Leptin was administered ip (10microg) and animals perfused. B, POMC35S-labeled neurons and C, STAT3 phosphorylation colocalized with POMC35S-labeled neurons were quantified in the rostral and caudal ARC (Paxinos levels 43 and 47) in littermate controls (white bars) and Pomc-Cre, Lepr^flox/flox IR^flox/flox mice (black bars). Values are presented as means +/− SE. See also figure S1.

A, Body weight curves of male Lepr^flox/flox IR^flox/flox mice (open squares, n = 12), Pomc-Cre, IR^flox/flox (filled grey triangles, n = 16), Pomc-Cre, Lepr^flox/flox (open black triangles and dashed line, n = 8), and Pomc-Cre, Lepr^flox/flox IR^flox/flox mice (filled black circles, n = 9), and body weight curves of female Lepr^flox/flox IR^flox/flox mice (open squares, n = 10), Pomc-Cre, IR^flox/flox (filled grey triangles, n = 18), Pomc-Cre, Lepr^flox/flox (open black triangles and dashed line, n = 8), and Pomc-Cre, Lepr^flox/flox IR^flox/flox mice (filled black circles, n = 8) on standard chow. B, Cumulative food intake in male and female Lepr^flox/flox IR^flox/flox mice (filled dark grey squares, n = 13,13) and Pomc-Cre, Lepr^flox/flox IR^flox/flox mice (open light grey circles, n = 9,10) over time. C, Fat mass in a separate cohort of 6 month old Lepr ^flox/flox, IR ^flox/flox (white bars), Pomc-Cre, IR ^flox/flox (grey bars), Pomc-Cre, Lepr ^flox/flox (striped bars), and Pomc-Cre, Lepr^flox/flox IR^flox/flox mice (black bars) as measured by NMR (n=7–15 per group). Values are means +/− SE. ANOVA: p<0.0001 males, p=0.0124 females. For entire figure, * = p< 0.05, ** = p<0.01, *** = p<0.0001, determined by Bonferroni’s Multiple Comparison Test following one-way ANOVA for each group or time point. D, Relative expression of POMC as measured by qPCR in Lepr^flox/flox IR^flox/flox (white bars, n=14–18), Lepr^flox/flox,POMC-Cre (striped bars, n=7–9), and Lepr^flox/flox IR^flox/flox POMC-Cre (black bars, n=7–8) mouse hypothalami. E, O2 consumption, F, ambulatory activity, and G respiratory exchange rate in 3 month old female mice lacking insulin and LepRs in POMC neurons (black bars, n=9), lacking only LepRs (striped bars, n=7), and littermate controls (white bars, n=12)

A, Basal and clamp insulin B, blood glucose C, Glucose infusion rate (GIR) D, HGP (EndoRa) and E, Glucose disposal rate (Rd) in conscious 2 month old female Lepr^flox/flox IR^flox/flox (white bars, n=5), and Pomc-Cre, Lepr^flox/flox IR^flox/flox (black bars, n=5) mice. Values are presented as means +/− SE. * p<0.05 See also figure S4.

A, Hypothalamic organotypic slices from FoxO1GFP-POMC reporter mice were treated with insulin (100 nM for 30 min) or vehicle and compared with slices from Pomc-Cre, Lepr^flox/flox IR^flox/flox mice carrying the FoxO1GFP reporter and subjected to anti-GFP immunohistochemistry. Scale bar, 20 μm. B, The percentage of neurons with cytoplasmic FoxO1GFP. C. Current-clamp recordings at resting membrane potential depicting an insulin-induced hyperpolarization and a leptin-induced depolarization in separate POMC neurons from POMC-GFP mice. Downward deflections are responses to rectangular current steps. D. Current-clamp recordings show insulin and leptin fail to influence the membrane potential of POMC neurons in Pomc-Cre, Lepr^flox/flox IR^flox/flox mice. E. Histogram of insulin- and leptin-induced responses in identified POMC neurons from POMC-GFP and Pomc-Cre, Lepr^flox/flox IR^flox/flox mice. Values are means +/− SE. ***p < 0.001, compared by 2-way ANOVA followed by Bonferroni post-hoc. See also figure S2.

A, Serum insulin levels were measured by ELISA following removal of food for 2 hours in 3 month old male (n=14–19) and female (n=7–21) Pomc-Cre, Lepr^flox/flox IR^flox/flox and Cre negative littermate controls and analyzed by one-way ANOVA followed by Tukey’s post-hoc test. B, D, Adult male and female Pomc-Cre, Lepr^flox/flox IR^flox/flox and littermates lacking POMC-cre (n = 7–8) were matched by weight and subjected to a GTT (1mg/kg). E, Male Pomc-Cre, Lepr^flox/flox IR^flox/flox and Lepr^flox/flox IR^flox/flox mice (n = 7) littermates 3 month of age were subjected to an ITT. Blood glucose levels were measured following injection of insulin (0.75U/kg). Values are presented as means +/− SE. Statistical significance as shown by p value was determined by comparison of area under the curve, and significant differences at individual time points were evaluated by t-test. F. Isolated islets (6 islets per well) from 3 month old male mice were incubated in 5mM or 17.5mM glucose for 1 hour and the secreted insulin in the media was harvested for insulin assay. Values are presented as means +/− SE. * p<0.05, ** p<0.01 See also figure S3.

A, Number of pups born to Lepr ^flox/flox, IR ^flox/flox (white bars), Pomc-Cre, IR ^flox/flox (grey bars), Pomc-Cre, Lepr ^flox/flox (striped bars), and Pomc-Cre, Lepr^flox/flox IR^flox/flox dams (black bars) that were 1.5–3 months of age (n=7–30), 4–5 months of age (n=7–14), or 6–8 months of age (n = 7–9). B, Percentage of Lepr ^flox/flox, IR ^flox/flox (white bars) and Pomc-Cre, Lepr^flox/flox IR^flox/flox dams (black bars) caged with control males that failed to produce a litter in two months. C, Pomc-Cre, Lepr^flox/flox IR^flox/flox mice (black bars, n=7) and littermate controls (Lepr ^flox/flox, IR ^flox/flox; white bars, n=7) were examined daily for 3 weeks for estrus length via vaginal cytology. Smears showing predominantly cornified cells were considered estrus-like. Data was analyzed by t-test. D, Blood was collected from female mice on diestrus as determined by vaginal cytology for assay of luteinizing hormone levels by RIA. Groups were compared by t-test. (n=16, 20) E, relative expression of GnRH as measured by qPCR in Lepr^flox/flox IR^flox/flox (white bars, n=7), Lepr^flox/flox,POMC-Cre (striped bars, n=7), and Lepr^flox/flox IR^flox/flox POMC-Cre (black bars, n=7) female mouse hypothalami. Values are means +/− SE. E,F, Sliced and H&E stained paraffin-embedded ovarian tissue from Pomc-Cre, Lepr^flox/flox IR^flox/flox mice (black bars, n=10) and littermate controls (Lepr ^flox/flox, IR ^flox/flox; white bars, n=10, 6) was examined for number of degenerating ova. G, Blood was collected from female mice on diestrus for assay of testosterone levels by RIA. (n=7) H, relative expression of murine 3β-HSD type I as measured by qPCR in Lepr^flox/flox IR^flox/flox (white bars, n=10), and Lepr^flox/flox IR^flox/flox POMC-Cre (black bars, n=11) female mouse ovaries by qPCR. Values are presented as means +/− SE. Groups were compared by t-test.