Type 2 corticotropin-releasing factor receptor in the ventromedial nucleus of hypothalamus is critical in regulating feeding and lipid metabolism in white adipose tissue

Abstract

Ventromedial nucleus of hypothalamus (VMH) plays a critical role in regulating feeding and energy metabolism. The nucleus expresses high levels of the type 2 corticotropin-releasing factor receptor (CRFR2) and receives prominent innervation of nerve fibers containing Urocortin 3 (Ucn 3), an endogenous ligand of the receptor. In the present study, we showed that mice deficient in Ucn 3 had elevated basal feeding and increased nocturnal food intake after overnight fasting compared with the wild-type (WT) littermates. The Ucn 3 null mice also had lower circulating insulin levels compared with those of the WT mice. Interestingly, the mutant mice maintained a comparable body weight with the WT littermates. Mice with reduced CRFR2 expression in the VMH by small hairpin RNA knockdown (KD) recapitulated feeding phenotypes observed in the Ucn 3 null mice. However, VMH CRFR2 KD mice gained significantly more weight than control mice. The weight gain was due to an accumulation of white adipose tissue (WAT) accompanied by reduced plasma free fatty acids and glycerol levels, increased respiratory quotients, and improved glucose tolerance. On the other hand, plasma insulin levels were comparable with the receptor KD and control mice. Furthermore, the expression of several genes, including hormone-sensitive lipase, was significantly reduced in the WAT of VMH CRFR2 KD mice compared with controls. These results indicate that Ucn 3 signaling through CRFR2 is a critical molecular mediator in the VMH in regulating feeding and lipid metabolism in WAT.

Fig. 1.

Characterization of Ucn 3 null mice. A, Body weight of male Ucn 3 KO (n = 10) and WT (n = 10) mice from wk 6 to 20. B, Average daily food intake of male Ucn 3 KO and WT littermates between wk 12 and 18. C, Accumulated food intake of Ucn 3 null mice and WT mice between wk 12 (first week) and 18 (sixth week). D, Overnight fasting-induced feeding in Ucn 3 null (KO) and WT littermates. Male Ucn 3 KO mice (n = 10) and WT mice (n = 10) were food deprived overnight and then food intake was measured 1, 2, 4, 6, 8, and 24 h after food was returned to the animals. E and F, GTT (E) and ITT (F) in Ucn 3 null (n = 7) and WT mice (n = 9). Glucose (2 g/kg body weight) or insulin (0.75 IU/kg body weight) was injected ip, and blood glucose was measured at various time points. Note the Ucn 3 null mice had an exaggerated hyperglycemic rebound compared with the WT mice. **, P < 0.01; *, P < 0.05 vs. WT littermates. o/n, Overnight.

Fig. 2.

Lentiviral shRNA KD CRFR2 expression in the VMH. A, Representative fluorescent image showing GFP expression in the VMH of a CRFR2 shRNA viral vector injected mouse. The location of the injection site is indicated by shaded area in B. C and D, Representative photomicrographs showing CRFR2 mRNA signals in control-injected (C) and CRFR2 shRNA vector-injected (D) mice in the VMH. E, Summary of CRFR2 mRNA levels in the VMH in mice injected with either control or CRFR2 shRNA viral vector into the VMH. *, P < 0.05. vs. control. Scale bar, 100 μm. 3V, Third ventricle; ARH, arcuate nucleus of hypothalamus; DMH, dorsomedial nucleus of hypothalamus; f, fornix; LH, lateral hypothalamus; mfb, medial forebrain bundle; ns, nigrostriatal bundle; VMHDM, dorsomedial part of VMH; VMHVL, ventrolateral part of VMH; AU, arbitrary unit.

Fig. 3.

Feeding response of mice with reduced CRFR2 expression in the VMH. A, Average daily food intake of mice injected with control (n = 7) or CRFR2 shRNA viral vector (CRFR2 KD; n = 6) in the VMH. B, Overnight fasting-induced feeding in control-injected (n = 6) and CRFR2 shRNA viral vector-injected (n = 6) mice. Mice were food deprived overnight, and then food intake was measured 1, 2, 4, 8, and 24 h after food was returned to the animals. o/n, Overnight. *, P < 0.05 vs. control.

Fig. 4.

Changes in body weight (A), fat mass (B), and fat pad weight (C) in mice injected with either control (n = 7) or CRFR2 shRNA (CRFR2 KD) viral vector (n = 6). D and E, Representative photomicrographs showing hematoxylin and eosin-stained eWAT section of control (D) or CRFR2 KD (E) mice. F, Summary of average fat cell size in eWAT of control and CRFR2 KD mice. *, P < 0.05 vs. control.

Fig. 5.

GTT (A) and ITT (B) in mice received either control (n = 6) or CRFR2 shRNA (CRFR2 KD) viral vector (n = 6) injection into the VMH. Glucose (2 g/kg body weight) or insulin (0.75 IU/kg body weight) was injected ip, and blood glucose was measured at various time points. *, P < 0.05 vs. control.