Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain

Abstract

Leptin secreted by adipocytes acts on the brain to reduce food intake by regulating neuronal activity in the mediobasal hypothalamus (MBH). Obesity is associated with resistance to high circulating leptin levels. Here, we demonstrate that peripherally administered leptin activates its receptor (LepR) in median eminence tanycytes followed by MBH neurons, a process requiring tanycytic ERK signaling and the passage of leptin through the cerebrospinal fluid. In mice lacking the signal-transducing LepRb isoform or with diet-induced obesity, leptin taken up by tanycytes accumulates in the median eminence and fails to reach the MBH. Triggering ERK signaling in tanycytes with EGF reestablishes leptin transport, elicits MBH neuron activation and energy expenditure in obese animals, and accelerates the restoration of leptin sensitivity upon the return to a normal-fat diet. ERK-dependent leptin transport by tanycytes could thus play a critical role in the pathophysiology of leptin resistance, and holds therapeutic potential for treating obesity.

Figure 1. Leptin transport into the MBH via the ME requires LepR signaling and is disrupted in DIO mice

(A) Representative photomicrographs showing pSTAT3-IR (red) in the ME of adult male WT mice 5, 15 and 45 min after i.p. leptin administration (3mg/kg). Arrowheads and empty arrowheads show pSTAT3-IR in tanycyte cell bodies lining the floor of the 3rd ventricle (3V) and tanycytic processes contacting the pial surface of the brain (asterisk), respectively. Arrows show neurons immunoreactive for HuC/D (green) and pSTAT3. (B) Graphs representing the mean number of ME tanycytes and arcuate neurons labeled for pSTAT3 per hemisection, 15 and 45 min after the i.p. administration of leptin (3mg/kg) or vehicle in control (CTL; white bars) and DIO mice (red bars). p<0.05 for a vs. b and c vs. d and e; ns: not statistically different. (C) Representative western blots (WB) and quantitative comparison of phosphorylated and total STAT3 and leptin in ME and MBH explants from adult male WT, DIO and db/db mice 15 and 45 min after i.p. leptin (3mg/kg) or vehicle administration (Veh.) (n=3-4 per group). p<0.001 for a vs. b; p<0.05 for d vs. e. ND: not detected. (D) Similar experiments as in B performed with WT mice 45 min after the i.p. administration of bioactive leptin or the mutated antagonist LAN (3mg/kg) (n=3-4 per group). (E) Representative photomicrographs showing tanycytic processes (arrows) and cell bodies (arrowheads) labeled by fluorescent leptin (25nmoles/animal; white labeling), but not fluorescent LAN (25nmoles/animal), 5 min after i.v. injection. However, fluorescent LAN labels blood vessels in the external zone of the ME and the ARH, as does fluorescent leptin (asterisk). (F) Representative photomicrographs and quantitative comparison of pSTAT3-IR (white) in the ARH, VMH and DMH of mice treated i.c.v. with vehicle or leptin-neutralizing antibodies (Leptin-Ab, n=5) 45 min after the i.p. administration of leptin (3mg/kg), or vehicle (n=3-5 per group). Scale bars: 20μm (A), 100μm (E) and 200μm (F). *: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001 leptin vs. vehicle treatment and between indicated groups. Values indicate means ± SEM. See also Figure S1, S2 and S4.

Figure 2. ME Tanycytes express functional LepR, internalize leptin through clathrin-coated vesicles and release captured leptin via an ERK-dependent signaling pathway

(A) RT-PCR analysis of the expression of mRNAs for LepR isoforms in primary cultures of tanycytes. (B) Representative WB and quantitative comparison of phosphorylated and total STAT3, Akt and ERK 15 min after leptin or vehicle treatment of cultured tanycytes (n=4 per group). (C) Representative confocal images for clathrin-IR (green) in tanycytes treated for 15 min with 50nM fluorescent leptin (red). Scale bar: 10μm. (D) Representative WB and quantitative comparison of leptin and clathrin in immunoprecipitated (IPP) clathrin-coated vesicles from tanycytes treated for 15 min with vehicle, LAN (1μg/ml) or leptin (1μg/ml) in the presence or absence of U0126 (10μM), LY294002 (30μM) and WP1066 (30μM). (E) Representative live-cell imaging of fluorescent leptin distribution within tanycytes over an acquisition time of 30 min. Top left panel, xy transmission image of the cultured tanycytes that were analyzed. Right panel, xz reconstruction of section 1 showing the dynamics of leptin transport from 15 to 45 min following leptin administration. The green and black arrowheads show individual cells that are releasing and taking up fluorescent leptin, respectively. Scale bar: 50μm. (F) Representative fluorescence profile in a single cell 15, 30 and 45 min after treatment. (G) Representative WB and quantitative comparison of leptin in cell lysates from leptin-loaded tanycytes and in the medium (released leptin) over 15 min, in the presence or absence of the pharmacological inhibitors of LepR signaling pathways used in D. STAT3 was used as a loading control for cell lysate samples. p<0.001 for a vs. b. *p<0.05, ***p<0.001 treated vs. control and indicated groups. Values indicate means ± SEM. See also Figure S3.

Figure 3. EGF-mediated activation of ERK signaling in the ME restores the transport of leptin into the MBH, increases energy expenditure and promotes locomotor activity in obese mice

(A) EGF (1mg/kg, 15 min) promotes ERK activation in the ME as shown by the quantitative comparison of WB (left panels; n=4-5 per group); right panels: immunofluorescence labeling of pERK (white) in tanycyte cell bodies (arrowheads) and processes (arrows) contacting the pericapillary space (asterisk) in the ME. 3V, 3rd ventricle; ARH, ARH. (B) Representative WB and quantitative comparison of leptin in ME and MBH explants from DIO (n=4) and db/db (n=3) mice after i.p. leptin administration (3mg/kg), with or without EGF treatment 15 min before sacrifice. (C) Representative photomicrographs and quantitation (n=4) of pSTAT3-IR immunofluorescence after i.p. administration of leptin (3mg/kg, 45 min) with or without EGF treatment 15 min before sacrifice. (D-F) Five-day food (D) and oxygen (E) consumption, and locomotor activity (F) monitoring in DIO mice injected once daily with EGF (1mg/kg/day, red) or vehicle (Veh, black) i.p. at 18:00 (n=7 mice per group). Scale bars: 100μm (A) and 200μm (C). ****: p<0.0001; ***: p<0.001; **: p<0.01 EGF and/or leptin vs. vehicle treatment. Values shown are means ± SEM.

Figure 4. EGF treatment accelerates the restoration of leptin sensitivity in obese mice once they are replaced on a normal diet

(A) Body weight change over 20 weeks in standard-chow-fed mice, DIO mice and DIO mice replaced on a standard diet after 1 week of EGF treatment (1mg/kg/24h; DIO-R+EGF, arrowheads) or no EGF treatment (DIO-R) (n=6-8 per group). (B) Representative WB and quantitative comparison of leptin in MBH explants from standard-chowfed, DIO, DIO-R and DIO-R+EGF mice (n=3-4 per group) after i.p. leptin administration (3mg/kg) at different time points. (C) Body weight change in standard-chow-fed, DIO, DIO-R and DIO-R+EGF mice after daily i.p. leptin administration for 3 days (n=6-8 per group). ***: p<0.001; **: p<0.01; *: p<0.05, EGF vs. vehicle treatment and between indicated groups. Values shown are means ± SEM.