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Department of Pediatrics, Second University of Naples, 80138 Napoli, Italy
Address all correspondence and requests for reprints to: Emanuele Miraglia del Giudice, Dipartimento di Pediatria, Seconda Università di Napoli, Via Luigi De Crecchio Number 2, 80138 Napoli, Italy. E-mail: emanuele.miraglia{at}unina2.it.
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Abstract |
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Allelic frequencies were comparable in our patients (0.053) and in 300 lean controls of Mediterranean descent (0.045). Interestingly, we showed that this polymorphism, in the obese patients, was associated with differences in fasting insulin levels; this finding persisted after correction for age, sex, and pubertal stage. Heterozygotes had 24% higher mean insulin levels than those homozygous for the wild allele and showed a stronger correlation between insulin and body mass index (P < 0.001).
These findings support the hypothesis that the melanocortin pathway may modulate glucose metabolism in obese subjects and suggest that this common POMC variant may be involved in the natural history of polygenic obesity in late adolescence and adulthood, contributing to the link between type 2 diabetes and obesity.
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Introduction |
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POMC is the precursor of a number of hormones and neuropeptides. It is cleaved tissue specifically to yield peptides with different functions, such as ACTH; 
-, ß-, and 
- MSH; ß-lipotropin; and ß-endorphin (9). -MSH acts on feeding behavior by binding its receptor melanocortin-4 receptor (MC4R) in the hypothalamus (4, 10).
Recently, some authors have reported a high prevalence of MC4R gene variants in obese children and adolescents (11, 12). Clinical features of obese children carrying MC4R mutations have been carefully described (12). These patients, although euglycemic, show significantly elevated plasma insulin concentrations compared with those observed in matched obese subjects carrying the MC4R wild type (12).
To examine the contribution of the POMC gene in the variability of serum insulin levels in obese children, we carried out an association study using a previously described polymorphism consisting of a 9-bp insertion, AGC AGC GGC, between nucleotides 6979 and 6998 (13, 14). This leads to the insertion of three amino acids (Ser-Ser-Gly) between codons 94 and 100 in the region of the 16-kDa fragment carboxy terminal to -MSH (Fig. 1
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Subjects and Methods |
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Genomic DNA was collected from nucleated white blood cells. Screening for the 9-bp insertional variant, 94-AGC AGC GGC-100, between nucleotides 6979 and 6998 of the POMC gene was performed by PCR amplification. A couple of primers, POMC-F (5'-CGAGAACCCCCGGAAGTACG-3') and POMC-R (5'-ACGTCCTCGCGCTTCTGCCC-3'), were used to amplify a 108-bp fragment (wild allele). Amplification of the mutated allele, on the contrary, produced a 117-bp fragment. PCRs were carried out using the following conditions: denaturation at 95 C for 5 min followed by 35 cycles of 30 sec at 94 C, 30 sec at 60 C, and 30 sec at 72 C. The amplified segments were analyzed on a 3% NouSieve (FMC BioProducts, Rockland, ME) agarose gel and stained with ethidium bromide (Fig. 2). In a few cases, to test the effectiveness of the separation with gel, PCR products were also sequenced with an automatic sequencer (ABI PRISM 310, Applied Biosystems, Foster City, CA) (Fig. 2).
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Results |
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BMI z-scores of the heterozygote patients for the insertional polymorphism and of the homozygotes for the wild allele were comparable (Table 1). No differences were observed concerning the distribution of sex (i.e. 19 girls of 40 patients and 166 girls of 340 patients in heterozygotes and wild-type homozygotes, respectively) and Tanner puberty stages between the two groups (data not shown).
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). The same finding was observed for IR-HOMA; heterozygotes showed 21% higher mean IR-HOMA values than wild-type homozygotes (Table 1). These differences persisted also when age, sex, and pubertal stage were used as covariates (F = 6.9; P = 0.01). The same finding was not observed in a subgroup of 150 of 300 controls (13 heterozygotes and 137 wild-type homozygotes). Insulin levels were not different between heterozygotes and wild-type homozygotes (26.5 ± 13.6 vs. 28 ± 15.8 pmol/l; F = 0.1; P = 0.7).
In the entire group of patients, as expected, BMI and insulin were correlated (r = 0.49; P < 0.001). However, the degree of hyperinsulinemia for a comparable BMI varied among patients, indicating that individual factors other than fatness might influence insulin levels. In fact, heterozygous children for the POMC 9-bp insertional polymorphism showed a stronger correlation between insulin and BMI than homozygotes for the wild allele (Fig. 3).
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Discussion |
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Many findings suggest that the central melanocortin system may be independently involved in the regulation of glucose homeostasis in addition to its regulation of energy intake and energy expenditure. Hyperinsulinemia and impaired glucose tolerance are the earliest metabolic alterations observed in murine melanocortin obesity models (MC4R knockout mice), appearing before the onset of hyperphagia or obesity (17). Furthermore, centrally administered melatonin II, a MC4-R/MC3-R agonist, produces a dose-dependent inhibitory effect on basal plasma insulin levels in lean control mice, and phentolamine, a nonspecific -adrenoreceptor antagonist, blocks the ability of centrally administered melanocortin to lower serum insulin in ob/ob mice (17). These experimental evidences suggest that melanocortin modulates insulin secretion by stimulating the sympathetic drive to the pancreas known to inhibit insulin release. Furthermore, a potent effect of melanocortinergic pathway on insulin-mediated glucose uptake and production has been demonstrated in the rat (18). In humans, it has been observed that subjects carrying mutations of the MC4R gene responsible for a loss or a reduction of function show higher plasma insulin levels than subjects carrying the wild allele (12).
Our findings, showing an influence of a POMC insertional polymorphism both on fasting insulin level and on insulin resistance evaluated by IR-HOMA, support the hypothesis for an involvement of the melanocortinergic system in the regulation of glucose metabolism in humans. One could hypothesize that obese children and adolescents carrying the POMC 9-bp insertion might experience both an increase of insulin secretion and a reduced insulin sensitivity.
No difference concerning fasting insulin levels between wild-type homozygous and heterozygous normal controls has been detected. This finding could be explained by the fact that the effect of POMC polymorphism on insulin levels may be noticed only when, as usually happens in obese children, an increasing insulin production in response to fat accumulation is required. Recently, Feng et al. (19) analyzed the association between the 9-bp insertion of the POMC gene and insulin levels in a multiracial cohort of obese children. The authors did not show a statistically significant difference concerning fasting insulin levels between wild-type homozygotes and obese subjects carrying the POMC variant. The discrepancy between our data and those of Feng et al. is likely due to a lower BMI in the patients of Feng et al. who were heterozygous for the 9-bp POMC variant compared with heterozygous patients reported in the present study (BMI SD score, 2.8 vs. 3.6).
This variant might produce a slight reduction of the gene function because of linkage with a mutation undetected until now in the POMC regulating region, but a direct action of the polymorphism cannot be excluded. In fact, a naturally deleted form of human POMC, characterized by three amino acid deletions (Ser-Ser-Gly) between residues 94–100, has been shown (20). The levels of expression of this variant were 5-fold decreased, suggesting that the integrity of this portion of the POMC 16-kDa fragment could play a primary role in POMC expression and/or secretion (20). This may be due to mRNA instability. Alternatively, the occurrence of two different POMC alleles in heterozygotes means that in these patients heterodimers of POMC occur, whereas homodimers occur in homozygotes (21). Therefore, heterozygotes may show defects in processing due to the structural configuration of the two subunits being different (20); for example, dibasic sites may show altered accessibility, resulting in less efficient cleavage.
We propose that, in the Italian population, the POMC variant characterized by the insertion of three amino acids (Ser-Ser-Gly) between codons 94 and 100, inducing variation of fasting insulin levels, may be involved in the natural history of polygenic obesity in late adolescence and adulthood, contributing to the link between type 2 diabetes and obesity.
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Footnotes |
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Received February 25, 2004.
Accepted July 15, 2004.
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