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NPY L7P polymorphism and metabolic diseases
Regulatory Peptides 149 (2008) 51–55
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Review
NPY L7P polymorphism and metabolic diseases Ullamari Pesonen ⁎ University of Turku, Department of Pharmacology, Drug Development and Therapeutics, FI-20014 Turun yliopisto, Finland
A R T I C L E
I N F O
A B S T R A C T
Article history: Received 29 March 2007 Accepted 22 August 2007 Available online 3 April 2008
Neuropeptide Y (NPY) is an abundant and widespread peptide in mammalian nervous system, both in the central and peripheral nervous systems. NPY is a multifunctional neurotransmitter with multiple modulator effects in the regulation of physiological functions and responses in the body. NPY is a potent orexigenic peptide, which has effects on energy balance at the level of energy intake, expenditure, and partition. There are many association studies between the NPY gene variants and cardiovascular and metabolic disease. Most of them are done by using p.L7P substitution as a marker. At the moment it seems that the p.L7P substitution of preproNPY protein causes altered NPY secretion, which leads to haemodynamic disturbances caused by sympathetic hyperactivity and to various effects caused by altered local signalling by NPY. SNP association studies using p.L7P polymorphism suggest that this functional substitution may be a strong independent risk factor for various metabolic and cardiovascular diseases. © 2008 Elsevier B.V. All rights reserved.
Keywords: Neuropeptide Y Cardiovascular disease Type 2 diabetes Obesity Lipid levels
Contents 1. Introduction . . . . . . . . . 2. Functional consequences of the 3. Lipid levels and atherosclerosis 4. BMI and obesity . . . . . . . 5. Impaired glucose tolerance and 6. Conclusion. . . . . . . . . . Note-added-in-proof . . . . . . . References . . . . . . . . . . . .
. . . . . . . . L7P alteration . . . . . . . . . . . . . . . . . type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Neuropeptide Y (NPY) is a 36-amino acid peptide belonging to the same structurally highly conserved peptide family (“PP-fold peptide family”) as pancreatic polypeptide (PP) and peptide YY (PYY). The members of the pancreatic polypeptide family are all evolved by gene duplications of the ancestral gene sequence [1], and they are known to have various effects on food intake, energy balance and on the function of the alimentary tract [2]. PP and PYY are released from the gastrointestinal tract and pancreas in response to meals, and they mediate anorexigenic signals from the gut to the central nervous system (CNS) [3]. Meanwhile, NPY is an abundant and widespread peptide in body, especially in the nervous system with multiple hormonal functions in the regulation of
⁎ Tel.: +358 2 333 7258; fax: +358 2 333 7216. E-mail address: ullamari.pesonen@utu.fi. 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.08.028
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HPA axis, growth, and cardiovascular functions. NPY also works as a neuropeptide transmitter in the sympathetic nervous system. NPY is a potent orexigenic peptide, which has multiple effects also on energy balance at the level of energy intake, expenditure, and partition. In CNS NPY is located in the areas known to regulate appetite, like hypothalamus and distinct brain stem areas. Administration of NPY directly into the CNS increases food intake and body adiposity leading to weight gain and obesity without clear tolerance formed to the stimulating effect even in chronic administration [4]. The weight gain is not only due to enhanced feeding, but also increased white fat (WAT) lipid storage by enlarged lipoprotein lipase (LPL) activity in liver and adipose tissue [5]. Additionally, the energy expenditure is decreased, since brown adipose tissue (BAT) thermogenesis is decreased due to lower sympathetic nerve activity to the brown fat. Thus, NPY favours positive energy balance by stimulating intake and decreasing consumption, which leads to obesity. Acute central NPY administration has also been shown to increase plasma insulin and corticosterone levels as well as to cause insulin resistance and stimulate glucose uptake levels in WAT
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independent of hyperphagia [6]. All these metabolic and hormonal changes are common features also in human obesity and in the metabolic syndrome X which are the major risk factors for type 2 diabetes (T2D) with serious complications and cardiovascular disease (CVD). There are many hormonal and metabolic signals known to have a perpetual interplay with NPY system. Leptin is a hormone secreted mainly by adipocytes and it signals of the amount of energy stored in adipocytes to the brain. Leptin controls energy balance, hormone, and glucose levels centrally through hypothalamic leptin receptors in hypothalamic appetite regulating neurons. Leptin decreases food intake in obese rats by inhibiting hypothalamic NPY synthesis and secretion. Also insulin and glucose are closely related to changes in leptin during feeding and fasting: Insulin directly stimulates leptin, and as feedback loop, leptin decreases glucose-stimulated insulin secretion. Leptin also acutely increases glucose metabolism by enhancing glucose turnover and uptake in peripheral tissues, without decreasing hepatic glycogen [7]. In human obesity levels of circulating leptin are high, but there seems to be a leptin resistance at the level of receptors and/or brain transport. Another important orexigenic shortterm signal is ghrelin, which has a crucial role in leptin, insulin, and NPY secretion and vice versa. Ghrelin is released mainly from the stomach and it connects the energy intake and expenditure in periphery and CNS. Stimulation of food intake by ghrelin is mediated by the stimulation of synthesis of NPY and other peptides in the hypothalamus [8]. Ghrelin is also expressed in the hypothalamus, where is has a direct action on appetite regulating pathways. The current hypothesis is that as adipose stores increase, both insulin and leptin levels increase, NPY synthesis and activity are inhibited, and food intake is reduced. Conversely, when animals are fasted (or when insulin or leptin levels are experimentally reduced and ghrelin levels increased), NPY synthesis and release are increased and the animal eats more food. Hence, considerable evidence supports the hypothesis that NPY is a key player in the negative feedback control of adiposity [9]. A primary physiological role of the NPY seems to be restoring normal energy balance and body fat stores under conditions of energy deficit or unbalance, which is signalled through alterations in leptin and/or insulin levels in periphery [10]. There are many human association studies which connect a single nucleotide polymorphism (SNP) of NPY gene or NPY receptor genes in a condition or disease [11]. Most of the candidate gene association studies on NPY are done using the non-synonymous sequence variant NM_000905.2:c.20TNC (p.L7P; 7th amino acid of the preproNPY, leucine, is changed to proline; rs16139) of the NPY gene. This SNP was originally discovered 1998 in Finnish and Dutch populations [12]. L7P substitution is located in the signal peptide part of preproNPY, and it is still the only documented, non-synonymous alteration in the NPY gene so far. The carrier frequency of P7 allele varies from 6% to 15% in the Caucasian populations, and it seems to be totally absent or extremely low in oriental populations (Fig. 1). Ding et al., 2003 has suggested that the L7P variant might originate in the north of Europe, since the highest allele frequencies were found in northern countries, and the allele frequency shows a geographical north to south gradient of decreasing frequency [13]. Additionally, in Europe there is a clear east–west gradient the allele frequency being higher in Eastern Europe compared to Western Europe. In animal studies the role of NPY in hyperphagia, energy expenditure, glucose, and insulin balance and in HPA-axis regulation is well documented. However, there may be major differences in the neuronal systems controlling food intake between rodents and humans, but these circuits are difficult to study directly in humans. This review summarises the current knowledge on predisposing role of the functional L7P polymorphism of NPY gene, which can be used to study the interactions of NPY system with various metabolic parameters in humans (Table 1). It seems that the p.P7 variant is clearly a major risk factor for significant health problems like type 2 diabetes (T2D) and cardiovascular diseases (CVD) when other risk factors are concomitant. This includes especially environmental factors (diet), dyslipidaemia, glucose intolerance, insulin
resistance, and hyperinsulinemia together with obesity (i.e. metabolic syndrome X). 2. Functional consequences of the L7P alteration The L7P alteration is a consequence of a single base substitution, c.20TNC, in the in the signal peptide part of preproNPY. Although this amino acid change causes a significant change in the tertiary structure of the signal peptide, it has been difficult to find the exact functional consequence at the molecular level [14]. At the cellular level the mutation causes different distribution of the NPY-related immunoreactivity in primary cultured HUVECs. In the cells carrying the p.P7 allele the amount of mature NPY without C-PON is prominent, while the homozygous [p.L7] + [p.L7] cells contain not fully processed proNPY i.e. NPY with C-PON [15]. This strongly suggests a difference in cellular storage and processing of the proNPY between the genotypes. Human subjects with p.P7 allele have different plasma NPY kinetics in rest and exercise than matched controls without the p.P7 allele: They have lower basal NPY secretion in rest, but greatly increased NPY release with sympathetic stimulation [15,16]. It seems that in rest, the L7P alteration leads to impaired release and intracellular retention of NPY, followed by an exaggerated release of NPY in high-intensity sympathetic stimulation. Additionally, the elimination of NPY from plasma by degradation or excretion through kidneys could be affected differently in rest and exercise in subjects carrying the p.P7 allele. However, changed intracellular kinetics of NPY synthesis and processing seems more plausible mechanism on the basis of known functions of signal peptides and cellular studies on HUVEC. Furthermore NPY being also a neurotransmitter is primarily regulated at the level of neuronal neurotransmitter content and release [17]. In conclusion, it seems that the L7P substitution causes altered synthesis, processing, and release of the active, mature NPY. 3. Lipid levels and atherosclerosis In the landmark study 1998 the p.P7 allele was associated with high serum cholesterol and low-density lipoprotein (LDL) cholesterol levels in obese, non-diabetic Finnish and Dutch subjects [12]. This work provided the first genetic evidence that NPY may be linked to altered cholesterol metabolism, and that the p.P7 allele in the NPY signalling peptide may be one of the strongest genetic factors influencing serum cholesterol and LDL levels in obese subjects. After the initial study the association was studied in several independent study populations. p.P7 allele is associated with higher mean serum TG values in the preschool aged boys and type 1 diabetic patients [18– 20]. In a large population-based sample of elderly Finnish men the P7 substitution was associated with accelerated four-year increase in the mean and maximal common carotid intima-media thickness (IMT) [21]. Men with P7 substitution had 31% greater increase in the mean IMT and 20% greater increase in the maximal IMT than homozygous [p.L7] + [p.L7] men. The P7 substitution was also in this study related to increased serum total cholesterol and LDL cholesterol in obese (body mass index (BMI) N 30 kg/m2) men. The association of p.P7 substitution and atherosclerosis was further confirmed in a independent population study, where the p.P7 substitution was associated with increased carotid IMT in type 2 diabetic patients, as well as with accelerated progression of carotid atherosclerosis during a four-year observation period in middle aged men [22]. Chronically high plasma lipid levels lead to an accumulation of TG in skeletal muscle and the liver, as well as increased intracellular concentrations of free fatty acids (FFAs), which are mainly derived from the TG rich lipoprotein particles. The physiological role of FFAs is to transport energy between the blood circulation and tissues and they are considered as a link between lipid and insulin metabolisms [23]. FFAs have a negative impact on insulin signalling which can lead to development of muscular insulin resistance which in turn is known
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Fig. 1. Frequency of P7 allele of L7P variation in NPY gene in different populations. Only those populations, where over 100 unrelated individuals are genotyped, have been included. The figures inside the columns indicate the pooled number of individuals genotyped in the literature (http://www.ncbi.nlm.nih.gov).
to promote directly or indirectly progression of atherosclerosis. In the exercise plasma levels of catecholamines, NPY, growth hormone, and FFA increase and insulin decrease. In the study with carefully matched pairs of different L7P genotypes exposed to strenuous exercise the group with heterozygote [p.L7] + [p.P7] had clearly lower postexercise FFA values [15], but not during resting conditions [24]. The inhibition of lipolysis in human adipocytes by NPY has been shown to be dosedependent [25], and therefore higher NPY release during exercise in subjects with the [p.L7] + [p.P7] genotype may explain the difference in FFA concentration between the genotypes. Low levels of FFAs in subjects with the p.P7 variant of preproNPY was observed in the presence of both low and high insulin levels, which indicates that this effect is independent of insulin's antilipolytic action [26]. This notion is also supported by our observations that the activities of LPL and hormone-sensitive LPL, which are regulated by insulin, are not changed in subjects with [p.L7] + [p.P7] genotype [27]. Most likely, insulin and NPY independent of each other lower a cAMP level, which in turn leads to inactivation of hormone-sensitive LPL and lower serum FFAs. NPY which reduces intracellular cAMP concentrations in human adipocytes can inhibit lipolysis and increase liver and WAT lipogenesis [28]. NPY increases centrally the expression of LPL and LPL enzymatic activity in WAT independent in food intake [5,29]. There is no significant difference between the L7P genotypes in the magnitude of postprandial lipemia induced by an oral fat tolerance test. In homozygous [p.L7] + [p.L7] genotype subjects the hormone-sensitive LPL-to-LPL ratio correlates with area of plasma total, VLDL and chylomicron TG, but not in subjects with heterozygous [p.L7] + [p.P7] genotype [27]. This suggest that there might be compositional differences in the lipid particles affecting postprandial lipid metabolism and the relationship of hormone-sensitive LPL and activities to lipid metabolism may differ between the L7P genotype groups. Most of the above mentioned positive association studies have been done in Finnish populations, but there are negative reports in different populations. In Brazilian population, non-diabetic individuals of European descent, p.P7 carriers had a tendency to have lower total cholesterol values, but the number of subjects with p.P7 substitution was very low (n = 18) to reach any statistical power [30]. In recent reports, the researchers could not find associations of plasma TG, total cholesterol, VLDL cholesterol, LDL cholesterol, and HDL cholesterol with L7P sequence variation in Swedish subjects nor in subgroups of obese and non-obese subjects analyzed separately [14] or in a Polish family study [31]. Some of
Table 1 Summary table of the studies on L7P polymorphism on different metabolic diseases and related conditions Disease or condition
Population
n
Reference
Higher serum TG concentrations
Young boys
[18,20]
Type 1 diabetes patients Obese adults
367/ 298/ 267 996 231
Women with CHD
414
[33]
Hypertensive subjects
638
[31]
266 966 199
[26] [21] [22]
247
[32]
1041
[39]
1040
[43]
98 27b
[46] [44]
309
[39]
86 253 94
[48] [47] [46]
1032
[49]
131 582 906 966 27b
[30] [38] [14] [21] [44]
Higher serum levels of total and LDL cholesterol Higher serum levels of total cholesterol Higher serum levels of LDL cholesterola Lower FFA levels Accelerated increase in intima-media thickness
Middle-aged subjects Middle-aged men Middle-aged, T2D patients and non-diabetic control subjects Higher FMD Middle-aged men, prepubertal children Development of T2D and IGT T2D, IGT and healthy subjects Hypertensive and healthy subjects Younger age of onset of T2D T2D patients Lower plasma insulin concentrations Healthy young subjects and insulin to glucose ratio during OGTT IGT, higher fasting plasma glucose T2D patients levels Development of diabetic retinopathy Middle-aged, T2D patients T2D patients Increased risk for nephropathy T2D patients with diabetic retinopathy Increased risk for myocardial Hypertensive subjects infarction and stroke Lower BMI Premenopausal women Higher BMI Adult men Non obese subjects Increased blood pressure Middle-aged men Higher heart rate variability Healthy young subjects
[19] [12]
TG = triglyceride, LDL = low-density lipoprotein, CHD = coronary heart disease, FFA = free fatty acids, T2D = type 2 diabetes, FMD = flow mediated endothelial dependent dilatation, IGT = impaired glucose tolerance, OGTT = oral glucose-tolerance test, BMI = body mass index. a Epistatic interaction with β2-adrenergic receptor polymorphism. b Matched pairs.
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the later studies on healthy, normal weight Finnish cohorts [15,32,33] have also suggested that p.P7 substitution might carry a risk factor to elevated plasma lipid levels only in combination with other major metabolic or environmental risk factors, like higher age, obesity, or diet rich in dairy products, which is very common in Eastern Finland populations [34]. 4. BMI and obesity NPY has been since its discovery the most potent and probably most studied neuropeptide which enhances body weight gain and obesity. Although the role of NPY in regulation of human feeding and energy balance is not totally clear, there are several human linkage studies, which connect NPY to human obesity and suggest that the NPY gene is a candidate gene for obesity related phenotypes [35–37]. Although there are many positive associations with p.L7P substitution and obesity, the association seems to depend on the study population and environmental factors. The largest study, which found a positive association, was done in Dutch men: subjects with the p.P7 substitution had higher BMI and higher risk on being overweight [38]. There are also some distinct subpopulations where p.L7P substitution of the NPY gene is independently associated with higher BMI or body weight: type 1 diabetic patients [19], newborn boys (birth weight) [18], normal weight subjects [14] and men with T2D [39]. There is one conflicting study, where the p.L7P substitution is associated with lower BMI in premenopausal women, but this may be due to a very low number of subjects with P7 variant (n = 7) in the study [30]. 5. Impaired glucose tolerance and type 2 diabetes One of the peripheral energy store signals integrated in hypothalamus by NPY containing nerves is insulin. In experimental animals central NPY administration leads to hyperinsulinemia and peripheral insulin resistance [40], but a tonic central effect of NPY is needed these metabolic abnormalities [41]. In periphery insulin and NPY have a different functional relationship: NPY inhibits glucose-stimulated insulin secretion from isolated pancreas. This may be a direct peripheral interaction, since liver contains NPYergic neurons, which are involved in the regulation of hepatic function and haemodynamics [42]. The L7P polymorphism has an effect on insulin secretion and glucose homeostasis. Healthy subjects with p.P7 allele have higher glucose, lower insulin and lower insulin/glucose ratio during rest and after meals [24,43] as well as during exercise [15] compared to subjects without p.P7 allele. This was also observed during the oral glucose-tolerance test with matched healthy subjects [44], but not in a large, heterogenous group of middle aged subjects [26]. The changes in the diurnal glucose and insulin levels may be reflected as altered insulin sensitivity, but this is still disputable due to large, contradictory studies [26,43]. If there is a true difference in the insulin levels, it could due to a central component regulating autonomic balance or peripherally increased local NPY levels in pancreas, where NPY has been shown to inhibit insulin secretion [45]. Two major abnormalities in T2D are β-cell dysfunction in pancreas and decreased insulin sensitivity in liver, skeletal muscle and adipose tissue. T2D patients have a loss of first-phase glucose-stimulated insulin response to food intake, which results in hyperglycaemia and hypertriglycaemia. Although there seems to be a genetic component in the T2D, it is often concomitant of obesity, insufficient physical activity, and excessive carbohydrate intake. L7P substitution has been associated with onset of T2D [39,43,46], impaired glucose tolerance [39] and with the worse glycaemic balance in type 1 diabetic patients as measured by HbA1c levels [19]. However, most of the association studies on metabolic and hormonal parameters found previously positive in healthy study subjects, have resulted negative associations when studied in diabetic patients. There is no association with serum lipid levels [22,47] or with plasma NPY levels [19] between the [p.L7] +
[p.L7] genotype and [p.L7] + [p.P7] genotype in diabetic patients. Instead, many studies have shown that diabetic patients with p.P7 variant are more prone to serious complications of the disease [19,47,48]. It seems that diabetes alters the metabolic status of the body such a way that, the ‘normal’ interactions of NPY are disturbed and the altered NPY balance further compromises the metabolic balance of the body. This may happen at the central autonomic level or at the peripheral, local level or at both levels. 6. Conclusion NPY is a multifunctional neurotransmitter with multiple modulator effects in the regulation of physiological functions and responses in the body. A change in the molecular structure of its precursor, the L7P substitution, has been shown to associate with many diseases and with many changes in sympathoadrenal, hormonal, and metabolic balance in healthy subjects. The exact molecular and cellular mechanisms by which the L7P polymorphism leads to the observed changes are not clear. At the moment it seems that L7P substitution of preproNPY protein causes altered NPY secretion, which leads to haemodynamic disturbances caused by sympathetic hyperactivity and to various effects caused by altered local signalling by NPY [11]. SNP association studies, or preferably haplotype analysis, on NPY gene can be used to guide functional studies and elucidate the pathophysiological NPYrelated mechanisms behind cardiovascular and metabolic disorders. A challenge with the association studies using L7P substitution is the extensive variation of the allele frequency in different populations. The effects of the L7P substitution on cardiovascular regulation and diseases are not likely to be relevant in populations other than European descent. However, this does not forbid us drawing conclusions of physiological and/or pathological roles of NPY in cardiovascular control and development of diseases. The study of DNA sequence variant that contributes to common disease risk offers one of the best opportunities for understanding the complex cause of disease in humans. The clinical relevance of L7P genetic variant in cardiovascular regulation and clinical prediction and treatment of cardiovascular and metabolic diseases remains to be determined. Note-added-in-proof In a recent paper Zhou et al. (Nature 452 (7190), 997-1001, 2008) show that NPY gene haplotypes predict NPY expression levels and thus plasma NPY concentrations. They claim that the majority of the variation in expression can be explained by a single SNP (rs16147) in the promoter area of NPY gene. The authors were not able to fully account the effects of p.L7P variant, because of the low frequency of this mutation in the study subjects. However, it seems that rs16147 T allele is in full linkage disequilibrium with Pro7 allele especially in Finnish population (Dr. Zhou, personal communication).
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[29] Billington CJ, Briggs JE, Harker S, Grace M, Levine AS. Neuropeptide Y in hypothalamic paraventricular nucleus: a center coordinating energy metabolism. Am J Physiol 1994;266:R1765–70. [30] Mattevi VS, Zembrzuski VM, Hutz MH. Association analysis of genes involved in the leptin-signaling pathway with obesity in Brazil. Int J Obes Relat Metab Disord 2002;26:1179–85. [31] Tomaszewski M, Charchar FJ, Lacka B, Pesonen U, Wang WY, ZukowskaSzczechowska E, et al. Epistatic interaction between b2-adrenergic receptor and neuropeptide Y genes influences LDL-cholesterol in hypertension. Hypertension 2004;44:689–94. [32] Järvisalo MJ, Jartti L, Karvonen MK, Pesonen U, Koulu M, Marniemi J, et al. Enhanced endothelium-dependent vasodilation in subjects with Proline7 substitution in the signal peptide of neuropeptide Y. Atherosclerosis 2003;167:319–26. [33] Erkkilä AT, Lindi V, Lehto S, Laakso M, Uusitupa MI. . Association of leucine 7 to proline 7 polymorphism in the preproneuropeptide y with serum lipids in patients with coronary heart disease. Mol Genet Metab 2002;75:260–4. [34] Menotti A, Kromhout D, Blackburn H, Fidanza F, Buzina R, Nissinen A, The Seven Countries Study Research Group. Food intake patterns and 25-year mortality from coronary heart disease: cross-cultural correlations in the Seven Countries Study. Eur J Epidemiol 1999;15:507–15. [35] Snyder EE, Walts B, Perusse L, Chagnon YC, Weisnagel SJ, Rankinen T, et al. The human obesity gene map: the 2003 update. Obes Res 2004;12:369–439. [36] Bray MS, Boerwinkle E, Hanis CL. Linkage analysis of candidate obesity genes among the Mexican-American population of Starr County, Texas. Genet Epidemiol 1999;16:397–411. [37] Bray MS, Boerwinkle E, Hanis CL. Sequence variation within the neuropeptide Y gene and obesity in Mexican Americans. Obes Res 2000;8:219–26. [38] van Rossum CTM, Pijl H, Adan RAH, Hoebee B, Seidell JC. Polymorphisms in the NPY and AGRP genes and body fatness in Dutch adults. Int J Obes 2006;30:1522–8. [39] Nordman S, Ding B, Ostenson CG, Karvestedt L, Brismar K, Efendic S, et al. Leu7Pro polymorphism in the neuropeptide Y (NPY) gene is associated with impaired glucose tolerance and type 2 diabetes in Swedish men. Exp Clin Endocrinol Diabetes 2005;113:282–7. [40] Zarjevski N, Cusin I, Vettor R, Rohner-Jeanrenaud F, Jeanrenaud B. Intracerebroventricular administration of neuropeptide Y to normal rats has divergent effects on glucose utilization by adipose tissue and skeletal muscle. Diabetes 1994;43:764–9. [41] Vettor R, Zarjevski N, Cusin I, Rohner-Jeanrenaud F, Jeanrenaud B. Induction and reversibility of an obesity syndrome by intracerebroventricular neuropeptide Y administration to normal rats. Diabetologia 1994;37:1202–8. [42] Moltz JH, McDonald JK. Neuropeptide Y: Direct and indirect action on insulin secretion in the rat. Peptides 1985;6:1155–9. [43] Ukkola O, Kesaniemi YA. Leu7Pro polymorphism of PreproNPY associated with an increased risk for type II diabetes in middle-aged subjects. Eur J Clin Nutr 2007. [44] Jaakkola U, Kuusela T, Jartti T, Pesonen U, Koulu M, Vahlberg T, et al. The Leu7Pro polymorphism of preproNPY is associated with decreased insulin secretion, delayed ghrelin suppression and increased cardiovascular responsiveness to norepinephrine during oral glucose-tolerance test. J Clin Endocrinol Metab 2005;90:3646–52. [45] Wang ZL, Bennet WM, Wang RM, Ghatei MA, Bloom SR. Evidence of a paracrine role of neuropeptide-Y in the regulation of insulin release from pancreatic islets of normal and dexamethasone-treated rats. Endocrinology 1994;135:200–6. [46] Jaakkola U, Pesonen U, Vainio-Jylhä E, Koulu M, Pöllönen M, Kallio J. The Leu7Pro polymorphism of neuropeptide Y is associated with younger age of onset of type 2 diabetes mellitus and increased risk for nephropathy in subjects with diabetic retinopathy. Exp Clin Endocrinol Diabetes 2006;114:147–52. [47] Koulu M, Movafagh S, Tuohimaa J, Jaakkola U, Kallio J, Pesonen U, et al. Neuropeptide Y and Y2-receptor are involved in development of diabetic retinopathy and retinal neovascularization. Ann Med 2004;36:232–40. [48] Niskanen L, Voutilainen-Kaunisto R, Terasvirta M, Karvonen MK, Valve R, Pesonen U, et al. Leucine 7 to proline 7 polymorphism in the neuropeptide Y gene is associated with retinopathy in type 2 diabetes. Exp Clin Endocrinol Diabetes 2000;108:235–6. [49] Wallerstedt SM, Skrtic S, Eriksson AL, Ohlsson C, Hedner T. Association analysis of the polymorphism T1128C in the signal peptide of neuropeptide Y in a Swedish hypertensive population. J Hypertens 2004;22:1277–81.
Contents lists available at ScienceDirect
Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Review
NPY L7P polymorphism and metabolic diseases Ullamari Pesonen ⁎ University of Turku, Department of Pharmacology, Drug Development and Therapeutics, FI-20014 Turun yliopisto, Finland
A R T I C L E
I N F O
A B S T R A C T
Article history: Received 29 March 2007 Accepted 22 August 2007 Available online 3 April 2008
Neuropeptide Y (NPY) is an abundant and widespread peptide in mammalian nervous system, both in the central and peripheral nervous systems. NPY is a multifunctional neurotransmitter with multiple modulator effects in the regulation of physiological functions and responses in the body. NPY is a potent orexigenic peptide, which has effects on energy balance at the level of energy intake, expenditure, and partition. There are many association studies between the NPY gene variants and cardiovascular and metabolic disease. Most of them are done by using p.L7P substitution as a marker. At the moment it seems that the p.L7P substitution of preproNPY protein causes altered NPY secretion, which leads to haemodynamic disturbances caused by sympathetic hyperactivity and to various effects caused by altered local signalling by NPY. SNP association studies using p.L7P polymorphism suggest that this functional substitution may be a strong independent risk factor for various metabolic and cardiovascular diseases. © 2008 Elsevier B.V. All rights reserved.
Keywords: Neuropeptide Y Cardiovascular disease Type 2 diabetes Obesity Lipid levels
Contents 1. Introduction . . . . . . . . . 2. Functional consequences of the 3. Lipid levels and atherosclerosis 4. BMI and obesity . . . . . . . 5. Impaired glucose tolerance and 6. Conclusion. . . . . . . . . . Note-added-in-proof . . . . . . . References . . . . . . . . . . . .
. . . . . . . . L7P alteration . . . . . . . . . . . . . . . . . type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Neuropeptide Y (NPY) is a 36-amino acid peptide belonging to the same structurally highly conserved peptide family (“PP-fold peptide family”) as pancreatic polypeptide (PP) and peptide YY (PYY). The members of the pancreatic polypeptide family are all evolved by gene duplications of the ancestral gene sequence [1], and they are known to have various effects on food intake, energy balance and on the function of the alimentary tract [2]. PP and PYY are released from the gastrointestinal tract and pancreas in response to meals, and they mediate anorexigenic signals from the gut to the central nervous system (CNS) [3]. Meanwhile, NPY is an abundant and widespread peptide in body, especially in the nervous system with multiple hormonal functions in the regulation of
⁎ Tel.: +358 2 333 7258; fax: +358 2 333 7216. E-mail address: ullamari.pesonen@utu.fi. 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.08.028
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HPA axis, growth, and cardiovascular functions. NPY also works as a neuropeptide transmitter in the sympathetic nervous system. NPY is a potent orexigenic peptide, which has multiple effects also on energy balance at the level of energy intake, expenditure, and partition. In CNS NPY is located in the areas known to regulate appetite, like hypothalamus and distinct brain stem areas. Administration of NPY directly into the CNS increases food intake and body adiposity leading to weight gain and obesity without clear tolerance formed to the stimulating effect even in chronic administration [4]. The weight gain is not only due to enhanced feeding, but also increased white fat (WAT) lipid storage by enlarged lipoprotein lipase (LPL) activity in liver and adipose tissue [5]. Additionally, the energy expenditure is decreased, since brown adipose tissue (BAT) thermogenesis is decreased due to lower sympathetic nerve activity to the brown fat. Thus, NPY favours positive energy balance by stimulating intake and decreasing consumption, which leads to obesity. Acute central NPY administration has also been shown to increase plasma insulin and corticosterone levels as well as to cause insulin resistance and stimulate glucose uptake levels in WAT
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independent of hyperphagia [6]. All these metabolic and hormonal changes are common features also in human obesity and in the metabolic syndrome X which are the major risk factors for type 2 diabetes (T2D) with serious complications and cardiovascular disease (CVD). There are many hormonal and metabolic signals known to have a perpetual interplay with NPY system. Leptin is a hormone secreted mainly by adipocytes and it signals of the amount of energy stored in adipocytes to the brain. Leptin controls energy balance, hormone, and glucose levels centrally through hypothalamic leptin receptors in hypothalamic appetite regulating neurons. Leptin decreases food intake in obese rats by inhibiting hypothalamic NPY synthesis and secretion. Also insulin and glucose are closely related to changes in leptin during feeding and fasting: Insulin directly stimulates leptin, and as feedback loop, leptin decreases glucose-stimulated insulin secretion. Leptin also acutely increases glucose metabolism by enhancing glucose turnover and uptake in peripheral tissues, without decreasing hepatic glycogen [7]. In human obesity levels of circulating leptin are high, but there seems to be a leptin resistance at the level of receptors and/or brain transport. Another important orexigenic shortterm signal is ghrelin, which has a crucial role in leptin, insulin, and NPY secretion and vice versa. Ghrelin is released mainly from the stomach and it connects the energy intake and expenditure in periphery and CNS. Stimulation of food intake by ghrelin is mediated by the stimulation of synthesis of NPY and other peptides in the hypothalamus [8]. Ghrelin is also expressed in the hypothalamus, where is has a direct action on appetite regulating pathways. The current hypothesis is that as adipose stores increase, both insulin and leptin levels increase, NPY synthesis and activity are inhibited, and food intake is reduced. Conversely, when animals are fasted (or when insulin or leptin levels are experimentally reduced and ghrelin levels increased), NPY synthesis and release are increased and the animal eats more food. Hence, considerable evidence supports the hypothesis that NPY is a key player in the negative feedback control of adiposity [9]. A primary physiological role of the NPY seems to be restoring normal energy balance and body fat stores under conditions of energy deficit or unbalance, which is signalled through alterations in leptin and/or insulin levels in periphery [10]. There are many human association studies which connect a single nucleotide polymorphism (SNP) of NPY gene or NPY receptor genes in a condition or disease [11]. Most of the candidate gene association studies on NPY are done using the non-synonymous sequence variant NM_000905.2:c.20TNC (p.L7P; 7th amino acid of the preproNPY, leucine, is changed to proline; rs16139) of the NPY gene. This SNP was originally discovered 1998 in Finnish and Dutch populations [12]. L7P substitution is located in the signal peptide part of preproNPY, and it is still the only documented, non-synonymous alteration in the NPY gene so far. The carrier frequency of P7 allele varies from 6% to 15% in the Caucasian populations, and it seems to be totally absent or extremely low in oriental populations (Fig. 1). Ding et al., 2003 has suggested that the L7P variant might originate in the north of Europe, since the highest allele frequencies were found in northern countries, and the allele frequency shows a geographical north to south gradient of decreasing frequency [13]. Additionally, in Europe there is a clear east–west gradient the allele frequency being higher in Eastern Europe compared to Western Europe. In animal studies the role of NPY in hyperphagia, energy expenditure, glucose, and insulin balance and in HPA-axis regulation is well documented. However, there may be major differences in the neuronal systems controlling food intake between rodents and humans, but these circuits are difficult to study directly in humans. This review summarises the current knowledge on predisposing role of the functional L7P polymorphism of NPY gene, which can be used to study the interactions of NPY system with various metabolic parameters in humans (Table 1). It seems that the p.P7 variant is clearly a major risk factor for significant health problems like type 2 diabetes (T2D) and cardiovascular diseases (CVD) when other risk factors are concomitant. This includes especially environmental factors (diet), dyslipidaemia, glucose intolerance, insulin
resistance, and hyperinsulinemia together with obesity (i.e. metabolic syndrome X). 2. Functional consequences of the L7P alteration The L7P alteration is a consequence of a single base substitution, c.20TNC, in the in the signal peptide part of preproNPY. Although this amino acid change causes a significant change in the tertiary structure of the signal peptide, it has been difficult to find the exact functional consequence at the molecular level [14]. At the cellular level the mutation causes different distribution of the NPY-related immunoreactivity in primary cultured HUVECs. In the cells carrying the p.P7 allele the amount of mature NPY without C-PON is prominent, while the homozygous [p.L7] + [p.L7] cells contain not fully processed proNPY i.e. NPY with C-PON [15]. This strongly suggests a difference in cellular storage and processing of the proNPY between the genotypes. Human subjects with p.P7 allele have different plasma NPY kinetics in rest and exercise than matched controls without the p.P7 allele: They have lower basal NPY secretion in rest, but greatly increased NPY release with sympathetic stimulation [15,16]. It seems that in rest, the L7P alteration leads to impaired release and intracellular retention of NPY, followed by an exaggerated release of NPY in high-intensity sympathetic stimulation. Additionally, the elimination of NPY from plasma by degradation or excretion through kidneys could be affected differently in rest and exercise in subjects carrying the p.P7 allele. However, changed intracellular kinetics of NPY synthesis and processing seems more plausible mechanism on the basis of known functions of signal peptides and cellular studies on HUVEC. Furthermore NPY being also a neurotransmitter is primarily regulated at the level of neuronal neurotransmitter content and release [17]. In conclusion, it seems that the L7P substitution causes altered synthesis, processing, and release of the active, mature NPY. 3. Lipid levels and atherosclerosis In the landmark study 1998 the p.P7 allele was associated with high serum cholesterol and low-density lipoprotein (LDL) cholesterol levels in obese, non-diabetic Finnish and Dutch subjects [12]. This work provided the first genetic evidence that NPY may be linked to altered cholesterol metabolism, and that the p.P7 allele in the NPY signalling peptide may be one of the strongest genetic factors influencing serum cholesterol and LDL levels in obese subjects. After the initial study the association was studied in several independent study populations. p.P7 allele is associated with higher mean serum TG values in the preschool aged boys and type 1 diabetic patients [18– 20]. In a large population-based sample of elderly Finnish men the P7 substitution was associated with accelerated four-year increase in the mean and maximal common carotid intima-media thickness (IMT) [21]. Men with P7 substitution had 31% greater increase in the mean IMT and 20% greater increase in the maximal IMT than homozygous [p.L7] + [p.L7] men. The P7 substitution was also in this study related to increased serum total cholesterol and LDL cholesterol in obese (body mass index (BMI) N 30 kg/m2) men. The association of p.P7 substitution and atherosclerosis was further confirmed in a independent population study, where the p.P7 substitution was associated with increased carotid IMT in type 2 diabetic patients, as well as with accelerated progression of carotid atherosclerosis during a four-year observation period in middle aged men [22]. Chronically high plasma lipid levels lead to an accumulation of TG in skeletal muscle and the liver, as well as increased intracellular concentrations of free fatty acids (FFAs), which are mainly derived from the TG rich lipoprotein particles. The physiological role of FFAs is to transport energy between the blood circulation and tissues and they are considered as a link between lipid and insulin metabolisms [23]. FFAs have a negative impact on insulin signalling which can lead to development of muscular insulin resistance which in turn is known
U. Pesonen / Regulatory Peptides 149 (2008) 51–55
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Fig. 1. Frequency of P7 allele of L7P variation in NPY gene in different populations. Only those populations, where over 100 unrelated individuals are genotyped, have been included. The figures inside the columns indicate the pooled number of individuals genotyped in the literature (http://www.ncbi.nlm.nih.gov).
to promote directly or indirectly progression of atherosclerosis. In the exercise plasma levels of catecholamines, NPY, growth hormone, and FFA increase and insulin decrease. In the study with carefully matched pairs of different L7P genotypes exposed to strenuous exercise the group with heterozygote [p.L7] + [p.P7] had clearly lower postexercise FFA values [15], but not during resting conditions [24]. The inhibition of lipolysis in human adipocytes by NPY has been shown to be dosedependent [25], and therefore higher NPY release during exercise in subjects with the [p.L7] + [p.P7] genotype may explain the difference in FFA concentration between the genotypes. Low levels of FFAs in subjects with the p.P7 variant of preproNPY was observed in the presence of both low and high insulin levels, which indicates that this effect is independent of insulin's antilipolytic action [26]. This notion is also supported by our observations that the activities of LPL and hormone-sensitive LPL, which are regulated by insulin, are not changed in subjects with [p.L7] + [p.P7] genotype [27]. Most likely, insulin and NPY independent of each other lower a cAMP level, which in turn leads to inactivation of hormone-sensitive LPL and lower serum FFAs. NPY which reduces intracellular cAMP concentrations in human adipocytes can inhibit lipolysis and increase liver and WAT lipogenesis [28]. NPY increases centrally the expression of LPL and LPL enzymatic activity in WAT independent in food intake [5,29]. There is no significant difference between the L7P genotypes in the magnitude of postprandial lipemia induced by an oral fat tolerance test. In homozygous [p.L7] + [p.L7] genotype subjects the hormone-sensitive LPL-to-LPL ratio correlates with area of plasma total, VLDL and chylomicron TG, but not in subjects with heterozygous [p.L7] + [p.P7] genotype [27]. This suggest that there might be compositional differences in the lipid particles affecting postprandial lipid metabolism and the relationship of hormone-sensitive LPL and activities to lipid metabolism may differ between the L7P genotype groups. Most of the above mentioned positive association studies have been done in Finnish populations, but there are negative reports in different populations. In Brazilian population, non-diabetic individuals of European descent, p.P7 carriers had a tendency to have lower total cholesterol values, but the number of subjects with p.P7 substitution was very low (n = 18) to reach any statistical power [30]. In recent reports, the researchers could not find associations of plasma TG, total cholesterol, VLDL cholesterol, LDL cholesterol, and HDL cholesterol with L7P sequence variation in Swedish subjects nor in subgroups of obese and non-obese subjects analyzed separately [14] or in a Polish family study [31]. Some of
Table 1 Summary table of the studies on L7P polymorphism on different metabolic diseases and related conditions Disease or condition
Population
n
Reference
Higher serum TG concentrations
Young boys
[18,20]
Type 1 diabetes patients Obese adults
367/ 298/ 267 996 231
Women with CHD
414
[33]
Hypertensive subjects
638
[31]
266 966 199
[26] [21] [22]
247
[32]
1041
[39]
1040
[43]
98 27b
[46] [44]
309
[39]
86 253 94
[48] [47] [46]
1032
[49]
131 582 906 966 27b
[30] [38] [14] [21] [44]
Higher serum levels of total and LDL cholesterol Higher serum levels of total cholesterol Higher serum levels of LDL cholesterola Lower FFA levels Accelerated increase in intima-media thickness
Middle-aged subjects Middle-aged men Middle-aged, T2D patients and non-diabetic control subjects Higher FMD Middle-aged men, prepubertal children Development of T2D and IGT T2D, IGT and healthy subjects Hypertensive and healthy subjects Younger age of onset of T2D T2D patients Lower plasma insulin concentrations Healthy young subjects and insulin to glucose ratio during OGTT IGT, higher fasting plasma glucose T2D patients levels Development of diabetic retinopathy Middle-aged, T2D patients T2D patients Increased risk for nephropathy T2D patients with diabetic retinopathy Increased risk for myocardial Hypertensive subjects infarction and stroke Lower BMI Premenopausal women Higher BMI Adult men Non obese subjects Increased blood pressure Middle-aged men Higher heart rate variability Healthy young subjects
[19] [12]
TG = triglyceride, LDL = low-density lipoprotein, CHD = coronary heart disease, FFA = free fatty acids, T2D = type 2 diabetes, FMD = flow mediated endothelial dependent dilatation, IGT = impaired glucose tolerance, OGTT = oral glucose-tolerance test, BMI = body mass index. a Epistatic interaction with β2-adrenergic receptor polymorphism. b Matched pairs.
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the later studies on healthy, normal weight Finnish cohorts [15,32,33] have also suggested that p.P7 substitution might carry a risk factor to elevated plasma lipid levels only in combination with other major metabolic or environmental risk factors, like higher age, obesity, or diet rich in dairy products, which is very common in Eastern Finland populations [34]. 4. BMI and obesity NPY has been since its discovery the most potent and probably most studied neuropeptide which enhances body weight gain and obesity. Although the role of NPY in regulation of human feeding and energy balance is not totally clear, there are several human linkage studies, which connect NPY to human obesity and suggest that the NPY gene is a candidate gene for obesity related phenotypes [35–37]. Although there are many positive associations with p.L7P substitution and obesity, the association seems to depend on the study population and environmental factors. The largest study, which found a positive association, was done in Dutch men: subjects with the p.P7 substitution had higher BMI and higher risk on being overweight [38]. There are also some distinct subpopulations where p.L7P substitution of the NPY gene is independently associated with higher BMI or body weight: type 1 diabetic patients [19], newborn boys (birth weight) [18], normal weight subjects [14] and men with T2D [39]. There is one conflicting study, where the p.L7P substitution is associated with lower BMI in premenopausal women, but this may be due to a very low number of subjects with P7 variant (n = 7) in the study [30]. 5. Impaired glucose tolerance and type 2 diabetes One of the peripheral energy store signals integrated in hypothalamus by NPY containing nerves is insulin. In experimental animals central NPY administration leads to hyperinsulinemia and peripheral insulin resistance [40], but a tonic central effect of NPY is needed these metabolic abnormalities [41]. In periphery insulin and NPY have a different functional relationship: NPY inhibits glucose-stimulated insulin secretion from isolated pancreas. This may be a direct peripheral interaction, since liver contains NPYergic neurons, which are involved in the regulation of hepatic function and haemodynamics [42]. The L7P polymorphism has an effect on insulin secretion and glucose homeostasis. Healthy subjects with p.P7 allele have higher glucose, lower insulin and lower insulin/glucose ratio during rest and after meals [24,43] as well as during exercise [15] compared to subjects without p.P7 allele. This was also observed during the oral glucose-tolerance test with matched healthy subjects [44], but not in a large, heterogenous group of middle aged subjects [26]. The changes in the diurnal glucose and insulin levels may be reflected as altered insulin sensitivity, but this is still disputable due to large, contradictory studies [26,43]. If there is a true difference in the insulin levels, it could due to a central component regulating autonomic balance or peripherally increased local NPY levels in pancreas, where NPY has been shown to inhibit insulin secretion [45]. Two major abnormalities in T2D are β-cell dysfunction in pancreas and decreased insulin sensitivity in liver, skeletal muscle and adipose tissue. T2D patients have a loss of first-phase glucose-stimulated insulin response to food intake, which results in hyperglycaemia and hypertriglycaemia. Although there seems to be a genetic component in the T2D, it is often concomitant of obesity, insufficient physical activity, and excessive carbohydrate intake. L7P substitution has been associated with onset of T2D [39,43,46], impaired glucose tolerance [39] and with the worse glycaemic balance in type 1 diabetic patients as measured by HbA1c levels [19]. However, most of the association studies on metabolic and hormonal parameters found previously positive in healthy study subjects, have resulted negative associations when studied in diabetic patients. There is no association with serum lipid levels [22,47] or with plasma NPY levels [19] between the [p.L7] +
[p.L7] genotype and [p.L7] + [p.P7] genotype in diabetic patients. Instead, many studies have shown that diabetic patients with p.P7 variant are more prone to serious complications of the disease [19,47,48]. It seems that diabetes alters the metabolic status of the body such a way that, the ‘normal’ interactions of NPY are disturbed and the altered NPY balance further compromises the metabolic balance of the body. This may happen at the central autonomic level or at the peripheral, local level or at both levels. 6. Conclusion NPY is a multifunctional neurotransmitter with multiple modulator effects in the regulation of physiological functions and responses in the body. A change in the molecular structure of its precursor, the L7P substitution, has been shown to associate with many diseases and with many changes in sympathoadrenal, hormonal, and metabolic balance in healthy subjects. The exact molecular and cellular mechanisms by which the L7P polymorphism leads to the observed changes are not clear. At the moment it seems that L7P substitution of preproNPY protein causes altered NPY secretion, which leads to haemodynamic disturbances caused by sympathetic hyperactivity and to various effects caused by altered local signalling by NPY [11]. SNP association studies, or preferably haplotype analysis, on NPY gene can be used to guide functional studies and elucidate the pathophysiological NPYrelated mechanisms behind cardiovascular and metabolic disorders. A challenge with the association studies using L7P substitution is the extensive variation of the allele frequency in different populations. The effects of the L7P substitution on cardiovascular regulation and diseases are not likely to be relevant in populations other than European descent. However, this does not forbid us drawing conclusions of physiological and/or pathological roles of NPY in cardiovascular control and development of diseases. The study of DNA sequence variant that contributes to common disease risk offers one of the best opportunities for understanding the complex cause of disease in humans. The clinical relevance of L7P genetic variant in cardiovascular regulation and clinical prediction and treatment of cardiovascular and metabolic diseases remains to be determined. Note-added-in-proof In a recent paper Zhou et al. (Nature 452 (7190), 997-1001, 2008) show that NPY gene haplotypes predict NPY expression levels and thus plasma NPY concentrations. They claim that the majority of the variation in expression can be explained by a single SNP (rs16147) in the promoter area of NPY gene. The authors were not able to fully account the effects of p.L7P variant, because of the low frequency of this mutation in the study subjects. However, it seems that rs16147 T allele is in full linkage disequilibrium with Pro7 allele especially in Finnish population (Dr. Zhou, personal communication).
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