PACAP neurons in the hypothalamic ventromedial nucleus are targets of central leptin signaling

Abstract

The adipose-derived hormone, leptin, was discovered over 10 years ago, but only now are we unmasking its downstream pathways which lead to reduced energy intake (feeding) and increased energy expenditure (thermogenesis). Recent transgenic models have challenged the long-standing supposition that the hypothalamic arcuate nucleus (Arc) is omnipotent in the central response to leptin, and research focus is beginning to shift to examine roles of extra-arcuate sites. Dhillon et al. (2006) demonstrated that targeted knock out of the signaling form of the leptin receptor (lepr-B) in steroidogenic factor 1 (SF-1) cells of the hypothalamic ventromedial nucleus (VMN) produces obesity of a similar magnitude to the pro-opiomelanocortin (POMC)-driven lepr-B deleted mouse, via a functionally distinct mechanism. These findings reveal that SF-1 cells of the VMN could be equally as important as POMC cells in mediating leptin's anti-obesity effects. However, the identification of molecular and cellular correlates of this relationship remains tantalizingly unknown. Here, we have shown that mRNA expression of the VMN-expressed neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated according to energy status and that it exerts catabolic effects when administered centrally to mice. Furthermore, we have shown that SF-1 and PACAP mRNAs are colocalized in the VMN, and that leptin signaling via lepr-B is required for normal PACAP expression in these cells. Finally, blocking endogenous central PACAP signaling with the antagonist PACAP(6-38) markedly attenuates leptin-induced hypophagia and hyperthermia in vivo. Thus, it appears that PACAP is an important mediator of central leptin effects on energy balance.

Figure 1.

Colocalization of SF-1 and PACAP in the VMN. Dual in situ hybridization histology was performed on mouse VMN. Gray arrow, SF-1 mRNA only; white arrows, PACAP mRNA only; black arrows, SF-1 and PACAP mRNA colocalization. A, SF-1 mRNA signal in the dorsomedial, central and ventrolateral subdivisions of the VMN. B, Dual detection of SF-1 (gray cell bodies) and PACAP (silver grains) mRNA in the dorsomedial VMN. C, Dual detection of SF-1 and PACAP mRNA in the ventrolateral VMN.

Figure 2.

Effects of metabolic stimuli on expression of PACAP mRNA. Semiquantitative in situ hybridization histology was performed on brain sections from wild-type and mutant mice that had undergone various metabolic stimuli. VMN and mHab PACAP mRNA expression levels were compared. Data are expressed as mean ± SEM. A, Representative autoradiograph showing PACAP oligoprobe hybridization. B, Saline-treated fed versus saline or leptin-treated 48-h-fasted CD-1 mice. C, Eight weeks of high-energy diet began at the age of 8 weeks. i, HED-fed versus chow-fed CD-1 mice. ii, iii, Correlation between body weight and PACAP expression in (ii) chow-fed and (iii) HED-fed mice. D, Saline-treated ob/wt versus saline or leptin-treated ob/ob mice. E, SF-1-Cre lepr^flox/flox versus lepr^flox/flox mice. *p < 0.05, ***p < 0.001.

Figure 3.

Effects of central PACAP injection on food intake and energy expenditure. PACAP or saline was administered intracerebroventricularly to CD-1 mice. A, Dose–response effect on daytime cumulative food intake following overnight food deprivation. B, Nocturnal food intake and 24 h change in body weight of PACAP-injected versus pair fed mice. C, Core body temperature of nonfasted mice. D, Oxygen consumption of nonfasted mice. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Coadministration of PACAP and PACAP_6-38. PACAP and the PAC₁ receptor antagonist PACAP_6-38 were administered intracerebroventricularly in a single injection to CD-1 mice. A, Food intake following overnight food deprivation. B, Core body temperature of nonfasted mice. *p < 0.05, **p < 0.01.

Figure 5.

Coadministration of leptin and PACAP_6-38. Leptin and PACAP_6-38 were administered intracerebroventricularly in a single injection to CD-1 mice. A, Nocturnal food intake and 24 h change in body weight. B, Core body temperature of nonfasted mice. *p < 0.05, **p < 0.01.