Specific amino acids inhibit food intake via the area postrema or vagal afferents

Abstract

To maintain nutrient homeostasis the central nervous system integrates signals that promote or inhibit eating. The supply of vital amino acids is tuned by adjusting food intake according to its dietary protein content. We hypothesized that this effect is based on the sensing of individual amino acids as a signal to control food intake. Here, we show that food intake was most potently reduced by oral L-arginine (Arg), L-lysine (Lys) and L-glutamic acid (Glu) compared to all other 17 proteogenic amino acids in rats. These three amino acids induced neuronal activity in the area postrema and the nucleus of the solitary tract. Surgical lesion of the area postrema abolished the anorectic response to Arg and Glu, whereas vagal afferent lesion prevented the response to Lys. These three amino acids also provoked gastric distension by differentially altering gastric secretion and/or emptying. Importantly, these peripheral mechanical vagal stimuli were dissociated from the amino acids' effect on food intake. Thus, Arg, Lys and Glu had a selective impact on food processing and intake suggesting them as direct sensory input to assess dietary protein content and quality in vivo. Overall, this study reveals novel amino acid-specific mechanisms for the control of food intake and of gastrointestinal function.

Figure 1. Intragastric Arg, Lys and Glu inhibited food intake most potently of all proteogenic amino acids

After 12 h food deprivation rats were gavaged with isomolar doses of individual amino acids (6.7 mmol kg⁻¹, which in the case of Glu corresponds to 1 g kg⁻¹ and to 27% of the daily average Glu intake) and their subsequent food intake measured for 1 (A), 2, 4, 24 and 48 h (B); n= 12, mean ±s.e.m.; (A) unpaired one-way ANOVA, Dunnett post-test; (B) unpaired two-way ANOVA, Bonferroni post-test; *P < 0.05, **P < 0.01, ***P < 0.001. (C–F) After 16 h food deprivation rats were gavaged with individual amino acids (6.7 mmol kg⁻¹) and their subsequent meal pattern analysed in an automated BioDaq system; n= 10, mean ±s.e.m.; unpaired one-way ANOVA, Dunnett post-test; *P < 0.05.

Figure 2. Intragastric Arg, Lys and Glu induced neuronal activity in the brainstem

After 16 h food deprivation, rats were gavaged with isomolar doses (6.7 mmol kg⁻¹) of individual amino acids and transcardially perfused with paraformaldehyde 2 h later. Animals had no access to food between gavage and transcardial perfusion. Different brain areas were analysed for cFOS expression. A, representative images showing cFOS-positive cells in the AP and the NTS (located –13.76 from bregma). Scale bar, 100 μm. Quantification of cFOS-positive cells in the AP (B) and NTS (C); n= 6–8, mean ±s.e.m.; unpaired one-way ANOVA, Dunnett post-test; **P < 0.01, ***P < 0.001. 4V, 4th ventricle; AP, area postrema; NTS, nucleus of the solitary tract; DMX, dorsal motor nucleus vagus nerve; GR, gracile nucleus.

Figure 3. AP mediated the anorectic effect of Arg and Glu, whereas abdominal vagal afferents are necessary for the anorectic effect of Lys

(A) Histological surgery validation showed complete removal of the AP and an intact vagus nerve, visualized by retrograded tracing of i.p. injected fluorogold to the NTS (primary vagal projection site), in AP-lesioned animals. Capsaicin treatment lesioned the vagus nerve and consequently less fluorogold reached the NTS in respective animals. The AP showed fluorogold labelling due to the absence of the blood–brain barrier. Location −13.76 from bregma. Scale bar 100 μm. After 16 h food deprivation AP-lesioned (B), capsaicin-treated (C) and the respective sham animals were gavaged with individual amino acids (6.7 mmol kg⁻¹) and their subsequent food intake measured for 1 h; n= 7–11, mean ±s.e.m.; repeated measures two-way ANOVA, Bonferroni post-test; *P < 0.05, **P < 0.01, ***P < 0.001. 4V, 4th ventricle; AP, area postrema; NTS, nucleus of the solitary tract; DMX, dorsal motor nucleus vagus nerve; GR, gracile nucleus.

Figure 4. Arg, Lys and Glu induced gastric distension by distinct mechanisms

A, after 16 h food deprivation rats were gavaged with an isovolumic (2 ml) dose of individual amino acids (6.7 mmol kg⁻¹); 30 min later their stomach was excised and weighted; n= 6, mean ±s.e.m.; unpaired one-way ANOVA, Dunnett post-test; *P < 0.05, ***P < 0.001. Representative images are shown; scale bar, 4 mm. B, after 16 h food deprivation rats were gavaged with individual amino acids (6.7 mmol kg⁻¹); 30 min later received access to 3 g rat chow and 1.5 h post-administration the gastrointestinal tract was excised and the wet weight measured; n= 6, mean ±s.e.m.; unpaired two-way ANOVA, Bonferroni post-test; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Intravenous Arg, Lys and Glu inhibited food intake, but only intravenous Glu delayed gastric emptying

A, after 16 h food deprivation rats were anaesthetized, received RLA or amino acid (2 mmol kg⁻¹) injection into the lateral tail vein, were gavaged with water or amino acid (HK, LK) and their subsequent food intake was measured for 1 h; n= 9–11, mean ±s.e.m.; unpaired one-way ANOVA, Dunnett post-test; *P < 0.05, **P < 0.01, ***P < 0.001. B, after 16 h food deprivation rats were anaesthetized, received RLA or amino acid (2 mmol kg⁻¹) injection into the lateral tail vein, were gavaged with water or amino acid (HK, LK), 30 min later their stomach was excised and weighted; n= 6, mean ±s.e.m.; unpaired one-way ANOVA, Dunnett post-test; *P < 0.05, ***P < 0.001. C, after 16 h food deprivation rats were anaesthetized, received RLA or amino acid (2 mmol kg⁻¹) injection into the lateral tail vein, were gavaged with water, 30 min later received access to 3 g rat chow and 1.5 h post-administration the gastrointestinal tract was excised and the wet weight measured; n= 6, mean ±s.e.m.; unpaired two-way ANOVA, Bonferroni post-test; *P < 0.05. HK, high l-lysine dose (6.7 mmol kg⁻¹ Lys); LK, low l-lysine dose (2 mmol kg⁻¹ Lys); RLA, Ringer lactate.

Figure 6. Conditioned taste aversion test following different routes of amino acid administration

A, rats received an amino acid (6.7 mmol kg⁻¹) or water gavage during the conditioning days. B, rats received an amino acid (2 mmol kg⁻¹) or RLA injection into the lateral tail vein and were gavaged with water or amino acid (HK) during the conditioning days. i.p. injected LiCl is the positive control. n= 6 (LiCl n= 3), mean ±s.e.m.; unpaired one-way ANOVA, Dunnett post-test; *P < 0.05, **P < 0.01, ***P < 0.001. HK, high l-lysine dose (6.7 mmol kg⁻¹ Lys); RLA, Ringer lactate.

Figure 7. Schematic representation of main findings

Arg, Lys and Glu were identified as the most potent oral anorectic amino acids. After gastric delivery, they specifically stimulated gastric secretion and/or delayed gastric emptying. After absorption only Glu inhibited gastric emptying. Importantly all three amino acids also inhibited food intake when administered intravenously but by a different neuronal mechanism. Lys is detected by vagal afferents projecting to the NTS, whereas Arg and Glu centrally in the AP – a brain area not protected by the blood barrier. Thus, Arg, Lys and Glu selectively affected food processing and intake suggesting a direct sensory input to assess dietary protein content and quality. AP, area postrema; NTS, nucleus of the solitary tract.