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Genetic variants of ghrelin in metabolic disorders
Peptides 32 (2011) 2319–2322
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Review
Genetic variants of ghrelin in metabolic disorders Olavi Ukkola a,b,∗ a b
Institute of Clinical Medicine, Department of Internal Medicine and Biocenter Oulu, University of Oulu, Finland Clinical Research Center, Oulu University Hospital, P.O. Box 20, FIN-90029 OYS, Oulu, Finland
a r t i c l e
i n f o
Article history: Received 14 January 2011 Received in revised form 8 April 2011 Accepted 11 April 2011 Available online 17 April 2011 Keywords: Ghrelin Gene Obesity Diabetes
a b s t r a c t An increasing understanding of the role of genes in the development of obesity may reveal genetic variants that, in combination with conventional risk factors, may help to predict an individual’s risk for developing metabolic disorders. Accumulating evidence indicates that ghrelin plays a role in regulating food intake and energy homeostasis and it is a reasonable candidate gene for obesity-related co-morbidities. In crosssectional studies low total ghrelin concentrations and some genetic polymorphisms of ghrelin have been associated with obesity-associated diseases. The present review highlights many of the important problems in association studies of genetic variants and complex diseases. It is known that population-specific differences in reported associations exist. We therefore conclude that more studies on variants of ghrelin gene are needed to perform in different populations to get deeper understanding on the relationship of ghrelin gene and its variants to obesity. © 2011 Elsevier Inc. All rights reserved.
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Variants in gene coding for ghrelin in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ghrelin gene–environment interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Ghrelin gene (GHRL, GeneID 51738) locates in the chromosome 3p26-25 and it contains four exons and three introns [29]. Ghrelin is synthesized in a precursor protein form that consists of 117 amino acids and is called prepro-ghrelin. The latter is modified by the action of protein convertase enzymes into mature ghrelin, consisting of 28 amino acids and another 23 amino acid-peptide hormone obestatin [35]. The nomenclature refers to the biological activities of this molecule that are opposite to those of ghrelin: it decreases appetite and inhibits weight gain. After post-translational modifications an acyl group, most commonly an octanoyl group, is attached to the third amino acid, serine (Ser3 ) of the mature ghrelin by an esteric-bond. The enzyme that catalyzes the reaction in which
∗ Correspondence address: Department of Internal Medicine and Biocenter Oulu, University of Oulu, Kajaanintie 50/P.O. Box 5000, Oulu FIN-90014, Finland. Tel.: +358 08 315 4121; fax: +358 08 315 4139. E-mail address: olavi.ukkola@oulu.fi 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.04.013
the octanoate is attached to the hydroxyl group of the Ser3 has been identified recently and given a name ghrelin-O-acyltransferase (GOAT) [34]. In a study conducted on identical twins plasma ghrelin concentration at baseline was more alike within pairs of twins than between pairs indicating a probable genetic effect underlying the variability in plasma ghrelin levels [21]. Similarly, in response to overfeeding, the decreases in ghrelin concentration were more similar within than between pairs. Taken together, these results indicate that genetic differences among people could play a role in the production and/or the clearance of this gastric peptide [21]. Several whole genome scans have suggested that certain areas of the chromosome 3, the same chromosome where ghrelin and ghrelin receptor gene are located, might be linked with obesity or metabolic syndrome [11,32]. These findings, in addition to the relevant biological functions of these proteins, increase the potential for the genes coding for ghrelin and its receptor to be ideal candidate genes in the genetic studies focusing on obesity and obesity related co-morbidities.
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2. Animal models To further study the role of endogenous ghrelin in energy balance ghrelin-deficient (ghrl(−/−)) mice have been generated. These studies have failed to show any significant defects in food intake, or body weight in adult Ghrl−/− mice. Wortley et al. reported that ghrl(−/−) [30] mice exhibited normal growth rates as well as normal spontaneous food intake patterns. However, endogenous ghrelin seems to play a prominent role in determining the type of metabolic substrate (i.e., fat vs. carbohydrate) that is used for maintenance of energy balance, particularly under conditions of high fat intake. Two recent studies have demonstrated that Ghrl−/− and Ghsr−/− mice are resistant to diet-induced obesity when fed a high-fat diet during the early post-weaning period [31,36]. De Smet et al. [7] studied energy homeostasis and gastric emptying in ghrelin knockout mice. They concluded that ghrelin is not an essential regulator of food intake and gastric emptying, but its loss may be compensated by other redundant inputs. In old mice, meal initiation triggered by the light/dark cue may be related to ghrelin while in young animals, ghrelin seems to be involved in the selection of energy stores. Therefore, ghrelin appears not to be an essential regulator of appetite in adult mice. The latter notion was supported by report of Sun et al. [24] suggesting that adult congenic ghrelin−/− and Ghsr−/− mice are not resistant to diet-induced obesity. However, they show under conditions of negative energy balance impairment in maintaining glucose homeostasis. The primary metabolic function of ghrelin in adult mice seems therefore to be to modulate glucose sensing and insulin sensitivity.
3. Variants in gene coding for ghrelin in humans Ghrelin gene has been a candidate gene in several studies focusing on the genetic variations underlying obesity and related co-morbidities. Several variations have been found to be associated with obesity related parameters. A genetic variation 152G > A in preproghrelin gene, that causes an amino acid change Arg51Gln of prepro-ghrelin, has been shown to be associated with obesity. Six subjects being heterozygous for such variation were found among obese but none in normal weight subjects [27]. However, after that report contrary results have been published, Gln51 allele has been shown to be equally distributed in obese and normal weight subjects [20,28]. Thus, its association with obesity is unknown. Interestingly, subjects with Gln51 allele were shown to be at higher risk for type 2 diabetes and hypertension in Finnish middle-aged subjects [19]. The variation changes the last amino acid of the mature peptide, but it is not known how this change might be linked to obesity in molecular level. It is possible, that the amino acid change at the last amino acid of the mature protein interferes with the proteases and disrupts the post-translational cleavage. This might lead to longer ghrelin molecules and might change the configuration or even the activity of the peptide. However, these hypothetic changes have not been demonstrated so far. Arg51Gln variation has been reported to be quite rare in several studies (the allelic distribution of the 51Gln being around 0–2%) [26]. Another variation of prepro-ghrelin gene, 214C > A, changes the amino acid leucine72 to methionine (Leu72Met). This amino acid 72 is outside the mature ghrelin peptide. The allelic frequency of this variant has been shown to vary around 1–9% in different populations [26]. The mechanism by which a variant outside the region coding for the mature peptide might be linked with certain phenotypes remains unknown, but some speculations that the variant might alter the stability of the mRNA or interfere with the splicing of the prepro-hormone have been raised. The 72Met allele has
been associated with obesity [12], earlier onset of [18,27] and a positive family history of obesity [28] and metabolic syndrome [33]. This variation has been shown to be associated with higher BMI in Japanese middle-aged men but not in women or older subjects [13]. However, the association of this variant with obesity and related parameters has not been replicated in all studies. For example, in the Finnish diabetes prevention study, Leu72Met polymorphism was not associated with BMI or waist circumference. Interestingly, in this study, the subjects with the 72Met allele were shown to have an increased risk for the development of type 2 diabetes [17]. Subjects with 72Met allele had decreased first-phase insulin secretion during OGTT, and this variation might contribute to defects in insulin and glucose metabolism [12]. Furthermore, this variant was shown to be associated with higher prevalence of metabolic syndrome, higher fasting glucose and triglyceride levels as well as lower HDL-cholesterol in subjects belonging to Amish Family diabetes study [23]. On the contrary, in a large study among Danish subjects, no association of Leu72Met variation with BMI, waist circumference or parameters of glucose and insulin metabolism were observed [4]. In addition, no association of Leu72Met polymorphism and type 2 diabetes was observed among Koreans [6]. It has also been reported that the Leu72Met polymorphism of the ghrelin gene was associated with a decreased risk for type 2 diabetes [2]. Leu72Met polymorphism has been associated with serum creatinine and Lp(a) levels in Finnish diabetic patients [25].All together, there is a lot of discrepancy in the results of different genetic association studies addressing the role of this polymorphism. In addition, a mutation 269A > T, which causes the Gln90Leu change in the prepro-ghrelin has been described. The 90Leu allele has been shown to be significantly more frequently detected among extremely obese than in normal weight subjects but when it was studied in a larger study population, the association disappeared [10]. Hinney et al. [10] found also a frameshift mutation in ghrelin gene, a 2 bp deletion at codon 34. This variant was not observed to have any effect on phenotype. Some other variants in the ghrelin gene have been found, but they have been studied less extensively. In Japanese women, an intron variant T3065C showing a strong linkage disequilibrium with Leu72Met, was found and the variant allele associated with higher body weight, BMI and waist circumference and lower serum HDL concentrations, whereas Leu72Met variants showed no association with these parameters. The authors suggested, that this intron variant might be even more important in respect to obesity than Leu72Met [1]. A recent review provides genetic-epidemiological evidence on genes, including ghrelin gene, associated with HDL cholesterol levels [5]. Ghrelin polymorphisms have shown inverse association with HDL-cholesterol in some studies. Recent sequencing data [9] suggest that common polymorphisms in ghrelin and its receptor genes are not major contributors to the development of polygenic obesity, although common variants may alter body weight and eating behavior and contribute to insulin resistance, in particular in the context of early-onset obesity. Similar negative findings have been observed in association studies exploring the relation between ghrelin gene variants and type 2 diabetes [8]. Influence of ghrelin gene polymorphisms on hypertension has also been studied and statistical evidence for association between GHRL SNPs and risk of hypertension has been observed in some studies [3,16]. In a recent study ghrelin gene expression correlated positively with the expressions of tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 during the weight loss/exercise intervention, but was not associated with the plasma ghrelin concentration in patients with metabolic syndrome [15]. Genotype-specific ghrelin gene expression in PBMCs was found for the −604G/A and the −501A/C polymorphisms in the ghrelin gene [15].
O. Ukkola / Peptides 32 (2011) 2319–2322
In conclusion, there is a growing body of evidence, that variations in the ghrelin gene might associate with obesity and its co-morbidities but there is discrepancy between different studies observed in different populations. 4. Ghrelin gene–environment interaction Total plasma ghrelin levels seem to be affected by acquired obesity independent of genetic background. Monozygotic twins discordant for obesity have the same genes and yet can show large differences in body mass. Leskelä et al. [14] determined fasting total ghrelin concentrations in a sample of monozygotic twin pairs discordant for obesity. The lean co-twins had higher ghrelin levels than the obese co-twins. In addition, the Leu72 allele was particularly common among monozygotic twins discordant for obesity, suggesting that this ghrelin allele is more permissive in the regulation of energy balance. The ghrelin gene may thus play a role in the regulation of variability of body weight, such that Leu72 allele carriers are more prone to weight variability in response to environmental factors. Robitaille et al. [22] assessed gene–diet interaction effects on cardiovascular disease (CVD) risk factors (waist circumference, plasma triacylglycerol, high-density lipoprotein-cholesterol and fasting glucose concentrations, and diastolic and systolic blood pressure) in the Quebec Family Study cohort. Results suggested that several alleles at candidate genes interact with dietary fat intake to modulate well-known CVD risk factors. Among them, the ghrelin Leu72Met polymorphism interacted with dietary fat in its relation to waist circumference and triacylglycerol concentrations. The identification of gene–diet interaction effects is thus likely to provide useful information concerning the etiology of cardiovascular disease. 5. Conclusions Obesity is a complex trait with several environmental, genetic and epigenetic factors and yet to mention, their interactions, playing a role in its etiology. It is likely, that genetic factors that affect obesity result from the additive effects of a combination of mutations in several genes at different loci rather than dominant or recessive effects of few genes or mutations. The role of one particular gene, such as gene coding for ghrelin, in the determination of BMI is likely to be very small. Studies exploring the functional significance of ghrelin SNP’s are needed since they are almost lacking in the earlier literature. In addition, the effects of ghrelin and its polymorphisms on metabolic parameters in a prospective study design are largely unknown. Several problems of these genetic association studies such as differences in allele frequencies between population, differences in study designs and differences in outcomes makes the role of ghrelin genetic variants in complex traits controversial. There are now more advanced methods available. We are in the era of sequencing genomes and the use of classic genetic study designs and the application of whole genome sequencing technologies to the understanding of the genetic influences of ghrelin in complex traits will be a main target in the future studies. This technology and the application to classic genetic studies design may overcome some of the contradictory findings in the genetic dissection of complex traits and the role of ghrelin. References [1] Ando T, Ichimaru Y, Konjiki F, Shoji M, Komaki G. Variations in the preproghrelin gene correlate with higher body mass index, fat mass, and body dissatisfaction in young Japanese women. Am J Clin Nutr 2007;86(1):25–32. [2] Berthold HK, Giannakidou E, Krone W, Mantzoros CS, Gouni-Berthold I. The Leu72Met polymorphism of the ghrelin gene is associated with a decreased risk for type 2 diabetes. Clin Chim Acta 2009;399(1–2):112–6.
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[3] Berthold HK, Giannakidou E, Krone W, Trégouët DA, Gouni-Berthold I. Influence of ghrelin gene polymorphisms on hypertension and atherosclerotic disease. Hypertens Res 2010;33(2):155–60. [4] Bing C, Ambye L, Fenger M, Jørgensen T, Borch-Johnsen K, Madsbad S, et al. Large-scale studies of the leu72met polymorphism of the ghrelin gene in relation to the metabolic syndrome and associated quantitative traits. Diabet Med 2005;22(9):1157–60. [5] Boes E, Coassin S, Kollerits B, Heid IM, Kronenberg F. Genetic-epidemiological evidence on genes associated with HDL cholesterol levels: a systematic indepth review. Exp Gerontol 2009;44:136–60. [6] Choi HJ, Cho YM, Moon MK, Choi HH, Shin HD, Jang HC, et al. Polymorphisms in the ghrelin gene are associated with serum high-density lipoprotein cholesterol level and not with type 2 diabetes mellitus in Koreans. J Clin Endocrinol Metab 2006;91(11):4657–63. [7] De Smet B, Depoortere I, Moechars D, Swennen Q, Moreaux B, Cryns K, et al. Energy homeostasis and gastric emptying in ghrelin knockout mice. J Pharmacol Exp Ther 2006;316(1):431–9. [8] Garcia EA, King P, Sidhu K, Ohgusu H, Walley A, Lecoeur C, et al. The role of ghrelin and ghrelin-receptor gene variants and promoter activity in type 2 diabetes. Eur J Endocrinol 2009;161(2):307–15. [9] Gueorguiev M, Lecoeur C, Meyre D, Benzinou M, Mein CA, Hinney A, et al. Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity. Obesity 2009;17(Silver Spring (4)):745–54. [10] Hinney A, Hoch A, Geller F, Schäfer H, Siegfried W, Goldschmidt H, et al. Ghrelin gene: identification of missense variants and a frameshift mutation in extremely obese children and adolescents and healthy normal weight students. J Clin Endocrinol Metab 2002;87(6):2716. [11] Kissebah AH, Sonnenberg GE, Myklebust J, Goldstein M, Broman K, James RG, et al. Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome. Proc Natl Acad Sci U S A 2000;97(26): 14478–83. [12] Korbonits M, Gueorguiev M, O’Grady E, Lecoeur C, Swan DC, Mein CA, et al. A variation in the ghrelin gene increases weight and decreases insulin secretion in tall, obese children. J Clin Endocrinol Metab 2002;87(8): 4005–8. [13] Kuzuya M, Ando F, Iguchi A, Shimokata H. Preproghrelin leu72met variant contributes to overweight in middle-aged men of a Japanese large cohort. Int J Obes (Lond) 2006;30(11):1609–14. [14] Leskelä P, Ukkola O, Vartiainen J, Rönnemaa T, Kaprio J, Bouchard C, et al. Fasting plasma total ghrelin concentrations in monozygotic twins discordant for obesity. Metabolism 2009;58(2):174–9. [15] Mager U, Kolehmainen M, de Mello VD, Schwab U, Laaksonen DE, Rauramaa R, et al. Expression of ghrelin gene in peripheral blood mononuclear cells and plasma ghrelin concentrations in patients with metabolic syndrome. Eur J Endocrinol 2008;158(April (4)):499–510. [16] Mager U, Kolehmainen M, Lindström J, Eriksson JG, Valle TT, Hämäläinen H, et al. Association between ghrelin gene variations and blood pressure in subjects with impaired glucose tolerance. Am J Hypertens 2006;19(9): 920–6. [17] Mager U, Lindi V, Lindström J, Eriksson JG, Valle TT, Hämäläinen H, et al. Association of the leu72met polymorphism of the ghrelin gene with the risk of type 2 diabetes in subjects with impaired glucose tolerance in the Finnish diabetes prevention study. Diabet Med 2006;23(6):685–9. [18] Miraglia del Giudice E, Santoro N, Cirillo G, Raimondo P, Grandone A, D’Aniello A, et al. Molecular screening of the ghrelin gene in Italian obese children: the leu72met variant is associated with an earlier onset of obesity. Int J Obes Relat Metab Disord 2004;28(3):447–50. [19] Pöykkö S, Ukkola O, Kauma H, Savolainen MJ, Kesäniemi YA. Ghrelin arg51gln mutation is a risk factor for type 2 diabetes and hypertension in a random sample of middle-aged subjects. Diabetologia 2003;46(4):455–8. [20] Pöykkö SM, Kellokoski E, Hörkkö S, Kauma H, Kesäniemi YA, Ukkola O. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes 2003;52(10):2546–53. [21] Ravussin E, Tschöp M, Morales S, Bouchard C, Heiman ML. Plasma ghrelin concentration and energy balance: overfeeding and negative energy balance studies in twins. J Clin Endocrinol Metab 2001;86(9):4547–51. [22] Robitaille J, Pérusse L, Bouchard C, Vohl MC. Genes, fat intake, and cardiovascular disease risk factors in the Quebec Family Study. Obesity 2007;15(Silver Spring (9)):2336–47. [23] Steinle NI, Pollin TI, O’Connell JR, Mitchell BD, Shuldiner AR. Variants in the ghrelin gene are associated with metabolic syndrome in the old order amish. J Clin Endocrinol Metab 2005;90(12):6672–7. [24] Sun Y, Butte NF, Garcia JM, Smith RG. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology 2007;149(2):843–50. [25] Ukkola O, Kesäniemi YA. Preproghrelin Leu72Met polymorphism in patients with type 2 diabetes mellitus. J Intern Med 2003;254(4):391–4. [26] Ukkola O, Ravussin E, Jacobson P, Pérusse L, Rankinen T, Tschöp M, et al. Role of ghrelin polymorphisms in obesity based on three different studies. Obes Res 2002;10(8):782–91. [27] Ukkola O, Ravussin E, Jacobson P, Snyder EE, Chagnon M, Sjöström L, et al. Mutations in the preproghrelin/ghrelin gene associated with obesity in humans. J Clin Endocrinol Metab 2001;86(8):3996–9. [28] Vivenza D, Rapa A, Castellino N, Bellone S, Petri A, Vacca G, et al. Ghrelin gene polymorphisms and ghrelin, insulin, igf-i, leptin and anthropometric data in children and adolescents. Eur J Endocrinol 2004;151(1):127–33.
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[29] Wajnrajch M, Ten I, Gretner JM, Leibel R. Genomic organization of the human ghrelin gene. The Journal of Endocrine genetics 2000;1:231–3. [30] Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, et al. Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc Natl Acad Sci U S A 2004;101(21):8227–32. [31] Wortley KE, del Rincon JP, Murray JD, Garcia K, Iida K, Thorner MO, et al. Absence of ghrelin protects against early-onset obesity. J Clin Invest 2005;115(12):3573–8. [32] Wu X, Cooper RS, Boerwinkle E, Turner ST, Hunt S, Myers R, et al. Combined analysis of genomewide scans for adult height: results from the nhlbi family blood pressure program. Eur J Hum Genet 2003;11(3):271–4.
[33] Xu LL, Xiang HD, Qiu CC, Xu Q. Association of ghrelin polymorphisms with metabolic syndrome in Han Nationality Chinese. Biomed Environ Sci 2008;21(3):188–92. [34] Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 2008;132(3):387–96. [35] Zhang JV, Ren P, Avsian-Kretchmer O, Luo C, Rauch R, Klein C, et al. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005;310(5750):996–9. [36] Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE, et al. Mice lacking ghrelin receptors resist the development of diet-induced obesity. J Clin Invest 2005;115(12):3564–72.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Review
Genetic variants of ghrelin in metabolic disorders Olavi Ukkola a,b,∗ a b
Institute of Clinical Medicine, Department of Internal Medicine and Biocenter Oulu, University of Oulu, Finland Clinical Research Center, Oulu University Hospital, P.O. Box 20, FIN-90029 OYS, Oulu, Finland
a r t i c l e
i n f o
Article history: Received 14 January 2011 Received in revised form 8 April 2011 Accepted 11 April 2011 Available online 17 April 2011 Keywords: Ghrelin Gene Obesity Diabetes
a b s t r a c t An increasing understanding of the role of genes in the development of obesity may reveal genetic variants that, in combination with conventional risk factors, may help to predict an individual’s risk for developing metabolic disorders. Accumulating evidence indicates that ghrelin plays a role in regulating food intake and energy homeostasis and it is a reasonable candidate gene for obesity-related co-morbidities. In crosssectional studies low total ghrelin concentrations and some genetic polymorphisms of ghrelin have been associated with obesity-associated diseases. The present review highlights many of the important problems in association studies of genetic variants and complex diseases. It is known that population-specific differences in reported associations exist. We therefore conclude that more studies on variants of ghrelin gene are needed to perform in different populations to get deeper understanding on the relationship of ghrelin gene and its variants to obesity. © 2011 Elsevier Inc. All rights reserved.
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Variants in gene coding for ghrelin in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ghrelin gene–environment interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2319 2320 2320 2321 2321 2321
1. Introduction Ghrelin gene (GHRL, GeneID 51738) locates in the chromosome 3p26-25 and it contains four exons and three introns [29]. Ghrelin is synthesized in a precursor protein form that consists of 117 amino acids and is called prepro-ghrelin. The latter is modified by the action of protein convertase enzymes into mature ghrelin, consisting of 28 amino acids and another 23 amino acid-peptide hormone obestatin [35]. The nomenclature refers to the biological activities of this molecule that are opposite to those of ghrelin: it decreases appetite and inhibits weight gain. After post-translational modifications an acyl group, most commonly an octanoyl group, is attached to the third amino acid, serine (Ser3 ) of the mature ghrelin by an esteric-bond. The enzyme that catalyzes the reaction in which
∗ Correspondence address: Department of Internal Medicine and Biocenter Oulu, University of Oulu, Kajaanintie 50/P.O. Box 5000, Oulu FIN-90014, Finland. Tel.: +358 08 315 4121; fax: +358 08 315 4139. E-mail address: olavi.ukkola@oulu.fi 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.04.013
the octanoate is attached to the hydroxyl group of the Ser3 has been identified recently and given a name ghrelin-O-acyltransferase (GOAT) [34]. In a study conducted on identical twins plasma ghrelin concentration at baseline was more alike within pairs of twins than between pairs indicating a probable genetic effect underlying the variability in plasma ghrelin levels [21]. Similarly, in response to overfeeding, the decreases in ghrelin concentration were more similar within than between pairs. Taken together, these results indicate that genetic differences among people could play a role in the production and/or the clearance of this gastric peptide [21]. Several whole genome scans have suggested that certain areas of the chromosome 3, the same chromosome where ghrelin and ghrelin receptor gene are located, might be linked with obesity or metabolic syndrome [11,32]. These findings, in addition to the relevant biological functions of these proteins, increase the potential for the genes coding for ghrelin and its receptor to be ideal candidate genes in the genetic studies focusing on obesity and obesity related co-morbidities.
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2. Animal models To further study the role of endogenous ghrelin in energy balance ghrelin-deficient (ghrl(−/−)) mice have been generated. These studies have failed to show any significant defects in food intake, or body weight in adult Ghrl−/− mice. Wortley et al. reported that ghrl(−/−) [30] mice exhibited normal growth rates as well as normal spontaneous food intake patterns. However, endogenous ghrelin seems to play a prominent role in determining the type of metabolic substrate (i.e., fat vs. carbohydrate) that is used for maintenance of energy balance, particularly under conditions of high fat intake. Two recent studies have demonstrated that Ghrl−/− and Ghsr−/− mice are resistant to diet-induced obesity when fed a high-fat diet during the early post-weaning period [31,36]. De Smet et al. [7] studied energy homeostasis and gastric emptying in ghrelin knockout mice. They concluded that ghrelin is not an essential regulator of food intake and gastric emptying, but its loss may be compensated by other redundant inputs. In old mice, meal initiation triggered by the light/dark cue may be related to ghrelin while in young animals, ghrelin seems to be involved in the selection of energy stores. Therefore, ghrelin appears not to be an essential regulator of appetite in adult mice. The latter notion was supported by report of Sun et al. [24] suggesting that adult congenic ghrelin−/− and Ghsr−/− mice are not resistant to diet-induced obesity. However, they show under conditions of negative energy balance impairment in maintaining glucose homeostasis. The primary metabolic function of ghrelin in adult mice seems therefore to be to modulate glucose sensing and insulin sensitivity.
3. Variants in gene coding for ghrelin in humans Ghrelin gene has been a candidate gene in several studies focusing on the genetic variations underlying obesity and related co-morbidities. Several variations have been found to be associated with obesity related parameters. A genetic variation 152G > A in preproghrelin gene, that causes an amino acid change Arg51Gln of prepro-ghrelin, has been shown to be associated with obesity. Six subjects being heterozygous for such variation were found among obese but none in normal weight subjects [27]. However, after that report contrary results have been published, Gln51 allele has been shown to be equally distributed in obese and normal weight subjects [20,28]. Thus, its association with obesity is unknown. Interestingly, subjects with Gln51 allele were shown to be at higher risk for type 2 diabetes and hypertension in Finnish middle-aged subjects [19]. The variation changes the last amino acid of the mature peptide, but it is not known how this change might be linked to obesity in molecular level. It is possible, that the amino acid change at the last amino acid of the mature protein interferes with the proteases and disrupts the post-translational cleavage. This might lead to longer ghrelin molecules and might change the configuration or even the activity of the peptide. However, these hypothetic changes have not been demonstrated so far. Arg51Gln variation has been reported to be quite rare in several studies (the allelic distribution of the 51Gln being around 0–2%) [26]. Another variation of prepro-ghrelin gene, 214C > A, changes the amino acid leucine72 to methionine (Leu72Met). This amino acid 72 is outside the mature ghrelin peptide. The allelic frequency of this variant has been shown to vary around 1–9% in different populations [26]. The mechanism by which a variant outside the region coding for the mature peptide might be linked with certain phenotypes remains unknown, but some speculations that the variant might alter the stability of the mRNA or interfere with the splicing of the prepro-hormone have been raised. The 72Met allele has
been associated with obesity [12], earlier onset of [18,27] and a positive family history of obesity [28] and metabolic syndrome [33]. This variation has been shown to be associated with higher BMI in Japanese middle-aged men but not in women or older subjects [13]. However, the association of this variant with obesity and related parameters has not been replicated in all studies. For example, in the Finnish diabetes prevention study, Leu72Met polymorphism was not associated with BMI or waist circumference. Interestingly, in this study, the subjects with the 72Met allele were shown to have an increased risk for the development of type 2 diabetes [17]. Subjects with 72Met allele had decreased first-phase insulin secretion during OGTT, and this variation might contribute to defects in insulin and glucose metabolism [12]. Furthermore, this variant was shown to be associated with higher prevalence of metabolic syndrome, higher fasting glucose and triglyceride levels as well as lower HDL-cholesterol in subjects belonging to Amish Family diabetes study [23]. On the contrary, in a large study among Danish subjects, no association of Leu72Met variation with BMI, waist circumference or parameters of glucose and insulin metabolism were observed [4]. In addition, no association of Leu72Met polymorphism and type 2 diabetes was observed among Koreans [6]. It has also been reported that the Leu72Met polymorphism of the ghrelin gene was associated with a decreased risk for type 2 diabetes [2]. Leu72Met polymorphism has been associated with serum creatinine and Lp(a) levels in Finnish diabetic patients [25].All together, there is a lot of discrepancy in the results of different genetic association studies addressing the role of this polymorphism. In addition, a mutation 269A > T, which causes the Gln90Leu change in the prepro-ghrelin has been described. The 90Leu allele has been shown to be significantly more frequently detected among extremely obese than in normal weight subjects but when it was studied in a larger study population, the association disappeared [10]. Hinney et al. [10] found also a frameshift mutation in ghrelin gene, a 2 bp deletion at codon 34. This variant was not observed to have any effect on phenotype. Some other variants in the ghrelin gene have been found, but they have been studied less extensively. In Japanese women, an intron variant T3065C showing a strong linkage disequilibrium with Leu72Met, was found and the variant allele associated with higher body weight, BMI and waist circumference and lower serum HDL concentrations, whereas Leu72Met variants showed no association with these parameters. The authors suggested, that this intron variant might be even more important in respect to obesity than Leu72Met [1]. A recent review provides genetic-epidemiological evidence on genes, including ghrelin gene, associated with HDL cholesterol levels [5]. Ghrelin polymorphisms have shown inverse association with HDL-cholesterol in some studies. Recent sequencing data [9] suggest that common polymorphisms in ghrelin and its receptor genes are not major contributors to the development of polygenic obesity, although common variants may alter body weight and eating behavior and contribute to insulin resistance, in particular in the context of early-onset obesity. Similar negative findings have been observed in association studies exploring the relation between ghrelin gene variants and type 2 diabetes [8]. Influence of ghrelin gene polymorphisms on hypertension has also been studied and statistical evidence for association between GHRL SNPs and risk of hypertension has been observed in some studies [3,16]. In a recent study ghrelin gene expression correlated positively with the expressions of tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 during the weight loss/exercise intervention, but was not associated with the plasma ghrelin concentration in patients with metabolic syndrome [15]. Genotype-specific ghrelin gene expression in PBMCs was found for the −604G/A and the −501A/C polymorphisms in the ghrelin gene [15].
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In conclusion, there is a growing body of evidence, that variations in the ghrelin gene might associate with obesity and its co-morbidities but there is discrepancy between different studies observed in different populations. 4. Ghrelin gene–environment interaction Total plasma ghrelin levels seem to be affected by acquired obesity independent of genetic background. Monozygotic twins discordant for obesity have the same genes and yet can show large differences in body mass. Leskelä et al. [14] determined fasting total ghrelin concentrations in a sample of monozygotic twin pairs discordant for obesity. The lean co-twins had higher ghrelin levels than the obese co-twins. In addition, the Leu72 allele was particularly common among monozygotic twins discordant for obesity, suggesting that this ghrelin allele is more permissive in the regulation of energy balance. The ghrelin gene may thus play a role in the regulation of variability of body weight, such that Leu72 allele carriers are more prone to weight variability in response to environmental factors. Robitaille et al. [22] assessed gene–diet interaction effects on cardiovascular disease (CVD) risk factors (waist circumference, plasma triacylglycerol, high-density lipoprotein-cholesterol and fasting glucose concentrations, and diastolic and systolic blood pressure) in the Quebec Family Study cohort. Results suggested that several alleles at candidate genes interact with dietary fat intake to modulate well-known CVD risk factors. Among them, the ghrelin Leu72Met polymorphism interacted with dietary fat in its relation to waist circumference and triacylglycerol concentrations. The identification of gene–diet interaction effects is thus likely to provide useful information concerning the etiology of cardiovascular disease. 5. Conclusions Obesity is a complex trait with several environmental, genetic and epigenetic factors and yet to mention, their interactions, playing a role in its etiology. It is likely, that genetic factors that affect obesity result from the additive effects of a combination of mutations in several genes at different loci rather than dominant or recessive effects of few genes or mutations. The role of one particular gene, such as gene coding for ghrelin, in the determination of BMI is likely to be very small. Studies exploring the functional significance of ghrelin SNP’s are needed since they are almost lacking in the earlier literature. In addition, the effects of ghrelin and its polymorphisms on metabolic parameters in a prospective study design are largely unknown. Several problems of these genetic association studies such as differences in allele frequencies between population, differences in study designs and differences in outcomes makes the role of ghrelin genetic variants in complex traits controversial. There are now more advanced methods available. We are in the era of sequencing genomes and the use of classic genetic study designs and the application of whole genome sequencing technologies to the understanding of the genetic influences of ghrelin in complex traits will be a main target in the future studies. This technology and the application to classic genetic studies design may overcome some of the contradictory findings in the genetic dissection of complex traits and the role of ghrelin. References [1] Ando T, Ichimaru Y, Konjiki F, Shoji M, Komaki G. Variations in the preproghrelin gene correlate with higher body mass index, fat mass, and body dissatisfaction in young Japanese women. Am J Clin Nutr 2007;86(1):25–32. [2] Berthold HK, Giannakidou E, Krone W, Mantzoros CS, Gouni-Berthold I. The Leu72Met polymorphism of the ghrelin gene is associated with a decreased risk for type 2 diabetes. Clin Chim Acta 2009;399(1–2):112–6.
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[3] Berthold HK, Giannakidou E, Krone W, Trégouët DA, Gouni-Berthold I. Influence of ghrelin gene polymorphisms on hypertension and atherosclerotic disease. Hypertens Res 2010;33(2):155–60. [4] Bing C, Ambye L, Fenger M, Jørgensen T, Borch-Johnsen K, Madsbad S, et al. Large-scale studies of the leu72met polymorphism of the ghrelin gene in relation to the metabolic syndrome and associated quantitative traits. Diabet Med 2005;22(9):1157–60. [5] Boes E, Coassin S, Kollerits B, Heid IM, Kronenberg F. Genetic-epidemiological evidence on genes associated with HDL cholesterol levels: a systematic indepth review. Exp Gerontol 2009;44:136–60. [6] Choi HJ, Cho YM, Moon MK, Choi HH, Shin HD, Jang HC, et al. Polymorphisms in the ghrelin gene are associated with serum high-density lipoprotein cholesterol level and not with type 2 diabetes mellitus in Koreans. J Clin Endocrinol Metab 2006;91(11):4657–63. [7] De Smet B, Depoortere I, Moechars D, Swennen Q, Moreaux B, Cryns K, et al. Energy homeostasis and gastric emptying in ghrelin knockout mice. J Pharmacol Exp Ther 2006;316(1):431–9. [8] Garcia EA, King P, Sidhu K, Ohgusu H, Walley A, Lecoeur C, et al. The role of ghrelin and ghrelin-receptor gene variants and promoter activity in type 2 diabetes. Eur J Endocrinol 2009;161(2):307–15. [9] Gueorguiev M, Lecoeur C, Meyre D, Benzinou M, Mein CA, Hinney A, et al. Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity. Obesity 2009;17(Silver Spring (4)):745–54. [10] Hinney A, Hoch A, Geller F, Schäfer H, Siegfried W, Goldschmidt H, et al. Ghrelin gene: identification of missense variants and a frameshift mutation in extremely obese children and adolescents and healthy normal weight students. J Clin Endocrinol Metab 2002;87(6):2716. [11] Kissebah AH, Sonnenberg GE, Myklebust J, Goldstein M, Broman K, James RG, et al. Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome. Proc Natl Acad Sci U S A 2000;97(26): 14478–83. [12] Korbonits M, Gueorguiev M, O’Grady E, Lecoeur C, Swan DC, Mein CA, et al. A variation in the ghrelin gene increases weight and decreases insulin secretion in tall, obese children. J Clin Endocrinol Metab 2002;87(8): 4005–8. [13] Kuzuya M, Ando F, Iguchi A, Shimokata H. Preproghrelin leu72met variant contributes to overweight in middle-aged men of a Japanese large cohort. Int J Obes (Lond) 2006;30(11):1609–14. [14] Leskelä P, Ukkola O, Vartiainen J, Rönnemaa T, Kaprio J, Bouchard C, et al. Fasting plasma total ghrelin concentrations in monozygotic twins discordant for obesity. Metabolism 2009;58(2):174–9. [15] Mager U, Kolehmainen M, de Mello VD, Schwab U, Laaksonen DE, Rauramaa R, et al. Expression of ghrelin gene in peripheral blood mononuclear cells and plasma ghrelin concentrations in patients with metabolic syndrome. Eur J Endocrinol 2008;158(April (4)):499–510. [16] Mager U, Kolehmainen M, Lindström J, Eriksson JG, Valle TT, Hämäläinen H, et al. Association between ghrelin gene variations and blood pressure in subjects with impaired glucose tolerance. Am J Hypertens 2006;19(9): 920–6. [17] Mager U, Lindi V, Lindström J, Eriksson JG, Valle TT, Hämäläinen H, et al. Association of the leu72met polymorphism of the ghrelin gene with the risk of type 2 diabetes in subjects with impaired glucose tolerance in the Finnish diabetes prevention study. Diabet Med 2006;23(6):685–9. [18] Miraglia del Giudice E, Santoro N, Cirillo G, Raimondo P, Grandone A, D’Aniello A, et al. Molecular screening of the ghrelin gene in Italian obese children: the leu72met variant is associated with an earlier onset of obesity. Int J Obes Relat Metab Disord 2004;28(3):447–50. [19] Pöykkö S, Ukkola O, Kauma H, Savolainen MJ, Kesäniemi YA. Ghrelin arg51gln mutation is a risk factor for type 2 diabetes and hypertension in a random sample of middle-aged subjects. Diabetologia 2003;46(4):455–8. [20] Pöykkö SM, Kellokoski E, Hörkkö S, Kauma H, Kesäniemi YA, Ukkola O. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes 2003;52(10):2546–53. [21] Ravussin E, Tschöp M, Morales S, Bouchard C, Heiman ML. Plasma ghrelin concentration and energy balance: overfeeding and negative energy balance studies in twins. J Clin Endocrinol Metab 2001;86(9):4547–51. [22] Robitaille J, Pérusse L, Bouchard C, Vohl MC. Genes, fat intake, and cardiovascular disease risk factors in the Quebec Family Study. Obesity 2007;15(Silver Spring (9)):2336–47. [23] Steinle NI, Pollin TI, O’Connell JR, Mitchell BD, Shuldiner AR. Variants in the ghrelin gene are associated with metabolic syndrome in the old order amish. J Clin Endocrinol Metab 2005;90(12):6672–7. [24] Sun Y, Butte NF, Garcia JM, Smith RG. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology 2007;149(2):843–50. [25] Ukkola O, Kesäniemi YA. Preproghrelin Leu72Met polymorphism in patients with type 2 diabetes mellitus. J Intern Med 2003;254(4):391–4. [26] Ukkola O, Ravussin E, Jacobson P, Pérusse L, Rankinen T, Tschöp M, et al. Role of ghrelin polymorphisms in obesity based on three different studies. Obes Res 2002;10(8):782–91. [27] Ukkola O, Ravussin E, Jacobson P, Snyder EE, Chagnon M, Sjöström L, et al. Mutations in the preproghrelin/ghrelin gene associated with obesity in humans. J Clin Endocrinol Metab 2001;86(8):3996–9. [28] Vivenza D, Rapa A, Castellino N, Bellone S, Petri A, Vacca G, et al. Ghrelin gene polymorphisms and ghrelin, insulin, igf-i, leptin and anthropometric data in children and adolescents. Eur J Endocrinol 2004;151(1):127–33.
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O. Ukkola / Peptides 32 (2011) 2319–2322
[29] Wajnrajch M, Ten I, Gretner JM, Leibel R. Genomic organization of the human ghrelin gene. The Journal of Endocrine genetics 2000;1:231–3. [30] Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, et al. Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc Natl Acad Sci U S A 2004;101(21):8227–32. [31] Wortley KE, del Rincon JP, Murray JD, Garcia K, Iida K, Thorner MO, et al. Absence of ghrelin protects against early-onset obesity. J Clin Invest 2005;115(12):3573–8. [32] Wu X, Cooper RS, Boerwinkle E, Turner ST, Hunt S, Myers R, et al. Combined analysis of genomewide scans for adult height: results from the nhlbi family blood pressure program. Eur J Hum Genet 2003;11(3):271–4.
[33] Xu LL, Xiang HD, Qiu CC, Xu Q. Association of ghrelin polymorphisms with metabolic syndrome in Han Nationality Chinese. Biomed Environ Sci 2008;21(3):188–92. [34] Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 2008;132(3):387–96. [35] Zhang JV, Ren P, Avsian-Kretchmer O, Luo C, Rauch R, Klein C, et al. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005;310(5750):996–9. [36] Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE, et al. Mice lacking ghrelin receptors resist the development of diet-induced obesity. J Clin Invest 2005;115(12):3564–72.