the allele to overweight or obese children from heterozygous parents was predicted (Rigoli et al., 2010). In another Caucasian population, 292 French morbidly obese subjects were recruited for sequence variability screen in the CART gene, where a few SNPs residing in the promoter region, with SNP-3608T\u003eC in particular, were suggested by haplotype analysis to prominently contribute to the genetic risk for obesity (Guerardel et al., 2005). The proposed association was further strengthened by the high prevalence of the specific allele in an expanded genotyping study, with additional populations of European Caucasian origin comprising 619 moderately obese French subjects and 385 morbidly obese Swiss subjects (Guerardel et al., 2005). Extended on the genetic studies, plausible functional effects of the SNP were also investigated by electrophoretic mobility shift assays in cellular system, where modulation of nuclear protein binding affinity was demonstrated to potentially correlate with the obesity phenotype (Guerardel et al., 2005). Besides Caucasians, a sequencing study in 528 Japanese subjects revealed a high level of polymorphisms in the 5′-flanking region of the CART gene housing the putative promoter region, wherein specific polymorphic sites or variants in linkage disequilibrium with each other were identified to associate with genetic predisposition to obesity (Yamada et al., 2002).\nAs mentioned, CART has recently been defined as a component of adipocytes involved in lipid substrate utilization in both human and rodents (Banke et al., 2013). Investigation in a large Caucasian population of approximately 1000 subjects in the United Kingdom identified two common polymorphisms in the 3′-untranslated region of CART, that were implicated to interfere with fat distribution and contribute to dyslipidaemia (Challis et al., 2000; Rogge et al., 2008) (Table 3). Despite a lack of correlation or consistent association with obesity through systematic mutational analysis (Lambert et al., 1998; Challis et al., 2000; Okumura et al., 2000; Rogge et al., 2008), a particular genetic variant 1475A\u003eG among the polymorphisms was illustrated to significantly affect waist-to-hip ratio as well as the levels of plasma insulin and triglycerides (Challis et al., 2000), suggesting a putative pivotal role of CART in glucose- and lipid-homeostasis. Consolidation of the proposition has been illustrated in subsequent haplotypic study in a general population of 840 subjects from northern France (Vasseur et al., 2007), a continuum from the former French project on CART promoter SNPs (Guerardel et al., 2005), where three of the previously identified SNPs were described to affect plasma low-density lipoprotein-cholesterol level and consequently associated with cholesterol metabolism and atherogenicity (Guerardel et al., 2005; Vasseur et al., 2007). Specifically, the functional SNP-3808C\u003eT was of particular interest, as plasma lipid profile traits protective against atherogenesis were displayed in cases bearing the allele, exemplifying the clinical potentials of CART in lipid metabolism and atherogenesis (Vasseur et al., 2007; Banke et al., 2013).\nTaken together, human studies based principally on genetic polymorphisms have provided evidence promoting a role of CART in body weight regulation in humans. Altered CART expression has generally been associated with an elevated genetic predisposition to overweight and obesity, indirectly substantiating the anorexigenic nature of the peptide, although results from the literature show both anorexigenic and orexigenic properties of CART in animal studies. It is also noteworthy to address the plausible challenges imposed on the translatability of results obtained from animal models to the human system, considering the discernible difference in the anatomy of central CART-containing neurons between the two, as discussed above. Furthermore, although overall support has been gained for the hypothesis that inherited variations in CART could influence the development of obesity, such genetic linkage was absent for some other sequence variants detected within the gene, where the polymorphisms have been speculated as insufficient to disturb the peptide structure or create topological and conformational changes in the protein that would ultimately affect the functional activity of the peptide (Echwald et al., 1999; Walder et al., 2000; Rogge et al., 2008). Indeed, recent studies conducting an alanine scan for assessing the importance of the structure-activity relationship of CART demonstrated the dependence of anorexigenic potency on individual disulfide bridges in the peptide (Maixnerova et al., 2007; Maletinska et al., 2007; Blechova et al., 2013). To elucidate the contribution of specific disulfide bridges to maintaining the stability and biological function of CART, analogs with only one or two among the three disulfide bridges in the intact peptide were synthesized, with which binding activities as well as metabolic effects were measured in both cell and animal systems (Maixnerova et al., 2007). Intriguingly, results from binding experiments in PC12 rat pheochromocytoma cells (Maixnerova et al., 2007; Maletinska et al., 2007; Lin et al., 2011) indicated that the preservation of two particular disulfide bridges as well as the full-length peptide was imperative for biological activity, where high affinity of the analog to PC12 cells in both states of native phenotype and differentiated into neurons was measured (Blechova et al., 2013). In mice subjected to i.c.v. administration of the same analog, strong and long-lasting anorexigenic potency was exhibited during food consumption and behavioral tests, further purporting that one particular disulfide bridge could be omitted without a loss of bioactive function (Blechova et al., 2013).\nIn summary, the familial nature of obesity is well-established to be interrelated with a prominent genetic component. The CART system has been evident to constitute a dominant player in feeding control, body weight regulation and energy metabolism, hence a promising candidate for the development of anti-obesity therapeutics. Respectively, population genetics have revealed the potential contributions of polymorphisms in the CART gene to abnormalities in feeding and body weight control, where effects on interactions between the transcription factors and regulatory elements binding to the polymorphic sites may exert phenotypic influence. However, elucidating the mechanisms of CART action as well as investigating and replicating the fine genetic mapping in further populations will be essential for unraveling the authentic role of CART in energy homeostasis and understanding obesity."}

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of CART in human\nAs introduced earlier, evolutionary conservation has been demonstrated for CART in the neuroendocrine system across various mammalian species in the contexts of isoform structure, expression distribution pattern as well as functional implications, including a role of CART in the regulation of energy balance in human (Hager et al., 1998; Challis et al., 2000; del Giudice et al., 2001; Yamada et al., 2002; Dominguez et al., 2004a; Guerardel et al., 2005; Yanik et al., 2006; Rigoli et al., 2010). First, a genome-wide scan for human obesity-susceptibility loci in obese French Caucasian families (Hager et al., 1998) revealed a clear linkage to the chromosomal locus of 5q13.2 where the human CART gene is encoded (Table 3). Respectively, the expression of CART transcripts and peptides has been characterized in various hypothalamic areas involved in appetite control (Charnay et al., 1999; Elias et al., 2001; Menyhért et al., 2007), as well as in the subcutaneous and visceral white adipose tissues central to the moderation of lipid homeostasis (Vasseur et al., 2007; Banke et al., 2013). Intriguingly, the aforementioned anatomical-functional implications provided by the expression patterns of CART in the human infundibular nucleus, which demonstrated colocalization with the orexigenic NPY/AgRP and segregation from the anorexigenic POMC neurons, had rendered a primary anorectic role of CART appealable (Menyhért et al., 2007).\nTable 3 Examples of human studies demonstrating the association between genetic variations in the CART gene and the development of obesity.\nPublication Genetic association study Ethnicity (sample size) Body weight (sample size) Genetic variation/susceptibility locus Occurrence Feeding behavior and body weight alterations Biochemical alteration Energy and glucose homeostasis\nHager et al., 1998 Genome-wide scan for human obesity-susceptibility loci using model-free multipoint linkage analysis French Caucasian (514) Overweight (72), obese (107), morbidly obese (196), and non-obese controls (139) Chromosomal locus 5q13.2 (CART gene) Higher allele frequencies in overweight and obese sibpairs N/A Linkage with ↑ serum leptin levels ↑ Fasting glucose and insulin levels\nChallis et al., 2000 Mutational analysis and population genetics British Caucasian (902) Morbidly obese (91) and non-obese (811) 1475A\u003eG SNP (3′-UTR of exon 3) NSD in allele frequency between obese and control subjects Potential link to early-onset obesity; ↓ waist-to-hip ratio in male heterozygotes Potential interference with fat distribution and contribution to dyslipidaemia ↓ Fasting plasma insulin and fasting triglycerides in male heterozygotes\ndel Giudice et al., 2001 Single-strand conformation polymorphism and automatic sequencing Italian (230) Obese (130) and non-obese controls (100) Leu34Phe missense mutation in pro-CART (729G\u003eC in exon 2) A large family of obese subjects across three generations Hyperphagia and severe early-onset obesity even when heterozygous for allele Altered post-translational processing; intracellular missorting of proCART; bioactive CART deficiency in the serum; ↑ serum leptin levels ↓ Resting metabolic rates; linked to type II diabetes\nYamada et al., 2002 Single-strand conformation polymorphism and direct sequencing Japanese (558) Overweight and obese (528), non-obese controls (30) 6 polymorphic sites at 5′-flanking region, e.g., −156A\u003eG [corresponds to −175A\u003eG (Guerardel et al., 2005)], −929G\u003eC Higher allele frequencies in obese subjects than controls ↑ Genetic predisposition to obesity when in linkage disequliibrium N/A Potential association with type II diabetes\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French Caucasian (660) Morbidly obese (292) and non-obese controls (368) 1475A\u003eG SNP (3′-UTR of exon 3) Higher allele frequencies in morbidly obese subjects than controls N/A N/A N/A\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French Caucasian (989) Morbidly obese (621) and non-obese controls (368) 5′ SNPs: −3608T\u003eC, −3607C\u003eT, −1702C\u003eT, −175A\u003eG; 3′UTR SNP: ΔA1457 Higher allele frequencies in morbidly obese subjects than controls; association enhanced with the SNP haplotype structure 3608T\u003eC (or 175A\u003eG) and −1702C\u003eT, combined to ΔA1457 N/A N/A N/A\nGuerardel et al., 2005 Sequence variability screen and haplotype analysis French (2340) and Swiss (385) Caucasian Moderately obese (619), morbidly obese (1006) and non-obese controls (1100) −3608T\u003eC SNP (promoter region) Higher allele frequencies ↑ Genetic predisposition to obesity Potential modulation of nuclear protein binding affinity N/A\nVasseur et al., 2007 Sequence variability screen and haplotype analysis French Caucasian (840) General population sample 5′ SNPs: −3608T\u003eC, −1702C\u003eT, −175A\u003eG (promoter region) NSD in allele frequency between subjects with different BMI; strong linkage disequilibrium between the SNPs, haplotypic effect attributed to −3608T\u003eC N/A ↓ Plasma LDL-cholesterol level and LDL/HDL ratio; potential protection against atherogenesis Potential association with lipid metabolism and atherogenicity\nRigoli et al., 2010 Family-based association methods Italian (320) Overweight (103), obese (30) and non-obese controls (187) 1475A\u003eG SNP (3′-UTR of exon 3) Higher allele frequencies in overweight (0.07) and obese (0.08) children compared to non-obese unrelated controls (children and/or adults) (0.02); preferential transmission of 1475G allele from heterozygous parents to overweight and obese offspring Early-onset obesity N/A N/A\nWith slight variation between different studies, body weight is categorized according to the body mass index (BMI): non-obese (\u003c25 kg/m2), overweight (25–30 kg/m2), moderately obese (30–40 kg/m2), morbidly obese (\u003e40 kg/m2). LDL, low-density lipoprotein; HDL, high-density lipoprotein; NSD, no significant difference; SNP, single nucleotide polymorphism; UTR, untranslated region; Δ, deletion. In human, alterations in CART have been associated with reduced metabolic rate, hyperphagia, obesity and elevated incidence of type II diabetes (Banke et al., 2013) (Table 3). For example, a Leu34Phe missense mutation in human proCART was discovered in obese members of an Italian family across three generations to affect post-translational processing, which coincided with CART peptide deficiency in the sera and reduced resting energy expenditure, ultimately leading to hyperphagia and severe early-onset obesity (del Giudice et al., 2001; Dominguez et al., 2004a; Yanik et al., 2006). In brief, the mutation was identified to neighbor a cluster of basic amino acids, hence presupposed to influence the specific processing of the proCART (1–89) in generating the biologically active CART I (42–89) and CART II (49–89) peptides (Kuhar and Yoho, 1999; Thim et al., 1999; del Giudice et al., 2001; Dominguez et al., 2004a). To simulate cellular effects of the mutation, subsequent investigations transfected human CART cDNA constructs representing either the wild-type or the mutant into corticotropic AtT-20 cells, a mouse pituitary-derived cell line often used for studying peptide processing and, more importantly, is known to express and process CART peptides (Dominguez et al., 2004a). Notably, in addition to reduced CART peptide levels in cells transfected with the mutated cDNA compared with controls, expression of the mutated proCART was also described to be mis-sorted, poorly processed and secreted, thus disarranging the cellular distribution of CART as a whole (Dominguez et al., 2004a). Other than discoveries that addressed the potentially crucial role of protein biosynthesis in the development of obesity, the majority of the studies directed at the association between CART and obesity focused on polymorphisms in CART within populations.\nPolymorphism studies in the CART gene conducted worldwide have established substantial linkage between a few specific single nucleotide polymorphisms (SNPs) to obesity (del Giudice et al., 2001; Yamada et al., 2002; Guerardel et al., 2005; Rigoli et al., 2010) (Table 3). For instance, a family-based association study of 133 Italian trios has identified significantly higher allele frequencies of the 1475A\u003eG SNP in the CART gene in overweight and obese children compared to non-obese unrelated controls consisting of both adults and children, while preferential transmission of the allele to overweight or obese children from heterozygous parents was predicted (Rigoli et al., 2010). In another Caucasian population, 292 French morbidly obese subjects were recruited for sequence variability screen in the CART gene, where a few SNPs residing in the promoter region, with SNP-3608T\u003eC in particular, were suggested by haplotype analysis to prominently contribute to the genetic risk for obesity (Guerardel et al., 2005). The proposed association was further strengthened by the high prevalence of the specific allele in an expanded genotyping study, with additional populations of European Caucasian origin comprising 619 moderately obese French subjects and 385 morbidly obese Swiss subjects (Guerardel et al., 2005). Extended on the genetic studies, plausible functional effects of the SNP were also investigated by electrophoretic mobility shift assays in cellular system, where modulation of nuclear protein binding affinity was demonstrated to potentially correlate with the obesity phenotype (Guerardel et al., 2005). Besides Caucasians, a sequencing study in 528 Japanese subjects revealed a high level of polymorphisms in the 5′-flanking region of the CART gene housing the putative promoter region, wherein specific polymorphic sites or variants in linkage disequilibrium with each other were identified to associate with genetic predisposition to obesity (Yamada et al., 2002).\nAs mentioned, CART has recently been defined as a component of adipocytes involved in lipid substrate utilization in both human and rodents (Banke et al., 2013). Investigation in a large Caucasian population of approximately 1000 subjects in the United Kingdom identified two common polymorphisms in the 3′-untranslated region of CART, that were implicated to interfere with fat distribution and contribute to dyslipidaemia (Challis et al., 2000; Rogge et al., 2008) (Table 3). Despite a lack of correlation or consistent association with obesity through systematic mutational analysis (Lambert et al., 1998; Challis et al., 2000; Okumura et al., 2000; Rogge et al., 2008), a particular genetic variant 1475A\u003eG among the polymorphisms was illustrated to significantly affect waist-to-hip ratio as well as the levels of plasma insulin and triglycerides (Challis et al., 2000), suggesting a putative pivotal role of CART in glucose- and lipid-homeostasis. Consolidation of the proposition has been illustrated in subsequent haplotypic study in a general population of 840 subjects from northern France (Vasseur et al., 2007), a continuum from the former French project on CART promoter SNPs (Guerardel et al., 2005), where three of the previously identified SNPs were described to affect plasma low-density lipoprotein-cholesterol level and consequently associated with cholesterol metabolism and atherogenicity (Guerardel et al., 2005; Vasseur et al., 2007). Specifically, the functional SNP-3808C\u003eT was of particular interest, as plasma lipid profile traits protective against atherogenesis were displayed in cases bearing the allele, exemplifying the clinical potentials of CART in lipid metabolism and atherogenesis (Vasseur et al., 2007; Banke et al., 2013).\nTaken together, human studies based principally on genetic polymorphisms have provided evidence promoting a role of CART in body weight regulation in humans. Altered CART expression has generally been associated with an elevated genetic predisposition to overweight and obesity, indirectly substantiating the anorexigenic nature of the peptide, although results from the literature show both anorexigenic and orexigenic properties of CART in animal studies. It is also noteworthy to address the plausible challenges imposed on the translatability of results obtained from animal models to the human system, considering the discernible difference in the anatomy of central CART-containing neurons between the two, as discussed above. Furthermore, although overall support has been gained for the hypothesis that inherited variations in CART could influence the development of obesity, such genetic linkage was absent for some other sequence variants detected within the gene, where the polymorphisms have been speculated as insufficient to disturb the peptide structure or create topological and conformational changes in the protein that would ultimately affect the functional activity of the peptide (Echwald et al., 1999; Walder et al., 2000; Rogge et al., 2008). Indeed, recent studies conducting an alanine scan for assessing the importance of the structure-activity relationship of CART demonstrated the dependence of anorexigenic potency on individual disulfide bridges in the peptide (Maixnerova et al., 2007; Maletinska et al., 2007; Blechova et al., 2013). To elucidate the contribution of specific disulfide bridges to maintaining the stability and biological function of CART, analogs with only one or two among the three disulfide bridges in the intact peptide were synthesized, with which binding activities as well as metabolic effects were measured in both cell and animal systems (Maixnerova et al., 2007). Intriguingly, results from binding experiments in PC12 rat pheochromocytoma cells (Maixnerova et al., 2007; Maletinska et al., 2007; Lin et al., 2011) indicated that the preservation of two particular disulfide bridges as well as the full-length peptide was imperative for biological activity, where high affinity of the analog to PC12 cells in both states of native phenotype and differentiated into neurons was measured (Blechova et al., 2013). In mice subjected to i.c.v. administration of the same analog, strong and long-lasting anorexigenic potency was exhibited during food consumption and behavioral tests, further purporting that one particular disulfide bridge could be omitted without a loss of bioactive function (Blechova et al., 2013).\nIn summary, the familial nature of obesity is well-established to be interrelated with a prominent genetic component. The CART system has been evident to constitute a dominant player in feeding control, body weight regulation and energy metabolism, hence a promising candidate for the development of anti-obesity therapeutics. Respectively, population genetics have revealed the potential contributions of polymorphisms in the CART gene to abnormalities in feeding and body weight control, where effects on interactions between the transcription factors and regulatory elements binding to the polymorphic sites may exert phenotypic influence. However, elucidating the mechanisms of CART action as well as investigating and replicating the fine genetic mapping in further populations will be essential for unraveling the authentic role of CART in energy homeostasis and understanding obesity."}