Skip to main content
. Author manuscript; available in PMC: 2014 Jul 17.
Published in final edited form as: Cell. 2014 Jan 23;156(3):495–509. doi: 10.1016/j.cell.2014.01.008

Figure 3. Effects of maternal HFD-feeding during lactation on hypothalamic neurocircuits.

Figure 3

All following analyses were performed in NCD/NCD and NCD/HFD male offspring. Quantitative real-time PCR analysis of hypothalamic (A) pro-opiomelanocortin (Pomc), agouti-related peptide (Agrp) and neuropeptide Y (Npy) and (B) thyrotropin-releasing hormone (Trh) mRNA expression at 3 (n=9vs11) and 20 weeks (n=8vs9) of age on NCD. (C) Analysis of POMC (left) and AgRP (right) neurons in the arcuate nucleus of the hypothalamus (ARH) in POMCeGFP and AgRPtdTomato mice, respectively, at 8 weeks of age (nPOMC=3vs3 and nAgRP=4vs5; scale bar = 100 μm). (D) Quantitative real-time PCR analysis of hypothalamic proprotein convertase subtilisin/kexin type 1 (Pcsk1), proprotein convertase subtilisin/kexin type 2 (Pcsk2) and carboxypeptidase E (Cpe) mRNA expression at 3 (n=9vs11) and 20 weeks (n=8vs9) of age on NCD. (E) MALDITOF mass spectra obtained by profiling extracts of the ARH at 20 weeks of age (n=4vs4). Prominent ion signals are labeled. i) Comparison of mass fingerprints showing nearly identical ion signals, including ions that are mass-identical with products of the POMC precursor (α-MSH, Di-Ac-MSH, joining peptide (JP)). Fragmentation experiments confirmed the sequences of all labeled peptides; the ion-signal at 1622.81 (asterisk) is composed of two substances (including α-MSH). The arrow marks processed and biologically more potent di-acetylated α-MSH. b) Isotopic pattern and signal intensity of di-acetylated α-MSH before (lower traces) and after Stage Tip concentration. ii) MALDI-TOF/TOF fragment spectrum of di-acetylated α-MSH purified and concentrated with Stage Tips. Y- and b-type fragment ions are labelled, which confirmed the amino acid sequence of di-acetylated α-MSH. iii) Gel view of mass spectra (n=4 each) from preparation of ARH and pituitary gland (Pit) demonstrating identical processing of Pomc-products in all samples. (F) Spontaneous spike frequency and (G) membrane potential of POMCeGFP neurons obtained by perforated patch clamp recordings (n=13 neurons obtained from n=3 animals for each group). (H) Whole-cell recordings showing the relative synaptic input on POMCeGFP neurons (n=13vs14 neurons obtained from n=5vs8 mice). Data are presented as mean ± SEM, *p < 0.05 versus all other groups at the same age. See also Figure S3 for hypothalamic mRNA expression of inflammatory markers at 3 and 20 weeks of age.