Angiotensin II reduces food intake by altering orexigenic neuropeptide expression in the mouse hypothalamus

Abstract

Angiotensin II (Ang II), which is elevated in many chronic disease states such as end-stage renal disease and congestive heart failure, induces cachexia and skeletal muscle wasting by increasing muscle protein breakdown and reducing food intake. Neurohormonal mechanisms that mediate Ang II-induced appetite suppression are unknown. Consequently, we examined the effect of Ang II on expression of genes regulating appetite. Systemic Ang II (1 μg/kg · min) infusion in FVB mice rapidly reduced hypothalamic expression of neuropeptide Y (Npy) and orexin and decreased food intake at 6 h compared with sham-infused controls but did not change peripheral leptin, ghrelin, adiponectin, glucagon-like peptide, peptide YY, or cholecystokinin levels. These effects were completely blocked by the Ang II type I receptor antagonist candesartan or deletion of Ang II type 1a receptor. Ang II markedly reduced phosphorylation of AMP-activated protein kinase (AMPK), an enzyme that is known to regulate Npy expression. Intracerebroventricular Ang II infusion (50 ng/kg · min) caused a reduction of food intake, and Ang II dose dependently reduced Npy and orexin expression in the hypothalamus cultured ex vivo. The reduction of Npy and orexin in hypothalamic cultures was completely prevented by candesartan or the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside. Thus, Ang II type 1a receptor-dependent Ang II signaling reduces food intake by suppressing the hypothalamic expression of Npy and orexin, likely via AMPK dephosphorylation. These findings have major implications for understanding mechanisms of cachexia in chronic disease states such as congestive heart failure and end-stage renal disease, in which the renin-angiotensin system is activated.

Fig. 1.

Reduction of food intake by Ang II is mediated via the AT1R. Body weight (A), SBP (B), and food intake (C) of FVB mice were measured after peripheral Ang II (1 μg/kg · min) or sham infusion. Ang II osmotic minipumps were implanted on d 0, and candesartan administration was started 1 d before the implantation. Gastrocnemius (gast), tibialis anterior (TA), and quadriceps (quad) muscle were collected and weighed on d 7 (D) (n = 6, mean ± sem). *, P < 0.05; **, P < 0.01. ns, Not significant.

Fig. 2.

Orexigenic/anorexigenic hypothalamic neuropeptide and tissue and circulating hormone expression after 4 d of Ang II infusion. FVB mice were infused peripherally with Ang II (1 μg/kg · min), and saline-infused animals were fed ad libitum (Ad-lib) or pair fed (PF) for 4 d and hypothalamus, epididymal white adipose tissue, stomach, intestine, and plasma were collected. A, Orexigenic/anorexigenic neuropeptide expression in the hypothalamus was quantified by quantitative RT-PCR. B, Tissue hormone expression was quantified in the adipose tissue (leptin), stomach (ghrelin), and intestine (GLP-1, Pyy, and Cck) by quantitative RT-PCR. C, Epididymal white adipose tissue mass after 4 d of infusion. D, Plasma leptin levels measured by ELISA. Animals were starved for 12 h before the animals were killed. Fold change of each gene in quantitative RT-PCR was calculated as changes from ad libitum control (n = 6, mean ± sem). *, P < 0.05; **, P < 0.01. Oxt, Oxytocin; POMC, proopiomelanocortin; CART, cocaine- and amphetamine-regulated transcript; MCH, melanin-concentrating hormone; GLP-1, glucagon-like peptide 1.

Fig. 3.

Orexigenic/anorexigenic hypothalamic neuropeptide and tissue and circulating hormone expression after 6 h of Ang II infusion. A, Candesartan was administered to FVB mice 1 d before minipump implantation, and mice were starved for 12 h. Mice were then implanted with Ang II (1 μg/kg · min) or saline (sham) minipumps sc, given access to food, and food intake was measured for 6 h. B–G, Candesartan was administered to FVB mice 1 d before minipump implantation, and mice were starved for 12 h. Mice were then implanted with Ang II (1 μg/kg · min) or saline (sham) minipumps sc (food was not given during this time period), and hypothalamus, epididymal white adipose tissue, stomach, intestine, and plasma were collected after 6 h. B, Orexigenic/anorexigenic neuropeptide expression in the hypothalamus was quantified by quantitative RT-PCR. C, Npy and orexin expression in the hypothalamus was quantified by quantitative RT-PCR after 6 h of Ang II infusion with or without candesartan administration. D, Tissue hormone expression was quantified in the adipose tissue (leptin) and intestine (GLP-1, Pyy, and Cck) by quantitative RT-PCR. Plasma leptin (E) and adiponectin (F) were measured by ELISA. G, Total and phosphorylated AMPK levels were quantified by immunoblotting of hypothalamic lysates. Fold change of each gene by quantitative RT-PCR was calculated as changes from sham infused control (n = 5–6, mean ± sem). *, P < 0.05; **, P < 0.01. Cand, Candesartan.

Fig. 4.

Intracerebroventricular administration of Ang II causes food intake and muscle weight reduction. A, Food intake of FVB mice was measured after icv Ang II infusion. Osmotic minipump infusing artificial cerebrospinal fluid was implanted on d 0 (white arrow) and replaced with Ang II or vehicle (sham) minipump on d 6 (black arrow). Epididymal fat (B) and gastrocnemius (gast), tibialis anterior (TA), and quadriceps (quad) muscles (C) were collected and weighed on d 13. Atrogin-1 and MuRF-1 expression was analyzed in gastrocnemius muscle on d 13 (D). Fold change of each gene by quantitative RT-PCR was calculated as changes from sham infused control (n = 5, mean ± sem). *, P < 0.05; **, P < 0.01. ns, Not significant.

Fig. 5.

Ang II induced reduction of orexigenic neuropeptides in the cultured hypothalamus. A, Whole hypothalamus was collected from FVB mice and cultured for 2 h in DMEM containing 1% FBS and 1 mm glucose, and then the medium was replaced by DMEM (supplemented with 1% FBS and 1 mm glucose) with increasing dose of Ang II or 10 mm glucose. Hypothalamus was collected 45 min after the replacement of medium, and Npy, AgRP, and orexin expression was analyzed by quantitative RT-PCR. B, Hypothalamus was preincubated for 2 h in DMEM (supplemented with 1% FBS and 1 mm glucose) with/without candesartan (100 nm), and vehicle (control) or Ang II (300 nm) was added. Npy, AgRP, and orexin expression was analyzed by quantitative RT-PCR after 45 min. C, Hypothalamus was preincubated for 2 h in DMEM (supplemented with 1% FBS and 1 mm glucose) with/without AICAR (2 mm), and vehicle (control) or Ang II (300 nm) was added. Npy, AgRP, and orexin expression was analyzed by quantitative RT-PCR after 45 min. Fold change of each gene by quantitative RT-PCR was calculated as changes from control (n = 5, mean ± sem). *, P < 0.05; **, P < 0.01.