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Altered intracellular processing and release of neuropeptide Y due to leucine 7 to proline 7 polymorphism in the signal peptide of preproneuropeptide Y in humans ¹

JAANA KALLIO^*2, ULLAMARI PESONEN^*, KATJA KAIPIO^*, MATTI K. KARVONEN^*, ULRIIKKA JAAKKOLA^*, OLLI J. HEINONEN, MATTI I. J. UUSITUPA and MARKKU KOULU^*

^* Department of Pharmacology and Clinical Pharmacology and
Paavo Nurmi Center, Department of Physiology, University of Turku, Turku, Finland; and
Department of Clinical Nutrition, University of Kuopio, Kuopio, Finland

²Correspondence: Department of Pharmacology and Clinical Pharmacology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: jaana.kallio{at}utu.fi

SPECIFIC AIMS

To study the functional role of the recently found leucine 7 to proline 7 (Leu7Pro) polymorphism in the signal peptide of preproneuropeptide Y, sympathetic responses, including neuropeptide Y (NPY) release, were compared during strenuous physical exercise between subjects with the Leu7/Pro7 genotype and their matched wild-type controls (Leu7/Leu7 genotype). To further evaluate the effect of this polymorphism on the intracellular processing of preproNPY, isolated human umbilical vein endothelial cells (HUVEC) of corresponding genotypes were compared using immunocytochemical methods to verify the presence and localization of NPY and proNPY in these cells by confocal microscopy.

PRINCIPAL FINDINGS

1. NPY-genotype influences heart rate level
Subjects with the Leu7/Pro7 genotype had a significantly higher mean heart rate over time than subjects with the Leu7/Leu7 genotype (Fig. 1A , P<0.05). No statistically significant differences were detected in mean heart rate at any separate time point between the groups, however. No differences were observed in systolic or diastolic blood pressure between the genotypes during the entire study period.

View larger version (15K): [in this window] [in a new window]   Figure 1. Nine subjects with the Leu7/Pro7 genotype (open triangles) had significantly higher heart rate (A) and plasma NPY concentrations (B) and significantly lower serum FFA concentrations (C) than 9 subjects with the Leu7/Leu7 genotype (solid triangles). The exercise time was 30 min (hatched area).

2. NPY-genotype influences exercise-induced increases in NPY and FFA concentrations
Subjects with the Leu7/Pro7 genotype had higher overall plasma NPY concentration (Fig. 1B ), with statistically significant differences at 20 min maximal NPY concentrations (P<0.05) and near significant differences at 30 min and 40 min postexercise NPY concentrations (P=0.05). The mean exercise-induced increase of NPY between 0 min and 20 min was 90.4 ± 12.7 pmol/l in the subjects with proline 7 (Pro7) in the preproNPY and 51.9 ± 5.4 pmol/l in the subjects without this substitution (P<0.05, t test). There were no statistically significant differences in the concentrations of epinephrine and norepinephrine (NE) in plasma between the groups. The mean NE/NPY ratio in plasma was 84.9 ± 9.7 in subjects with the Leu7/Leu7 genotype and 56 ± 5.3 in subjects with the Leu7/Pro7 genotype (P<0.05, t test).

A clear difference was observed in overall free fatty acid (FFA) concentrations between the groups (Fig. 1C ): subjects with the Leu7/Pro7 genotype had significantly lower FFA concentrations than subjects with the Leu7/Leu7 genotype, with the largest difference in postexercise 40 min concentration (P<0.05). There were lower overall insulin concentrations in subjects with the Leu7/Pro7 genotype and no clear exercise-induced reduction in insulin levels in this group; the statistically significant differences in concentrations were detected before the exercise at 0 min (P<0.05) and after exercise at 60 min (P<0.05). Lactate levels were identical in the two genotype groups throughout the study period.

3. NPY-genotype determines the pattern of NPY- and proNPY-immunoreactivity (ir) in endothelial cells
Clear intracellular punctate staining (Fig. 2 ) could be seen in HUVECs with immunocytochemical detection of proNPY and NPY. These clustered immunoreactive puncta were considered to represent peptides concentrated in some intracellular compartments of the cells. As shown in Fig. 2 , there was a marked difference in the pattern of staining between HUVECs with the Leu7/Leu7 and the Leu7/Pro7 genotype. HUVECs with the Leu7/Leu7 genotype (Fig. 2A ) exhibited profound vesicle-like structures with proNPY-ir (yellow), and these cells had only sparse NPY (red) staining. In contrast, prominent NPY-ir (red) was detected in Leu7/Pro7 cells (Fig. 2B ), which also had some proNPY-ir. No evident staining related to cleaved C-pon (green) could be detected in either cell type.

View larger version (79K): [in this window] [in a new window]   Figure 2. Scanning confocal images of HUVECs with the Leu7/Leu7 genotype (A) and the Leu7/Pro7 genotype (B) double-immunostained with rabbit polyclonal anti-C-ponderal peptide (C-pon) antibody and goat polyclonal anti-NPY antibody. The images are summations of 8 midsections of permeabilized cells. Prominent overlapping staining (yellow) of NPY and C-pon was seen in cells with the Leu7/Leu7 genotype (A), indicating the overwhelming presence of proNPY. NPY without C-pon (red), processed NPY, was abundant in vesicle-like structures in HUVECs with the Leu7/Pro7 genotype (B), these cells also had some proNPY staining.

CONCLUSIONS

Recent studies have linked the Leu7Pro polymorphism of preproNPY to risk factors and development of atherosclerosis in adults and children. This polymorphism is likely to influence the intracellular processing of the synthesized preproNPY peptide as the mutation is located in the signal peptide part, which guides the newly formed peptide into ER, where it is cleaved off; the ensuing further processing of proNPY leads to the formation of the mature secretory NPY. As a result of changed processing of the prohormone, the storage or kinetics of NPY release could be modified in subjects having this polymorphism. This hypothesis was tested in the present study in matched healthy volunteers having either the Leu7/Pro7 or the Leu7/Leu7 genotype.

The results show similar timing of exercise-induced NPY release in the two genotype groups but significantly higher NPY concentrations in subjects with the Leu7/Pro7 genotype, the maximal values showing the largest difference compared to controls. There was no difference in the basal NPY concentrations, but a > 40% larger increase from the basal to the maximal NPY concentration in the subjects with Pro7 in the preproNPY. This suggests that either more NPY is produced by the sympathetic nerves or NPY is more easily released by sympathetic stimulation (Fig. 3 ).

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram about the functional consequences of the leucine 7 to proline 7 polymorphism in the signal peptide part of preproNPY in humans.

Subjects with the NPY polymorphism had a higher heart rate; the difference in the mean heart rate was 5 to10 beats/min continuously during the study period and the 30-min pre-exercise resting period. This suggests that the NPY genotype influences heart rate. As the catecholamine levels were not different between the study groups, the higher heart rate is not explained by their action.

The two genotype groups had strikingly different exercise-induced FFA concentrations, with the group of Leu7/Pro7 genotype having clearly lower postexercise values. The inhibition of lipolysis in human adipocytes by NPY has been shown to be dose dependent; therefore, higher NPY release in subjects with the Leu7/Pro7 genotype during exercise may explain the difference in FFA concentration between the genotypes.

The immunocytochemical studies of isolated and genotyped HUVECs revealed clearly different distribution of the NPY-related ir between the genotypes. With double-labeling techniques, we could demonstrate that in human endothelial cells with the Leu7/Pro7 genotype the amount of NPY without C-pon (shown in red) was prominent. Endothelial cells with the Leu7/Leu7 genotype contained almost exclusively NPY with C-pon (yellow), which is proNPY. This strongly suggests a difference in processing of the preproNPY between the genotypes.

These are the first reported functional consequences of the Leu7Pro substitution in the signal peptide of NPY. Earlier studies linked this polymorphism to increased levels of total cholesterol, LDL cholesterol, and triglycerides in blood and to accelerated development of atherosclerosis, all of which may be the ultimate consequences of increased NPY contents in blood and/or tissues of subjects with the Leu7/Pro7 genotype (Fig. 3 ). Therefore, the present observations may provide new insights for strategies for prevention and treatment of these diseases.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0437fje ; to cite this article, use FASEB J. (March 12, 2001) 10.1096/fj.00-0437fje

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