Figure 8: Epo regulation of hypothalamic neuropeptide expression.

(a) Absolute quantification of EpoR mRNA in spleen, WAT and hypothalamus (Hypo) from WT- (open bar) and Tg-mice (closed bar) at age of 6 months (female, n=8) was determined by Taqman PCR. (b) Relative EpoR expression in Hypo from WT- and Tg-mice (male, n=5) at age of 6 months, and ob/ob-mice (OB) and lean littermates (OL) (male, n=6) at age of 4 months was determined by SYBR green RT-PCR. (c) Expression of NPY (NY), Pro-MCH (pM), prepro-orexin (pO), AGRP (Ag) and POMC in Hypo from WT-mice (open bar) and Tg-mice (closed bar) (female, n=8) at age of 6 months was determined by SYBR green RT–PCR. (d) Expression of NY, pM, pO, Ag and POMC in Hypo from 6-month-old WT-mice treated with Epo (closed bar) or saline (open bar) (male, n=8) for 3 weeks was similarly determined. (e) Double fluorescent immunostaining for EpoR (red) and POMC (green) on coronal Hypo sections was combined with DAPI staining (blue) for nuclei. The top panel shows the staining without adding primary antibodies as negative control. Scale bar in the top and middle panels: 100 μm. Scale bar in the bottom panel: 50 μm (3 V indicates third ventricle). (f) Adult neurons isolated from WT Hypo were cultured for 24 h and then stimulated with Epo (5 U ml⁻¹) or saline for 24 h. Total RNA was isolated after Epo or saline treatment and analysed for NY, pM, pO, Ag and POMC expression using quantitative real time PCR. Error bars indicate s.e.m. and significance is indicated as *P<0.05, **P<0.01 and ***P<0.001 determined by Student's t-test in c,d and f. Statistical analyses in b are relative to WT-WAT by one-way analysis of variance (Dunnet's multiple comparison test) and significance is indicated as *P<0.05 and ***P<0.001.