Elongator regulates the melanocortin satiety pathway

Abstract

This study investigates the function of Elp1 and Elongator in the pituitary gland. Two conditional knockout models were generated where Elp1 was selectively deleted in either somatotropes of the anterior pituitary or Pomc-expressing cells of the anterior and intermediate pituitary. Although loss of Elp1 in somatotropes did not significantly impact murine growth or development, its loss in Pomc-expressing cells resulted in dramatically reduced levels of α-MSH, hyperphagia and obesity. This report provides the first evidence that Elongator plays an essential role in regulating the melanocortin satiety pathway.

Keywords: Elongator; Melanocortin; Pituitary; α-MSH.

Fig. 1.. Elp1 expression within the HP axis.

A-G) β-gal staining in P0 Elp1-Lacz reporter mice. shown in blue in A-D and black in E-G. A, B) Elp1 expression is robust in the AP and IP. B-D) Elp1 is less prominent in the PP (B), in the ARC (C, D), and within the main lobes of the hypothalamus (asterisks in C). D shows an enlargement of the boxed region in C. E-G) Combination IHC and β-gal staining demonstrates that Elp1 is specifically expressed in GH1-expressing somatotropes and Pomc-expressing corticotropes in the AP (arrows in E and F), as well as in Pomc-expressing melantotropes in the IP (arrows in G). AP, anterior pituitary; ARC, arcuate nucleus; H. hypothalamus; IP, intermediate pituitary; MO, medulla oblongata; P, pituitary; PP, posterior pituitary; T. thymus. Scale bar: A, 350 μm; B, C, 100 μm; D, 60 μm; E-G, 10 μm.

Fig. 2.. Generation of cell-type specific Elp1 CKO mouse models.

A-L) E17.5 Rosa^mT-mG embryos. Red cells expressing a tomato fluorophore (red) convert to GFP expression (green) in the presence of Cre recombinase. A-D) In Gh1-Cre; Rosa^mT-mG embryos, Cre is active in the AP and not active in the IP or PP (A). GFP positive cells are also positive for GH1, confirming selective expression of Cre in somatotropes (B–D). E-L) In Promc-Cre; Rosa^mT-mC embryos, Cre is active in both the AP and IP (E). GFP postivie cells in the anterior lobe (F–H) and intermediate lobe (I–K) are positive for POMC, confirming selective expression in corticotropes and melanotropes, respectively. L) GFP is barely detectable in the ARC indicating minimal Cre activity. M. N) ELP1 levels are reduced in pooled pituitaries (3 pituitaries per genotype) from 6-week old male and female CKO mice compared to controls. M,N) Control 1 (C1) (Elp1^LoxP/LoxP), Control 2 (C2) (Cre; Elp1^+/LoxP), CKO (Cre; Elp1^LoxP/LoxP). The small amount of ELP1 protein in the CKO lanes corresponds to cell types within the pituitary where Elp1 expression remains intact. Sclae bar: A, E, L, 100 μm; B-D, F–K, 10 μm.

Fig. 3.

Obesity and hyperphagia in Pomc-Cre; Elp1^LoxP/LoxP mice. A, B) The masses of Pomc-Cre; Elp1^LoxP/LoxP mice begin to diverge from controls in both sexes at approximately six weeks. Weight gain begins to level off in male and female controls at approximately nine weeks, while the CKOs continue to gain weight through 27 weeks. A-D) At week 27, the average masses of male and female Pomc-Cre; Elp1^LoxP/LoxP mice are 33% and 40% higher, respectively, than age- and sex-matched controls. P value < 0.05 in both sexes at the nine-controls. G) Pomc-Cre; Elp1^LoxP/LoxP mice consistently consume more food than controls. For A, B, E, F, n = six male controls, six male CKOs, four female controls, five female CKOs. C1 (Elp1^LoxP/LoxP), C2 (Promc-Cre; Elp1^+/LoxP), CKO (Promc-Cre; Elp1^LoxP/LoxP). Bars = standard error.

Fig. 4.. POMC, ACTH, and α-MSH are diminished in Pomc-Cre; Elp1^LoxP/LoxP mice.

A-C) Western blot shows that levels of both the precursor peptide POMC and ACTH are reduced in Pomc-Cre; Elp1^LoxP/LoxP mice, with ACTH more dramatically reduced than POMC. Anti-POMC antibody 66358-1-Ig (Proteintech) recognizes POMC (29 kDa) and glycosylated ACTH (13 kDa). Each lane/bar represents three pooled pituitaries per genotype from mice six weeks of age. D) qRTPCR using individual pituitaries from three male C1 and three male CKOs demonstrates reduced expression of the Pomc gene. P = 0.010 (unpaired t-test). Bars represent SD. E-I) Each lane/bar represents three pooled pituitaries per genotype from mice six weeks of age. E) Mass spectrometry shows that α-MSH levels are most reduced in Pomc-Cre; Elp1^LoxP/LoxP mice (see also Fig. S3). F, G) PC1 is elevated in adult males and normal in females. H, I) PC2 is reduced in both C2 and CKO male and female mice. EIC, extracted-ion chromatogram. C1 (Elp1^LoxP/LoxP), C2 (Pomc-Cre; Elp1^+/LoxP) CKO (Pomc-Cre; Elp1^LoxP/LoxP).