Prolylcarboxypeptidase regulates food intake by inactivating α-MSH in rodents
Nicholas Wallingford, Bertrand Perroud, Qian Gao, Anna Coppola, Erika Gyengesi, Zhong-Wu Liu, Xiao-Bing Gao, Adam Diament, Kari A. Haus, Zia Shariat-Madar, Fakhri Mahdi, Sharon L. Wardlaw, Alvin H. Schmaier, Craig H. Warden, Sabrina Diano
Nicholas Wallingford, Bertrand Perroud, Qian Gao, Anna Coppola, Erika Gyengesi, Zhong-Wu Liu, Xiao-Bing Gao, Adam Diament, Kari A. Haus, Zia Shariat-Madar, Fakhri Mahdi, Sharon L. Wardlaw, Alvin H. Schmaier, Craig H. Warden, Sabrina Diano
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Research Article

Prolylcarboxypeptidase regulates food intake by inactivating α-MSH in rodents

Abstract

The anorexigenic neuromodulator α-melanocyte–stimulating hormone (α-MSH; referred to here as α-MSH1–13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of α-MSH1–13 maturation and inactivation is incompletely understood. Here we have provided insight into α-MSH1–13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of α-MSH1–13, producing α-MSH1–12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where α-MSH1–13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of α-MSH1–13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet–induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active α-MSH1–13 levels.

Authors

Nicholas Wallingford, Bertrand Perroud, Qian Gao, Anna Coppola, Erika Gyengesi, Zhong-Wu Liu, Xiao-Bing Gao, Adam Diament, Kari A. Haus, Zia Shariat-Madar, Fakhri Mahdi, Sharon L. Wardlaw, Alvin H. Schmaier, Craig H. Warden, Sabrina Diano

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Figure 4

α-MSH levels in Prcpgt/gt mice.

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α-MSH levels in Prcpgt/gt mice. (A and B) Mass spectrometry analysis...
(A and B) Mass spectrometry analysis of products of the in vitro PRCP reaction with α-MSH1–13. (A) Control (T0) analysis. (B) Liquid chromatography–mass spectrometry (LC-MS) analysis for the 1-hour incubation reaction (T1). The HPLC chromatogram base peaks are shown in the top panels for the retention time (RT) ranging from 25 to 40 minutes. A major peak eluting at 32 minutes (A, top panel, gray) has an MS spectrum perfectly matching the triply charged ion isotopic masses (middle panel) and α-MSH1–13 theoretical size (MW, 1,665 Da). A new peak eluting at 30 minutes (B, top panel, gray) appears in the 1-hour reaction. Its MS spectrum matches the triply charged ion isotopic masses of α-MSH1–12 standard (B, bottom panel) and its theoretical size (MW, 1,566 daltons). α-MSH1–12 was not detected in the control samples (A, bottom panel). Standard spectra are shown overlaid in gray inserts. (C) α-MSH levels (expressed as fmol α-MSH/mg protein) in the hypothalamus of Prcpgt/gt mice (n = 29) compared with wild-type controls (n = 14). Data represent the mean ± SEM. *P = 0.046. (D) α-MSH/ACTH ratio in the hypothalamus of Prcpgt/gt mice compared with wild-type controls. Data represent the mean ± SEM. †P = 0.047.