Spexin Regulates Hypothalamic Leptin Action on Feeding Behavior

Abstract

Spexin (SPX) is a recently identified neuropeptide that is believed to play an important role in the regulation of energy homeostasis. Here, we describe a mediating function of SPX in hypothalamic leptin action. Intracerebroventricular (icv) SPX administration induced a decrease in food intake and body weight gain. SPX was found to be expressed in cells expressing leptin receptor ObRb in the mouse hypothalamus. In line with this finding, icv leptin injection increased SPX mRNA in the ObRb-positive cells of the hypothalamus, which was blocked by treatment with a STAT3 inhibitor. Leptin also increased STAT3 binding to the SPX promoter, as measured by chromatin immunoprecipitation assays. In vivo blockade of hypothalamic SPX biosynthesis with an antisense oligodeoxynucleotide (AS ODN) resulted in a diminished leptin effect on food intake and body weight. AS ODN reversed leptin's effect on the proopiomelanocortin (POMC) mRNA expression and, moreover, decreased leptin-induced STAT3 binding to the POMC promoter sequence. These results suggest that SPX is involved in leptin's action on POMC gene expression in the hypothalamus and impacts the anorexigenic effects of leptin.

Keywords: appetite regulation; energy homeostasis; hypothalamus; spexin.

Figure 1

Effects of icv SPX administration on food intake, body weight and POMC and AgRP mRNA levels. Eight-week-old male mice were icv-injected with saline or SPX (10 μg). Food intake (A) and body weight gain (B) were measured for 24 h after injection. Body temperature (C) and locomotor activity (D) were observed for 48 h using a vital-view telemetry system. SPX was injected at the beginning of light period as indicated with arrows. Shaded areas represent the dark period. No difference was observed between injection with saline and SPX (n = 4/group). POMC (E) and AgRP (F) mRNA levels were assessed by qRT-PCR analysis. Data are presented as mean ± S.E.M. *, p < 0.05; ***, p < 0.001.

Figure 2

Effect of leptin on SPX expression via STAT3 activation. To determine the effect of leptin on SPX expression, eight-week-old male mice were fasted for 24 h and icv-injected with leptin (2.5 μg); tissue samples were collected 1 h after leptin injection. (A,B) Total RNA was isolated from the mediobasal hypothalamus (MBH) (A) and arcuate nucleus (ARC) (B) and analyzed with qRT-PCR. (C) Leptin was icv-injected into ObRb-Cre;Rpl22^HA mice to analyze leptin-induced SPX mRNA level in ObRb-expressing cells. SPX mRNA was determined using qRT-PCR of MBH RNA samples immunoprecipitated with HA antibody. (D) Mice were icv-injected with 100 μM of S3I-201, an inhibitor of STAT3 activation, and then after 3 h, icv-injected with leptin. After 1 h, MBH SPX mRNA was analyzed by qRT-PCR. (E) ChIP assays were performed to verify whether STAT3 directly binds to the SPX promoter and whether leptin affects this binding. Mice were icv-injected with either saline (CTL) or leptin (LEP) and nuclear DNA samples from their MBH were immunoprecipitated with anti-STAT3 antibody or mouse IgG. Immunoprecipitates were analyzed by PCR amplification using a primer set specific to the mouse SPX promoter. Input represents the DNA extracted from the mouse MBH before immunoprecipitation. Data are presented as mean ± S.E.M. *, p < 0.05; ***, p < 0.001.

Figure 3

Effect of SPX synthesis blockade on the anorexigenic effects of leptin. To inhibit SPX synthesis in the brain, eight-week-old male mice were icv-injected with 2 μg of antisense (AS) or scrambled (SCR) oligodeoxynucleotide (ODN) once a day for 2 days. (A) MBH samples were collected at 24 h after the second ODN injection and SPX mRNA levels were determined using qRT-PCR. (B,C) Mice injected twice with AS ODN were fasted for 24 h before icv leptin (2.5 μg) injection. Food intake (B) and body weight gain (C) were measured for 24 h after leptin injection. Data are presented as mean ± S.E.M. *, p < 0.05; **, p < 0.01.

Figure 4

Effect of SPX synthesis blockade on leptin-induced changes in POMC and AgRP expression. Eight-week-old male mice were icv-injected with 2 μg of the AS or SCR ODN once a day for 2 days and fasted for 24 h after the second ODN injection. Then, mice were icv-injected with leptin (2.5 μg) and MBH samples were collected 3 h after leptin injection. (A,B) RNA samples were isolated from MBH tissues and were analyzed to determine POMC (A) and AgRP (B) mRNA levels using qRT-PCR. (C) ChIP assays showing the effect of AS ODN-mediated SPX synthesis blockade on the leptin (LEP)-induced increase in STAT3 binding onto the POMC and AgRP promoters. Nuclear DNA samples from MBH tissues were immunoprecipitated with anti-STAT3 antibody or mouse IgG and were analyzed by PCR amplification using primers specific to mouse POMC and AgRP promoters. Input represents the DNA extracted from the MBH before immunoprecipitation. Data are presented as mean ± S.E.M. *, p < 0.05.

Figure 5

Anorexigenic effect of leptin-induced SPX via GALR2. (A,B) Eight-week-old male mice were fasted for 24 h and icv-injected with M871 (GALR2 antagonist, 5 nM) or SNAP (GALR3 antagonist, 5 nM) 1 h before icv leptin administration. Food intake (A) and body weight gain (B) were measured for 24 h after leptin injection. (C) Luciferase reporter constructs containing the 5′-flanking region of POMC (300 ng) were transfected into mHypoA cells. After 24 h, cells were pre-treated with M871 (1 μM) or SNAP (1 μM) at 30 min before SPX (300 ng/mL) treatment. Then, cells were harvested for luciferase and β-galactosidase assays at 1 h after SPX treatment. Data are presented as mean ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6

Proposed model for SPX action in leptin-mediated control of food intake within the hypothalamic ARC. SPX is synthesized under a control of leptin in the ObRb expressing cells and acts on POMC expression via GALR2 receptor signaling. Dashed lines represent autocrine and/or paracrine SPX action.