Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin - PubMed (original) (raw)

. 2013 Oct 17;3(1):64-72.

doi: 10.1016/j.molmet.2013.10.001. eCollection 2014 Feb.

Chen Liu 1, Aki Uchida 1, Jen-Chieh Chuang 1, Angela Walker 2, Tiemin Liu 1, Sherri Osborne-Lawrence 1, Brittany L Mason 1, Christina Mosher 1, Eric D Berglund 1, Joel K Elmquist 1, Jeffrey M Zigman 2

Affiliations

Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin

Qian Wang et al. Mol Metab. 2013.

Abstract

The hormone ghrelin stimulates eating and helps maintain blood glucose upon caloric restriction. While previous studies have demonstrated that hypothalamic arcuate AgRP neurons are targets of ghrelin, the overall relevance of ghrelin signaling within intact AgRP neurons is unclear. Here, we tested the functional significance of ghrelin action on AgRP neurons using a new, tamoxifen-inducible AgRP-CreER(T2) transgenic mouse model that allows spatiotemporally-controlled re-expression of physiological levels of ghrelin receptors (GHSRs) specifically in AgRP neurons of adult GHSR-null mice that otherwise lack GHSR expression. AgRP neuron-selective GHSR re-expression partially restored the orexigenic response to administered ghrelin and fully restored the lowered blood glucose levels observed upon caloric restriction. The normalizing glucoregulatory effect of AgRP neuron-selective GHSR expression was linked to glucagon rises and hepatic gluconeogenesis induction. Thus, our data indicate that GHSR-containing AgRP neurons are not solely responsible for ghrelin's orexigenic effects but are sufficient to mediate ghrelin's effects on glycemia.

Keywords: ARC, arcuate nucleus; AgRP; AgRP, Agouti-related peptide; BAC, bacterial artificial chromosome; Blood glucose homeostasis; CNS, central nervous system; DG, dentate gyrus; DVC, dorsal vagal complex; Food intake; Foxo1, Forkhead box protein O1; G6p, glucose-6 phosphatase; GABA, gamma-aminobutyric acid; GHRH, Growth-hormone-releasing hormone; GHSR, growth hormone secretagogue receptor, ghrelin receptor; GOAT, ghrelin O-acyltransferase; Ghrelin; Ghrelin receptor; Hnf4α, hepatocyte nuclear factor 4α; NAc, nucleus accumbens; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; Pcx, pyruvate carboxylase; Pepck, phosphoenolpyruvate carboxykinase; Phox2b, paired-like homeobox 2b; VGAT, vesicular GABA transporter; VTA, ventral tegmental area.

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Figures

Figure 1

Figure 1

Generation of a mouse model with selective GHSR expression in AgRP neurons. (A) Schematic diagram of the derivation of AgRP-CreERT2 mice. The coding sequence of AgRP, found in exons III and IV, was replaced by the coding sequence of a CreERT2 fusion protein. (B) The desired spatiotemporal expression of cre recombinase in AgRP-CreERT2 mice was validated by a cross to Rosa26-lox-STOP-lox-tdTomato reporter mice; tdTomato expression (white neurons) was detected selectively in the ARC of tamoxifen (TM)-injected mice but not in vehicle-injected mice (_n_=3 per group; representative photomicrographs shown; scale bar 100 µm). (C) Ghsr mRNA is expressed in the ARC and VTA of wild-type and wild-type/AgRP-CreERT2 mice but not in GHSR-null animals as expected, as determined using qRT-PCR; tamoxifen-induced Cre-mediated re-expression of Ghsr mRNA is only observed in the ARC of GHSR-null/AgRP-CreERT2 mice, but not other regions (mean±SEM, _n_=4–6 per group).

Figure 2

Figure 2

GHSR-expressing AgRP neurons partially mediate ghrelin's orexigenic action. (A) Ghrelin-induced 2 h-food intake is partially restored in male GHSR-null/AgRP-CreERT2 mice with tamoxifen administration (mean±SEM, _n_=7–12 per group). (B) Ghrelin-induced 2 h-food intake is also partially restored in female GHSR-null/AgRP-CreERT2 mice with tamoxifen administration (mean±SEM, _n_=7 per group). (C) Ghrelin-induced acute food intake is not restored in male GHSR-null/AgRP-CreERT2 mice with vehicle injection (mean±SEM, _n_=3–7 per group). (D) Induction of c-fos (dark orange cell bodies) in response to ghrelin in ARC of wild-type/AgRP-CreERT2 and GHSR-null/AgRP-CreERT2 mice but not in GHSR-null mice or in response to saline (_n_=3 per group, representative photomicrographs shown; scale bar 100 µm). *P<0.05; **P<0.01; ***P<0.001.

Figure 3

Figure 3

GHSR-expressing AgRP neurons mediate the glucoregulatory action of ghrelin. (A) Fasting blood glucose levels are restored to wild-type levels in male GHSR-null/AgRP-CreERT2 mice with tamoxifen administration (mean±SEM, _n_=7–12 per group). (B) Fasting blood glucose levels are also restored to wild-type levels in female GHSR-null/AgRP-CreERT2 mice with tamoxifen administration (mean±SEM, _n_=7 per group). (C) Fasting blood glucose levels are not restored in male GHSR-null/AgRP-CreERT2 mice with vehicle (Veh) injection (mean±SEM, _n_=3–7 per group). (D) Blood glucose levels during severe calorie restriction are restored to wild-type levels in male tamoxifen-treated GHSR-null/AgRP-CreERT2 mice (mean±SEM, _n_=5–12 per group). *P<0.05; **P<0.01; ***P<0.001.

Figure 4

Figure 4

AgRP neuronal expression of GHSR restores fasting glucagon levels and increases fasting-induced liver gluconeogenesis. (A) Fasting glucagon levels are restored in male GHSR-null/AgRP-CreERT2 mice with tamoxifen administration (mean±SEM, _n_=8–14 per group). (B) Fasting insulin levels are similar for all genotypes (mean±SEM, _n_=8–14 per group). (C) Expression of hepatic gluconeogenesis genes after overnight fasting, as determined using qRT-PCR, is increased in male GHSR-null/AgRP-CreERT2 mice with tamoxifen injection (mean±SEM, _n_=4–8 per group). ∧_P_=0.07; *P<0.05.

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