Int J Obes. 2015 Jun;39(6):986-93.

Novel lipidized analogs of prolactin-releasing peptide have prolonged half-lives and exert anti-obesity effects after peripheral administration

 

Lenka Maletínská1, Veronika Nagelová1, Anežka Tichá1, Jana Zemenová1,4, Zdeno Pirník1,5,6, Martina Holubová1, Andrea Špolcová1, Barbora Mikulášková1, Miroslava Blechová1, David Sýkora4, Zdena Lacinová3, Martin Haluzík3, Blanka Železná1 and Jaroslav Kuneš2

1Institute of Organic Chemistry and Biochemistry, AS CR, Prague, Czech Republic

2Institute of Physiology, AS CR, Prague, Czech Republic

3Third Department of Medicine, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic

4Institute of Chemical Technology, Department of Analytical Chemistry, Prague, Czech Republic

5Laboratory of Functional Neuromorphology, Institute of Experimental Endocrinology, SAS, Bratislava, Slovak Republic

6Department of Human and Clinical Pharmacology, University of Veterinary Medicine, Košice, Slovak Republic

 

Abstract

Obesity is a frequent metabolic disorder but an effective therapy is still scarce. Anorexigenic neuropeptides produced and acting in the brain have the potential to decrease food intake and ameliorate obesity but are ineffective after peripheral application. We have designed lipidized analogs of prolactin-releasing peptide (PrRP) which is involved in energy balance regulation as demonstrated by obesity phenotypes of both PrRP- and PrRP-receptor-knockout mice. Lipidized PrRP analogs showed binding affinity and signaling in PrRP receptor-expressing cells similar to natural PrRP. Moreover, these analogs showed high binding affinity also to anorexigenic neuropeptide FF (NPFF)-2 receptor. Peripheral administration of myristoylated and palmitoylated PrRP analogs to fasted mice induced strong and long-lasting anorexigenic effects and neuronal activation in the brain areas involved in food intake regulation. Two-week-long subcutaneous administration of palmitoylated-PrRP31 and myristoylated-PrRP20 lowered food intake and body weight and improved metabolic parameters and positively affected lipid metabolism in mice with diet-induced obesity. In conclusion, our data suggest that the lipidization of PrRP enhances stability and mediates its crossing of the blood-brain barrier. Strong anorexigenic and body-weight-reducing effects make lipidized PrRP an attractive candidate for anti-obesity treatment.

PMID: 25771926

 

Supplement:

Prolactin-releasing peptide (PrRP) was originally discovered as an endogenous ligand of an orphan G-protein-coupled receptor (1). PrRP and its receptor named GPR10 were detected in several hypothalamic nuclei (2) suggesting an involvement of PrRP in the control of food intake and body weight (3).

The intracerebroventricular administration of PrRP inhibited food intake and body weight gain in rats but did not cause conditioned taste aversion. Furthermore, Fos immunoreactivity was enhanced after PrRP administration in the brain areas associated with food intake regulation (4).

PrRP receptor knockout mice had significantly higher body weight at 15 weeks of age compared to wild-type mice, and this late-onset obesity was much more pronounced in female mice, which also exhibited a significant decrease in energy expenditure (5). Similarly, PrRP-deficient mice displayed late-onset obesity, increased food intake and attenuated responses to the anorexigenic signals cholecystokinin and leptin (6).

Taken together, these findings suggest that PrRP and other anorexigenic neuropeptides involved in food intake regulation (7,8) may have a potential in the development of future anti-obesity agents. Nevertheless, because these peptides normally regulate food intake directly in the hypothalamus, their anorexigenic potential after peripheral administration is hampered by their inability to cross the blood-brain barrier (BBB) and to reach the target brain receptors.

For the design of peptide drugs, the lipidization of peptides, i.e. the attachment of fatty acid chain to peptides through ester or amide bond is advantageous. Such modification results in an increased stability and half-life of the peptide, and it is possible that these modifications allow peptide to cross the BBB after a peripheral administration (reviewed by (9-11)). PrRP seems to be a suitable candidate for lipidization because of its linear, one-chain peptide structure.

The aim of this study was to achieve the direct central anorexigenic activity of PrRP via its peripheral route of administration employing the lipidization of its N-terminus. We tested a series of PrRP analogs modified with fatty acids of various lengths both in vitro and in vivo. The data showed that myristoylated PrRP20 and palmitoylated PrRP31 retained the biological activity of PrRP while significantly decreasing food intake and body weight and improving metabolic parameters upon peripheral administration in mice with diet-induced obesity. Thus, the lipidization of neuropeptides involved in food intake regulation might serve as a tool to retain their ability to act centrally after peripheral administration.

In conclusion, we have demonstrated that the lipidization of PrRP enabled its central anorexigenic effect after peripheral administration in both acute and chronic settings by enhancing its stability in the blood and improving its ability to cross the blood brain barrier. Our data also confirmed that GPR10 and/or NPFF2 receptors are suitable targets for the treatment of obesity. Collectively, our data suggest that lipidized PrRP analogs have potential as a possible future anti-obesity drugs.


 LM FIG1Figure 1:Palmitoylated PrRP31 and myristoylated PrRP20 attenuate food intake after acute peripheral administration in fasted mice. Cumulative food intake of 17 h fasted mice after acute subcutaneous (SC) administration of myristoylated PrRP20, palmitoylated PrRP31 and natural PrRP31 at a dose of 5mg kg−1. Food intake is expressed in grams of food consumed (n=6–8 mice per group). ***P<0.001 vs saline-treated group. The significance concerns the whole time course.

 

 

 LM FIG2Figure 2:Palmitoylated PrRP31 and myristoylated PrRP20 increase neuronal activity in food intake-regulating areas of mouse brain after peripheral administration. Fos immunoreactivity: the qualitative as well as quantitative assessment of Fos-immunostained cells in coronal section of PVN (a–d), Arc (e–h) and NTS (i–l) 90 min after SC application of saline (a, e, i), PrRP31 (b, f, j), myr-PrRP20 (c, g, k) and palm-PrRP31 (d, h, l) at a dose of 5mg kg− 1 in fasted mice (n=4). *P<0.05, **P<0.01 vs saline. PVN—paraventricular hypothalamic nucleus, Arc—arcuate hypothalamic nucleus, NTS—solitary tract nucleus, 3v—third brain ventricle. Scale bar is 50 μm for a–d, 100 μm for e–l.

 


 LM FIG3

LM FIG3B

Figure 3: Palmitoylated PrRP31 and myristoylated PrRP20 reduce food intake and body weight of diet-induced obese mice. Effect of 14-day administration of palm-PrRP31 and myr-PrRP20 on (a) food intake and (b) body weight of DIO mice. Mice were SC administered by saline or peptides at a dose of 5mg/ kg twice daily (n= 10). The data were analyzed by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001 vs saline-treated group.

 

 

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