Aged PROP1 Deficient Dwarf Mice Maintain ACTH Production

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1null (Prop1-/-) and the Ames dwarf (Prop1df/df) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism

<i>Prop1</i>-defiency results in low blood glucose levels.

<p>Blood glucose levels were measured in normal and <i>Prop1</i> mutant mice at four ages. (A) Basal glucose levels in 3.5 to 5 week <i>Prop1^-/-</i> mice (n = 6) were lower than <i>Prop1^+/+</i> (n = 10) and <i>Prop1^+/−</i> (n = 10) mice from mixed genetic backgrounds, but the difference was not statistically significant at this age. (B) On mixed genetic backgrounds the blood-glucose measurements from 5 to 6.5 wk old <i>Prop1^+/+</i> (n = 6) and <i>Prop1</i>^df/df (n = 6) were normal, but <i>Prop1</i>^df/- (n = 3), <i>Prop1^-/-</i> healthy (n = 12) and <i>Prop1^-/-</i> wasting (n = 7) mice had significantly decreased blood-glucose levels. Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.01; **, <i>P</i><0.005. (C) The low glucose levels in mutants shown in panel B are associated with elevated corticosterone levels (ng corticosterone/ml blood +/− SE.) (D) Blood-glucose levels were measured in 8 to 10 week old mice of the N4 B6 background prior to (white bars) and following restraint stress (black bars). <i>Prop1^-/-</i> (n = 8) mice had decreased basal and post-stress blood-glucose levels compared to <i>Prop1^+/+</i> (n = 9) and <i>Prop1^+/−</i> (n = 11). Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.0001; **, <i>P</i><0.0005. (E) Blood-glucose levels in 34 to 52 wk old mice on mixed genetic background were decreased in all genotypes of <i>Prop1</i> mutants, <i>Prop1^df/df</i> (n = 4), <i>Prop1^df/-</i> (n = 11), <i>Prop1^-/-</i> (n = 7), compared to normals, <i>Prop1^+/+</i> (n = 4). Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.005; **, <i>P</i><0.0005; ***, <i>P</i><0.0001.</p

Elevated basal corticosterone levels in young adult <i>Prop1</i> deficient mice become higher in response to restraint stress.

<p>RIA analysis of circulating corticosterone was carried out on serum from 8 to 10 week males (A) and females (B) of segregating the <i>Prop1</i> null allele at N4 B6 prior to (white bars) and following restraint stress (black bars). Male <i>Prop1</i>^-/- (n = 6) had significantly elevated basal and post-stress levels of corticosterone compared to <i>Prop1^+/−</i> (n = 7) and <i>Prop1^+/+</i> (n = 3). Values represent the mean corticosterone (ng/mL of blood) ± SE. *, <i>P</i><0.0001. Female <i>Prop1^-/-</i> (n = 3) mice had both elevated basal and post-stress levels of corticosterone compared to <i>Prop1^+/−</i> (n = 5) and <i>Prop1^+/+</i> (n = 6). Values represent the mean corticosterone (ng/mL of blood) ± SE. *, <i>P</i><0.005.</p

Adrenal glands of <i>Prop1</i> deficient mice are not hypotrophic.

<p>Adrenal glands were dissected from 5 and 8 week old female N4 B6 <i>Prop1^+/+</i> and <i>Prop1^-/-</i> mice, fixed, embedded, sectioned, and stained with hemotoxylin and eosin (Panels A, C, E, G) and immunostained for 20α-hydroxysteroid dehydrogenase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028355#pone.0028355-Hershkovitz1" target="_blank">[59]</a> and developed with diaminobenzidine (brown, Panels B, D, F, H) to visualize the X-zone (brackets). The ratio of adrenal weight to body weight (Panel I) was increased in <i>Prop1</i>^-/- (n = 5) compared to <i>Prop1^+/−</i> (n = 6) or <i>Prop1^+/+</i> (n = 3) N4 B6 male mice at 8 to 10 wks. Values represent the mean adrenal weight (mg) per body weight (g) ± SE. *, <i>P</i><0.0001; **, <i>P</i><0.0005.</p

No evidence for evolving hypocortisolism in <i>Prop1</i> deficient animals.

<p>Blood plasma was collected from 3.5 to 5 wk N4B6 (Panel A) and 34 to 52 wk mixed genetic background (Panel B) animals from and the circulating ACTH levels were determined by RIA. Males and females were included together because the individual analysis showed no difference in the ACTH levels of aged-matched animals of the same genotype. At 3.5 to 5 weeks <i>Prop1^-/-</i> (n = 6) animals tended to have higher circulating levels of ACTH than <i>Prop1^+/</i>⁻ (n = 10) or <i>Prop1^+/+</i> (n = 10) animals, but the difference was not statistically significant (top). At 34 to 52 weeks three different genotypes of <i>Prop1</i> mutant animals, <i>Prop1</i>^-/- (n = 8), <i>Prop1</i>^df/- (n = 20), and <i>Prop1</i>^df/df (n = 12), exhibited an increase in circulating ACTH levels compared to <i>Prop1^+/+</i> (n = 9) (bottom). Values represent the mean ACTH production (pg/mL) ± SE. *, <i>P</i><0.01; **, <i>P</i><0.005; ***, <i>P</i><0.0005. Corticosterone levels were measured in serum from aged male <i>Prop1^+/+</i> (n = 4), <i>Prop1</i>^df/df (n = 4), <i>Prop1</i>^df/- (n = 11), and <i>Prop1</i>^-/- (n = 7) mice (Panel C). All three genotypes of <i>Prop1</i> deficient mice show elevated basal levels of corticosterone compared to wild type. <i>Prop1</i>^df/df mice have statistically higher basal levels of corticosterone compared to <i>Prop1</i>^df/- or <i>Prop1</i>^-/- mice. Values represent the mean corticosterone (ng/mL of blood) ± SE. *, <i>P</i><0.005; **, <i>P</i><0.0005; ***, <i>P</i><0.05.</p