Hypothalamic malonyl-CoA as a mediator of feeding behavior - PubMed (original) (raw)

Hypothalamic malonyl-CoA as a mediator of feeding behavior

Zhiyuan Hu et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Previous studies showed that i.p. administration of C75, a potent inhibitor of fatty acid synthase (FAS), blocked fasting-induced up-regulation of orexigenic neuropeptides and down-regulation of anorexigenic neuropeptides in the hypothalami of mice. As a result, food intake and body weight were drastically reduced. Here we provide evidence supporting the hypothesis that hypothalamic malonyl-CoA, a substrate of FAS, is an indicator of global energy status and mediates the feeding behavior of mice. We use a sensitive recycling assay to quantify malonyl-CoA to show that the hypothalamic malonyl-CoA level is low in fasted mice and rapidly (< or = 2 h) increases (approximately 5-fold) on refeeding. Intracerebroventricular (i.c.v.) administration of C75 to fasted mice rapidly (< or = 2 h) increased (by 4-fold) hypothalamic malonyl-CoA and blocked feeding when the mice were presented with food. Moreover, prior i.c.v. administration of an acetyl-CoA carboxylase inhibitor, 5-(tetradecyloxy)-2-furoic acid, rapidly (although only partially) prevented the C75-induced rise of hypothalamic malonyl-CoA and prevented the C75-induced decrease of food intake. These effects correlated closely with the rapid (< or = 2 h) and reciprocal effects of i.c.v. C75 on the expression of hypothalamic orexigenic (NPY and AgRP) and anorexigenic (proopiomelanocortin) neuropeptide mRNAs. Previous results showing that C75 administered i.c.v. rapidly activates hypothalamic neurons of the arcuate and paraventricular nuclei are consistent with the results reported in this paper. Together these findings suggest that level of hypothalamic malonyl-CoA, which depends on the relative activities of acetyl-CoA carboxylase and FAS, is an indicator of energy status and mediates feeding behavior.

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Figures

Fig. 1.

Fig. 1.

Enzymatic recycling assay for malonyl-CoA. (A) Protocol for determination of hypothalamic malonyl-CoA by using bacterial malonyl-CoA decarboxylase. Acetyl-CoA in the tissue extract was first eliminated with citrate synthase in the presence of oxaloacetate. Then the cycling reaction was initiated by addition of excess malonate, ATP, and malonate decarboxylase, followed by the addition of acetate kinase. The malonate decarboxylase multicomponent enzyme first decarboxylates malonyl-CoA to produce acetyl-CoA and then transfers CoA from acetyl-CoA to malonate to regenerate malonyl-CoA. Repetition of these events amplifies accumulation of the ultimate product, acetate, which is then converted to acetyl-phosphate. After addition of hydroxylamine, the acetyl-hydroxamate formed is quantified at _A_540. (B) Standard curve for the malonyl-CoA assay. (C) Verification that contaminating acetyl-CoA does not interfere with quantification of malonyl-CoA in the recycling assay. An excess (60 pmol) of acetyl-CoA (to mimic acetyl-CoA contaminant in tissue extracts) was added (or not) to the reaction mixture with or without 2.5 pmol of malonyl-CoA in the presence or absence of citrate synthase. The amount of acetyl-hydroxamate formed (as indicated by _A_540) was not affected by added acetyl-CoA, verifying lack of interference in the recycling assay.

Fig. 2.

Fig. 2.

Long-term effects of C75 administered i.p. on blood glucose, hypothalamic malonyl-CoA, and food intake. Male BALB/c mice were acclimated for 1 week to a 12-h light/12-h dark cycle at 22°C and fed standard laboratory chow ad libitum. C75 or vehicle was administered by i.p. injection 2 h before the start of the dark cycle. Cumulative food intake over the next 24 h was measured. At that point, blood samples were drawn and hypothalami were quickly removed for malonyl-CoA analysis. Another group of mice was given mock injection and pair-fed the same quantity of food consumed by C75-treated mice. (A) Effect of C75 administered i.p. on 24-h food intake. (B) Effect of C75 administered i.p. on hypothalamic malonyl-CoA level. (C) Effect of C75 administered i.p. on blood glucose level. There were five mice per treatment. *, P < 0.001 vs. ad libitum; **, P < 0.001 vs. both ad libitum and C75.

Fig. 3.

Fig. 3.

Short-term effects of C75 administered i.c.v. on blood glucose, hypothalamic malonyl-CoA, and food intake. Male BALB/c mice were acclimated for 1 week to a 12-h light/12-h dark cycle at 22°C and fed standard laboratory chow ad libitum. Mice were fasted for 23 h and then given 10 μg of C75 or vehicle by i.c.v. injection at 1.5 h before the start of the dark cycle. Thirty minutes after i.c.v. injection, food was presented to the mice and cumulative food intake was measured for 2 h. Hypothalami were then quickly removed for malonyl-CoA analysis. Another group of mice previously fasted for 23 h was given an i.c.v. injection of vehicle, and 2.5 h later hypothalami were removed for analysis. (A) Effect of C75 administered i.c.v. on 2-h food intake. (B) Effect of C75 administered i.c.v. on blood glucose level. (C) Effect of C75 administered i.c.v. on hypothalamic malonyl-CoA level. There were four mice per treatment. *, P < 0.001 vs. refed; **, P < 0.001 vs. both refed and C75.

Fig. 4.

Fig. 4.

Short-term effects of C75 administered i.c.v. on the expression of orexigenic and anorexigenic neuropeptides in the hypothalamus. Mice were treated as described in Fig. 3. Two and a half hours after i.c.v. injection of C75, hypothalami were removed, and RNA was isolated and subjected to RNase protection analysis by using antisense probes specific for each neuropeptide mRNA. (A) NPY. (B) AgRP. (C) POMC. There were four mice per treatment. *, P < 0.001 vs. both refed and C75. +, P < 0.01 vs. C75 and P < 0.001 vs. refed. Results are presented as percentage relative to the level in refed mice.

Fig. 5.

Fig. 5.

i.c.v. administration of an ACC inhibitor rapidly blocks i.c.v. C75-induced up-regulation of hypothalamic malonyl-CoA and suppression of food intake. Male BALB/c mice were first fasted for 24 h. Where indicated, TOFA (2 μg), an ACC inhibitor, or vehicle was injected i.c.v. 2.5 h before the dark cycle. Where indicated, C75 (10 μg) or vehicle was injected i.c.v. 1 h before the dark cycle. In some cases, both TOFA and C75 were injected sequentially. (A) To determine the effects of TOFA or C75 or both (TOFA, then C75) on food intake, mice were given food 0.5 h before the dark cycle, after which food consumption was measured during the next 2 h. (B) Hypothalami were removed for malonyl-CoA analysis 2.5 h after C75 or 4 h after TOFA injection. Mice for analysis of hypothalamic malonyl-CoA did not receive food. There were four mice per treatment. *, P < 0.001 vs. control or TOFA alone; +, P < 0.001 vs. C75.

Fig. 6.

Fig. 6.

Model for hypothalamic malonyl-CoA as mediator of expression of orexigenic and anorexigenic neuropeptides and food intake.

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