Angiopoietin-Like Growth Factor Involved in Leptin Signaling in the Hypothalamus

Figures

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  Figure 1. Angiopoietin-like growth factor (AGF) is expressed in the hypothalamus. (A) Immunohistochemical detection of AGF in the human brain. The boxed area within the image indicates a magnified field. (B) Agf mRNA expression in the hypothalamus of ad libitum-fed, overnight fasted or refed mice, confirmed by qPCR analysis (n = 5/group). Data represent means ± SEM (* p < 0.05). Scale bars: 50 μm.

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  Figure 2. AGF is colocalized to proopiomelanocortin (POMC) neurons rather than glial fibrillary acidic protein (GFAP)-positive cells in the hypothalamus. (A,B) Immunofluorescence staining of AGF (green) and NeuN (red), a neuronal nuclei marker and GFAP (magenta), an astrocyte marker in the hypothalamus section. (C) POMC neurons in the hypothalamus, detected based on red fluorescence in POMC-specific tandem dimer Tomato (tdTomato) expressing reporter mice, and AGF immunofluorescence staining. Brain slices were prepared by cryosectioning (25 μm/section). Sections were incubated overnight at 4 °C with Alexa 488-conjugated mouse anti-AGF antibody (1:100). Fluorescence was visualized by confocal microscopy. A yellow-colored neuron represents the colocalization of AGF with POMC. (D) Number of AGF and POMC double-positive neurons in the ARC (between −1.7 and −3.4 mm from bregma) presented as means ± SD (n = 10/slide). Scale bars: 50 μm (upper).

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  Figure 3. AGF expression is induced by leptin-stimulated signal transducer and activator of transcription 3 (STAT3) activation. (A) AGF level in the hypothalamus of C57BL/6 mice after intraperitoneal injection of recombinant mouse leptin (3 mg/kg) or vehicle, determined by Western blotting. AGF, pSTAT3 (Tyr705), STAT3 and α-tubulin were detected in tissue lysates containing equal amounts of protein from the hypothalamus of each group. (B,C) Band intensities of AGF and pSTAT3 measured by the ImageJ program; values are normalized to vehicle-injected samples. (D) AGF and DAPI (4′,6-diamidino-2-phenylindole) co-staining in brain sections from leptin- or vehicle-injected mice. Immunoreactivity was detected by confocal microscopy. (E) Number of AGF-positive neurons in the ARC. Data represent means ± SEM (* p < 0.05, ** p < 0.01).

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  Figure 4. AGF promoter activity is increased by leptin. (A) AGF protein level in N1 cells, determined after treatment with 100 ng/ml leptin for 0, 30, 45 and 60 min. (B,C) AGF and pSTAT3 band intensities, measured using the ImageJ program. (D,E) AGF mRNA expression level in the hypothalamus (D) and in N1 cells (E) after leptin administration, normalized to 18s rRNA level, as determined by qPCR (n = 5/group). (F) Location of STAT3 binding sequences in the AGF promoter (−1072 to −52 bp). (G) AGF promoter activity, measured in N1 cells transfected with an AGF promoter-luciferase reporter plasmid after a 45 min incubation with leptin (100 ng/ml). Cells were pre-incubated in the presence or absence of the JAK2 inhibitor, ruxolitinib (1 μM) for 1 h. N1 cells were harvested and luminescence was measured. Data represent means ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001).