VGF is required for obesity induced by diet, gold thioglucose treatment, and agouti and is differentially regulated in pro-opiomelanocortin- and neuropeptide Y-containing arcuate neurons in response to fasting

Abstract

Targeted deletion of the gene encoding the neuronal and neuroendocrine secreted polypeptide VGF (nonacronymic) produces a lean, hypermetabolic mouse. Consistent with this phenotype, VGF mRNA levels are regulated in the hypothalamic arcuate nucleus in response to fasting. To gain insight into the site(s) and mechanism(s) of action of VGF, we further characterized VGF expression in the hypothalamus. Double-label studies indicated that VGF and pro-opiomelanocortin were coexpressed in lateral arcuate neurons in the fed state, and that VGF expression was induced after fasting in medial arcuate neurons that synthesize neuropeptide Y (NPY). Like NPY, VGF mRNA induction in this region of the hypothalamus in fasted mice was inhibited by exogenous leptin. In leptin-deficient ob/ob and receptor-mutant db/db mice, VGF mRNA levels in the medial arcuate were elevated. To identify neural pathways that are functionally compromised by Vgf ablation, VGF mutant mice were crossed with obese A(y)/a (agouti) and ob/ob mice. VGF deficiency completely blocked the development of obesity in A(y)/a mice, whereas deletion of Vgf in ob/ob mice attenuated weight gain but had no impact on adiposity. Hypothalamic levels of NPY and agouti-related polypeptide mRNAs in both double-mutant lines were dramatically elevated 10- to 15-fold above those of wild-type mice. VGF-deficient mice were also found to resist diet- and gold thioglucose-induced obesity. These data and the susceptibility of VGF mutant mice to monosodium glutamate-induced obesity are consistent with a role for VGF in outflow pathways, downstream of hypothalamic and/or brainstem melanocortin 4 receptors, that project via the autonomic nervous system to peripheral metabolic tissues and regulate energy homeostasis.

Fig. 1.

Colocalization of VGF with α-MSH and NPY in the arcuate nucleus of fed and fasted rats. Low-power micrographs of double-labeled immunofluorescence preparations (A–C) demonstrate the colocalization (yellow) of VGF (green) and α-MSH (red) in the retrochiasmatic region (A) and in the rostral (B) and caudal (C) part of the arcuate nucleus of fed colchicine-treated rats (n = 3). Low-power micrographs (D, E) show the colocalization (yellow) of POMC mRNA (labeled with AMCA, pseudocolored red) and VGF mRNA (dark-field image of the silver grains, pseudocolored green) in the arcuate nucleus of fed (D) (n= 4) and fasted (E) (n = 4) rats. In colchicine-treated fed rats (F), VGF immunoreactivity (green) and NPY immunoreactivity (red) are localized in different populations of arcuate neurons. In contrast, ∼50% of NPY-immunoreactive neurons (yellow), localized dorsally, cocontained VGF in the arcuate nucleus of fasted colchicine-treated rats (G) (n = 3).

Fig. 2.

Hypothalamic expression of VGF is regulated by leptin. VGF expression was examined by in situhybridization in the hypothalami of ad libitum fed and 48 hr fasted wild-type, ob/ob, and db/dbmice (A–H). Wild-type mice were injected with saline (sal) or leptin (lep) as indicated (A–D). Photomicrographs of film autoradiograms, representative of coronal sections examined from three independent animals for each treatment group, are shown. Scale bar, 100 μm. VGF mutant mice respond to exogenous leptin (I). VGF-deficient mice were treated with leptin (20 μg/gm body weight, twice daily i.p. for 2 d), and weight loss, feeding, hypothalamic POMC mRNA, and adipose leptin mRNA were measured. Histograms identified by asterisks are significantly different from one another (mean ± SE;p < 0.05; ANOVA with Tukey–Kramer post hoc comparisons; n = 4–6 mice per treatment group).

Fig. 3.

VGF mutant mice resist diet-induced obesity. Wild-type (WT) (+/+) and VGF mutant (−/−) mice were placed either on regular laboratory diets (Chow) or high-fat diets (HF) for 5 weeks. Mice were then weighed (A) and anesthetized, adipose tissues were removed and weighed (B), and total RNA was isolated and leptin mRNA levels were quantified by Northern blotting (C). Histograms identified by differentletters are significantly different from one another (mean ± SE; p < 0.05; ANOVA with Tukey–Kramer post hoc comparisons;n = 9–15 mice per treatment group).

Fig. 4.

VGF mutant mice resist obesity caused by GTG- but not MSG-induced chemical lesions. Wild-type (+/+) and VGF-deficient (−/−) mice were treated with GTG, and body weights (A) and food consumption (B) were determined (mean ± SE) at 16 weeks of age. Wild-type and heterozygous and homozygous VGF-deficient mice were treated with MSG, and body weights were measured (mean ± SE) at 36 weeks of age (C). Histograms identified by different letters are significantly different from one another (p < 0.05; ANOVA with Tukey–Kramerpost hoc comparisons; n = 4–6 mice per treatment group).

Fig. 5.

VGF is required for the development of obesity in agouti mice. Representative agouti (A^y/a) and double-mutant (Vgf−/Vgf−,A^y/a) mice are shown (A). Body weight (B) and body composition (C) measurements for the indicated genotypes are shown (mean ± SE). Histograms identified by differentletters are significantly different from one another (B) (p < 0.05; ANOVA with Tukey–Kramer post hoc comparisons;n = number of mice analyzed of the indicated genotype).

Fig. 6.

VGF is required for the development of hyperphagia but not increased adiposity in ob/ob mice. Representative males of the following genotypes are shown: VGF mutant (Vgf−/Vgf−), double-mutant (Vgf−/Vgf−,ob/ob),ob/ob, and wild type (A). Body weight (B), body composition (C), and daily food intake (D) were measured for the indicated genotypes (mean ± SE). Histograms identified by differentletters are significantly different from one another (B, D) (p < 0.05; ANOVA with Tukey–Kramer post hoc comparisons;n = number of mice analyzed of the indicated genotype).

Fig. 7.

Schematic model indicating the sites where VGF may regulate energy balance, based on genetic, histochemical, and lesion data. Arcuate orexigenic and anorexigenic projections are shown asgray and white, respectively. Regions of the nervous system in which VGF mRNA or protein is abundantly expressed and/or regulated by feeding or GI manipulation are indicated by aspeckled pattern. The dashed linesidentify areas susceptible to GTG- or MSG-induced injury.Dark-gray projections outlined inblack, downstream of agouti effects on the MC4-R, represent possible candidate circuits that might be regulated by locally synthesized VGF (speckled areas).ARC, Arcuate nucleus; DRG, dorsal root ganglia; LH, lateral hypothalamus; VMN, ventromedial nucleus.