Functional implications of limited leptin receptor and ghrelin receptor coexpression in the brain

Abstract

The hormones leptin and ghrelin act in apposition to one another in the regulation of body weight homeostasis. Interestingly, both leptin receptor expression and ghrelin receptor expression have been observed within many of the same nuclei of the central nervous system (CNS), suggesting that these hormones may act on a common population of neurons to produce changes in food intake and energy expenditure. In the present study we explored the extent of this putative direct leptin and ghrelin interaction in the CNS and addressed the question of whether a loss of ghrelin signaling would affect sensitivity to leptin. Using histological mapping of leptin receptor and ghrelin receptor expression, we found that cells containing both leptin receptors and ghrelin receptors are mainly located in the medial part of the hypothalamic arcuate nucleus. In contrast, coexpression was much less extensive elsewhere in the brain. To assess the functional consequences of this observed receptor distribution, we explored the effect of ghrelin receptor deletion on leptin sensitivity. In particular, the responses of ad libitum-fed, diet-induced obese and fasted mice to the anorectic actions of leptin were examined. Surprisingly, we found that deletion of the ghrelin receptor did not affect the sensitivity to exogenously administrated leptin. Thus, we conclude that ghrelin and leptin act largely on distinct neuronal populations and that ghrelin receptor deficiency does not affect sensitivity to the anorexigenic and body weight-lowering actions of leptin.

Figure 1

GHSR and LepRb coexpressing neurons are mainly localized to the medial aspect of the hypothalamic Arc. Dual-label histochemistry was performed on coronal sections of mouse brains. Neurons with GFP-IR, which reports on LepRb expression, are stained brown. Neurons expressing GHSR mRNA have overlying punctuate black sliver granules. Strong coexpression of LepRb and GHSR is observed in the medial basal Arc (A–C, examples indicated by red arrows). Medially, few cells were singly labeled for GHSR mRNA (example indicated by asterisk) or GFP-IR (example indicated by black arrow), while laterally a significant number of LepRb singly labeled neurons predominated (C, black arrow). Panel I further demonstrates the segregation of coexpression, with singly labeled cells laterally (asterisk, black arrows) and significant coexpression medially (red arrow). Coexpression in other regions of the hypothalamus was minimal, as in the DMH, with the majority of cells showing segregated expression of LepRb and GHSR (D, black arrows, asterisks). Scattered LepRb neurons exhibited coexpression in the PMV (J, red arrow), yet most were singly labeled (black arrow and asterisk). Distribution of LepRb and GHSR expression in the VTA also exhibited minimal overlap (E–G, red arrows). In the rostral VTA, occasional double-positive cells (E, red arrows) were observed. Large numbers of singly labeled GHSR neurons intermixed with scattered LepRb-positive cells (F, asterisks and black arrows). Caudally in the VTA, few GHSR-positive cells were seen, and LepRb-expressing neurons predominated (G, black arrow). Elsewhere in the caudal midbrain, segregation of GHSR and LepRb expression was observed in the Edinger Westphal (EW) and caudal linear nucleus (Cli) (K). Lastly, similar to other sites showing expression of both LepRb and GHSR, hindbrain expression of the two receptors showed minimal overlap (H, asterisks and arrows). Scale bar = 50 μm.

Figure 2

Wildtype and GHSR-null mice maintained on regular chow are equally sensitive to the anorectic actions of acute or chronic leptin treatment. A single injection of leptin (2 μg/g BW, i.p.) reduced body weight A) and overnight food intake (B) similarly in both ad libitum-fed GHSR-null and wildtype littermates. A chronic treatment with twice daily injections of leptin (1 μg/g BW, i.p.) for 3 successive days reduced body weight (C) and average daily food intake during the injection periods (D) similarly in both ad libitum-fed GHSR-null and wildtype littermates. Data represent the mean ± SEM. **P < 0.01.

Figure 3

GHSR-null mice maintained on HFD are insensitive to the anorectic actions of acute or chronic leptin treatment. A single injection of leptin (2 μg/g BW, i.p.) failed to reduce body weight (A) and overnight food intake (B) in both GHSR-null and wildtype littermates. A chronic treatment with twice-daily injections of leptin (1 μg/g BW, i.p.) for 3 successive days also failed to reduce body weight (C) and overnight food intake (D) in GHSR-null and wildtype littermates. Data represent the mean ± SEM.

Figure 4

Fasted wildtype and GHSR-null mice are equally sensitive to the anorectic actions of leptin. Overnight fasted GHSR-null and wildtype littermates have a similar increase in plasma ghrelin, as compared to ad libitum-fed mice (A). A single injection of leptin (2 μg/g BW, i.p.) to overnight fasted GHSR-null and wildtype littermates significantly reduced the refeeding response (B) and the refeeding-induced increase of body weight in both groups of mice (C). Data represent the mean ± SEM. *P < 0.05; **P < 0.01.