Effect of centrally administered C75, a fatty acid synthase inhibitor, on ghrelin secretion and its downstream effects

Abstract

The central administration of the fatty acid synthase (FAS) inhibitor, C75, rapidly suppresses the expression of orexigenic neuropeptides [neuropeptide Y (NPY) and agouti-related protein (AgRP)] and activates expression of anorexigenic neuropeptides [proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART)] in the hypothalamus. The combined actions of these changes inhibit food intake and decrease body weight. Intracerebroventricular injection of C75 appears to rapidly inhibit the secretion of ghrelin by hypothalamic explants ex vivo and by the stomach in vivo. Ghrelin administered intracerebroventricularly reverses the anorexic effect of C75, suggesting that C75 acts upstream of ghrelin. Because ghrelin-producing neurons are known to form synapses onto NPY/AgRP neurons, we suggest that the reversal of C75-induced anorexia by ghrelin may be mediated by NPY/AgRP neurons. This hypothesis is supported by the finding that ghrelin reverses the C75-induced inactivation (assessed by c-Fos expression) of neurons in the arcuate nucleus that express NPY (assessed by immunohistochemical costaining). These effects closely correlate with appropriate changes downstream in the expression of the hypothalamic neuropeptides that regulate feeding behavior, i.e., down-regulation of the expression of NPY and AgRP and up-regulation of the expression of proopiomelanocortin/alpha-melanocyte-stimulating hormone, provoked by C75 and reversed by ghrelin. We propose a model in which ghrelin secretion plays an intermediary role between malonyl-CoA, the substrate of fatty acid synthase, and the neural circuitry regulating energy homeostasis.

Figures

Fig. 1.

Separation and analysis of octanoyl- and des-octanoyl-ghrelin. (A) Pure octanoyl- and des-octanoyl-ghrelin were separated by HPLC on a C18 reverse-phase column by using a linear gradient. (B) Pooled or individual fractions from the HPLC run were subjected to SDS/PAGE (15% acrylamide), after which the gels were immunoblotted with antibody (Phoenix) that recognized both forms of ghrelin. (C) Washed stomach tissue from mice was extracted and separated by HPLC. (D) Pooled or individual fractions were subjected to SDS/PAGE and then immunoblotted with antibody as in B.

Fig. 2.

Distribution of octanoyl- and des-octanoyl-ghrelin in the stomach and hypothalamus. Stomach (A) and hypothalamic (B) tissue was extracted and subjected to HPLC to separate octanoyl- and des-octanoyl-ghrelin. HPLC fractions were assayed for total and active octanoyl-ghrelin by RIA.

Fig. 3.

Effect of centrally administered C75 on blood and stomach ghrelin levels. (A) Treatment and analysis protocol (i.c.v., 10 μg of C75). (B) Food intake. The tissue content of total and active octanoyl-ghrelin and ghrelin mRNA were determined 2 h after C75 injection. (C) Ghrelin levels in plasma. (D) Ghrelin protein in stomach tissue. (E) Ghrelin mRNA level in stomach tissue. *, P < 0.01 vs. refed or C75-treated.

Fig. 4.

Effect of centrally administered C75 on the secretion of ghrelin by hypothalamic explants. (A) Treatment and analysis protocol. Mice were treated as described in the legend for Fig. 3, after which hypothalamic explants were prepared and incubated as described in Materials and Methods.(B) Amount of ghrelin secreted into the medium during the incubation. (C) Ghrelin content of explants at the end of incubation. (D) Relative ghrelin mRNA content of the hypothalamic tissue before incubation of the explants. *, P < 0.01 vs. refed or C75-treated; +, P < 0.05 vs. refed or C75-treated.

Fig. 5.

Effect of central administration of ghrelin on C75-induced changes in food intake and hypothalamic neuronal activity and neuropeptide mRNA expression. (A) Treatment and analysis protocol. The preliminary treatment of the mice was similar to that described in the legend for Fig. 3. (B) Food intake was measured 2–3 h after the i.c.v. administration of 10 μg of C75, i.e., 1 h after i.c.v. administration of 0.2 μg of ghrelin. (C and D) Immunostaining and quantitation of c-Fos in the Arc and PVN. (Magnification: ×10 objective). 3V, third ventricle; ME, median eminence. Bar graphs show the quantification of c-Fos staining (n = 4) in the Arc and PVN. Values are the mean ± SEM. Differences between treatment groups in each region were assessed by Student's t test. *, P < 0.01 vs. none; +, P < 0.01 vs. C75. (E) Brain sections in the region of Arc in the hypothalamus and nuclear c-Fos [3,3′-diaminobenzidine (DAB) plus nickel, shown in purple] and cytoplasmic NPY (DAB, shown in brown) in the Arc. (Scale bars, 20 μm.) (F) Expression of neuropeptide mRNAs in the hypothalamus. *, P < 0.01 vs. none; +, P < 0.05 vs. C75.

Fig. 6.

Possible events leading to decreased secretion of ghrelin and suppression food intake. AMPK, AMP kinase; Ac-CoA, acetyl-CoA; FA-CoA, fatty acetyl-CoA; Mal-CoA, malonyl-CoA.