Fig 2: Differential enrichment of genes after leptin treatment. PhosphoTRAP was performed on the samples indicated below, and the RNA abundance determined by the number of RNA-seq reads was plotted in pair-wise comparisons showing (A) differential enrichment of genes in pS6 immunoprecipitates determined by RNA-seq in 14 d leptin treatment of ob/ob mice vs. PBS in (A) hypothalamus and (B) brainstem. TaqMan assays were then performed for the pS6-precipitated polysomes after 14 d PBS treatment of ob/ob mice after leptin treatment (for 2, 4, 7, and 14 d) of ob mice and after 14 d PBS treatment of WT mice. The genes shown are those whose level of enrichment had changed in the comparison between 14 d of leptin vs. PBS (specific genes are shown in A and B): (C) Ppp1r17, (D) POMC, (E) AGRP, (F) Tph2, (G) Npw, and (H) CRH. Data are expressed as the ratio of fold enrichment (IP per input) for each group of mice divided by the fold enrichment (IP per input) for ob/ob mice treated with PBS for 14 d. Homogenates of pooled hypothalami from 15 to 20 mice are used for each group.

Fig 3: EYFP immunoreactivity in the INL is localized to nGnG amacrine cells but not to GABAergic or glycinergic amacrine cells. Retinal sections were co-stained with EYFP and biomarkers for three major classes of ACs, including: GABAergic makers GABA (A1-A3) and GAD65 (B1-B3), glycinergic markers GlyT1 (C1-C3), and glycine (D1-D3), and PPP1R17, a specific marker for nGnG ACs (E1-E3). From the merged images (right panels), it is clear that virtually no immunosignals for GABAergic or glycinergic markers are seen in EYFP-positive cells (arrows) in the INL. By contrast, all EYFP-positive cells exhibit robust PPP1R17 immunoreactivity (arrowheads). Scale bar = 20 µm.

Fig 4: pS6 expression after leptin treatment of ob mice. Dual ISH and IHC in hypothalamus and brainstem was performed for the marker genes and pS6 (244 and 247) as indicated in sections from ob mice treated with leptin (14 d) vs. PBS. (A) POMC: ISH for Pomc and IHC for pS6 in arcuate nucleus. Pomc neurons in Arc are activated by 14 d leptin treatment of ob/ob mice. (B) Tph2: ISH for Tph2 and IHC for pS6 in dorsal raphe. Tph2 neurons in dorsal raphe are activated by 14 d leptin treatment of ob/ob mice. (C) CRH: ISH for Crh and IHC for pS6 in inferior colliculus. Crh neurons in inferior colliculus are activated by 14 d leptin treatment of ob/ob mice. (D) Ppp1r17: ISH for Ppp1r17 and IHC for pS6 in DMH. Ppp1r17 neurons in DMH are inhibited by 14 d leptin treatment of ob/ob mice. (Scale bars, 50 µm.)

Fig 5: cFos expression in Ppp1r17 neurons in ob mice and during scheduled feeding. (A) IHC for Ppp1r17 and TdTomato in ObRb TdTomato mice. DMHPpp1r17 neurons do not express Leprb. (B) cFos expression in DMHPpp1r17 neurons in ob/ob mice is reduced by 14 d pair-feeding. (C) cFos expression in DMHPpp1r17 neurons in scheduled feeding during the 3-h feeding window in response to different amounts of chow. Food is provided to mice between Circadian Time (CT)4 and CT7: (Upper) 4 g of food, the amount normally consumed during the 3-h feeding window; (Middle) 2 g of food, the amount mice consume in 3 h after a fast; and (Lower) 1 g, the amount consumed during the dark phase within 3 h. (Scale bars, 50 µm.)