Injection of Urocortin 3 into the ventromedial hypothalamus modulates feeding, blood glucose levels, and hypothalamic POMC gene expression but not the HPA axis

Abstract

Urocortin 3 (Ucn 3) is a corticotropin-releasing factor (CRF)-related peptide with high affinity for the type 2 CRF receptor (CRFR2). Central administration of Ucn 3 stimulates the hypothalamic-pituitary-adrenal axis, suppresses feeding, and elevates blood glucose levels, suggesting that activation of brain CRFR2 promotes stress-like responses. Several CRFR2-expressing brain areas, including the ventromedial hypothalamus (VMH) and the posterior amygdala (PA), may be potential sites mediating the effects of Ucn 3. In the present study, Ucn 3 or vehicle was bilaterally injected into the VMH or PA, and food intake and plasma levels of ACTH, corticosterone, glucose, and insulin were determined. Food intake was greatly reduced in rats following Ucn 3 injection into the VMH. Ucn 3 injection into the VMH rapidly elevated plasma levels of glucose and insulin but did not affect ACTH and corticosterone secretion. Injection of Ucn 3 into the PA did not alter any of the parameters measured. We determined that the majority of CRFR2-positive neurons in the VMH were excitatory glutamatergic, and a subset of these neurons project to the arcuate nucleus of the hypothalamus (ARH). Importantly, stimulation of CRFR2 in the VMH increased proopiomelanocortin mRNA expression in the ARH. In conclusion, the present study demonstrates that CRFR2 in the VMH mediates some of the central effects of Ucn 3, and the ARH melanocortin system may be a downstream target of VMH CRFR2 neurons.

Fig. 1.

A: representative photomicrograph showing cannula placement in the ventromedial hypothalamus (VMH). Tips of cannulas are indicated by arrowheads. B–D: schematic drawings showing the placement of cannulas in the VMH of rats included in this study. 3V, third ventricle; ARH, arcuate nucleus of hypothalamus; DMH, dorsomedial nucleus of hypothalamus; ME, median eminence; VMH, ventromedial nucleus of hypothalamus; VMHDM, dorsomedial division of VMH; VMHVL, ventrolateral division of VMH.

Fig. 2.

Effect of Urocortin 3 (Ucn 3) injection into the VMH on plasma levels of glucose (A), insulin (B), ACTH (C), and corticosterone (D). Note that both vehicle and Ucn 3 injection resulted in a small elevation of ACTH and corticosterone levels compared with the baseline levels (time 0). *P < 0.05 vs. vehicle control.

Fig. 3.

Food intake of rats receiving VMH injection of Ucn 3 or vehicle. Ucn 3 or vehicle was injected into free-moving rats 15 min before the lights were turned off, and food intake was monitored for 4 h. Ucn 3-injected rats ate significantly less than vehicle-treated rats in the first 2 h after the injection; total food intake measured during the 4-h postinjection period was also reduced. *P < 0.05 vs. vehicle control.

Fig. 4.

Representative photomicrographs showing proopiomelanocortin (POMC) mRNA signals in vehicle (A)- and Ucn 3 (B)-treated rats in the arcuate nucleus of the hypothalamus (ARH). C: summary of POMC mRNA levels in the ARH in rats injected with either vehicle or Ucn 3 into the VMH. Scale bar = 10 μm. 3V, 3rd ventricle. *P < 0.05. vs. vehicle control.

Fig. 5.

Double-label in situ hybridization showing the colocalization of type 2 corticotropin-releasing factor receptor (CRFR2) and vesicular glutamate transporter isoform-2 (VGLUT2) in the VMH. Representative photomicrographs showing brain sections probed with digoxigenin-labeled VGLUT2 cRNA probe (A) and ³³P-labeled CRFR2 cRNA probe (B). C and D: high-magnification brightfield photomicrograph of the boxed area in A and B showing colocalization of VGLUT2 mRNA signals (C, dark cells) and CRFR2 mRNA signals (D, black dot clusters) in the VMH. Representative double-label neurons are indicated by arrowheads. Scale bar = 150 μm (A and B) and 20 μm (C and D). VMHc, ventromedial nucleus of hypothalamus central division; VMHdm, dorsomedial division of VMH; VMHvl, ventrolateral division of VMH.

Fig. 6.

Afferent input into the ARH from CRFR2 neurons in the VMH. A: representative fluorescence micrograph showing fluorogold (FG) injection site (white arrow) in the ARH. B and C: representative photomicrographs showing FG-positive cells (B, dark brown color cells) and CRFR2 mRNA-positive signals (C, silver grain clusters) in the VMH. D and E: high-power magnification of boxed areas in B showing colocalization of FG immunoreactivity (brown color cells) and CRFR2 mRNA signals (black dot clusters). Representative double-labeled cells are indicated by red arrows. Scale bar = 125 (A), 100 (B), 25 (D) μm. ME, median eminence.