Leucine acts in the brain to suppress food intake but does not function as a physiological signal of low dietary protein

Abstract

Intracerebroventricular injections of leucine are sufficient to suppress food intake, but it remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in dietary and circulating levels of leucine, or all three branched-chain amino acids (BCAAs), contribute to the detection of reduced dietary protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 μg) was sufficient to suppress food intake. Isocaloric low- (9% protein energy; LP) or normal- (18% protein energy) protein diets induced a divergence in food intake, with an increased consumption of LP beginning on day 2 and persisting throughout the study (P < 0.05). Circulating BCAA levels were reduced the day after LP diet exposure, but levels subsequently increased and normalized by day 4, despite persistent hyperphagia. Brain BCAA levels as measured by microdialysis on day 2 of diet exposure were reduced in LP rats, but this effect was most prominent postprandially. Despite these diet-induced changes in BCAA levels, reducing dietary leucine or total BCAAs independently from total protein was neither necessary nor sufficient to induce hyperphagia, while chronic infusion of EAAs into the brain of LP rats failed to consistently block LP-induced hyperphagia. Collectively, these data suggest that circulating BCAAs are transiently reduced by dietary protein restriction, but variations in dietary or brain BCAAs alone do not explain the hyperphagia induced by a low-protein diet.

Keywords: branched-chain amino acids; food intake; hypothalamus; macronutrient; protein restriction.

Fig. 1.

Low-protein diets increase food intake. Rats were transitioned from chow diet to either an isocaloric low-protein (LP) or normal-protein (NP) diet, and daily food intake was measured for 7 days. Starting on day 2, food intake in the LP vs. NP groups significantly diverged, with intake remaining significantly different for the remainder of the study (*P < 0.01).

Fig. 2.

Amino acids have variable effects on food intake. A: rats bearing third ventricular cannula were fasted for 24 h and subsequently injected with a single amino acid (AA; 10 μg, 2 μl) ∼2 h prior to lights off, and 24-h food intake was subsequently recorded. Amino acids tested were tryptophan (TRP), methionine (MET), lysine (LYS), threonine (THR), leucine (LEU), and serine (SER). Only leucine significantly suppressed food intake (*P < 0.05). B: rats bearing third ventricular cannula were placed on LP diet for 4 days. Rats were injected with leucine (10 μg, 2 μl) ∼2 h prior to lights off (rats were not fasted), and 24-h food intake was recorded. Leucine significantly suppressed food intake in animals consuming the LP diet (*P < 0.027).

Fig. 3.

Plasma amino acids in rats acutely placed on low-protein diets. Rats were transitioned from chow diet to either an isocaloric LP or NP diet, with individual groups killed on day 0 (NP), or after 1, 2, or 4 days of being placed on the low-protein and normal-protein diets. Trunk blood was collected at death, and plasma amino acids were measured via HPLC. Individual branched-chain amino acids (BCAAs) (A: leucine, B: isoleucine, C: valine), total BCAAs (D), and total essential amino acids (EAAs; E) were affected by the LP diet (*P < 0.05).

Fig. 4.

Preprandial and postprandial brain and postprandial plasma amino acids in rats placed on a LP diet. Microdialysis in the third cerebral ventricle was performed in rats consuming either NP or LP diet for 2 days. Microdialysis samples were collected at 30-min intervals from 1 h before to 1 h after feeding of a controlled test meal, with amino acid concentrations averaged together for the two preprandial and two postprandial samples. One hour after the test meal, animals were killed, and trunk blood was collected to assess plasma amino acid levels. Food intake (A) in LP and NP rats is shown prior to microdialysis. Brain concentrations of individual BCAAs (B) and total EAAs (C) both before (pre) and following (post) a fixed NP or LP meal are shown. Plasma BCAAs (D) and total EAAs (E) were measured at the end of the experiment. F: schematic drawing presents experimental design (MS, microdialysis sample). *P < 0.05. NP vs. LP at each time point.

Fig. 5.

Dietary leucine does not contribute to the hyperphagia induced by low-protein diets. Rats were placed on one of five isocaloric diets that varied in protein and leucine content. Control diets included a NP, LP, or a modified NP diet, in which half of the protein was provided as casein and the other half was provided as free amino acids (LP+All). The fourth diet (LP+LEU) consisted of the LP diet with leucine added back to equal the leucine content of the NP diet. The fifth diet (NP−LEU) consisted of the LP+All diet minus leucine, such that leucine levels were equal to the LP diet. Left: daily food intake. Right: daily intake averaged across days 3–12. Placing animals on LP induced a significant increase in food intake (*P < 0.05), regardless of leucine content.

Fig. 6.

Dietary BCAAs do not contribute to the hyperphagia induced by LP diets. Rats were placed on one of five isocaloric diets that varied in protein and BCAA content, similar to above. Control diets included a NP, a LP, or a modified NP diet in which half of the protein was provided as casein and the other half was provided as free amino acids (LP+All). The fourth diet (LP+BCAA) consisted of the LP diet with leucine, isoleucine, and valine added back to equal the BCAA content of the NP diet. The fifth diet (NP-BCAA) consisted of the LP+All diet with leucine, isoleucine, and valine reduced to levels equal to that of the LP diet. Left: daily food intake. Right: daily intake averaged across days 3–7. Placing animals on the LP diet induced a significant increase in food intake (*P < 0.05), regardless of BCAA content.

Fig. 7.

Brain infusion of amino acids did not block LP-induced hyperphagia. Rats bearing lateral ventricular cannula were continuously infused (via osmotic minipump) with saline or increasing concentrations of a cell culture-based amino acid mixture. Coincident with initiation of intracerebroventricular infusion, rats were also placed on either control (NP) or LP diet to determine whether brain amino acid infusion is sufficient to block LP-induced hyperphagia. LP+Saline rats exhibited a significant increase in food intake relative to NP+Saline, beginning on day 2 of diet. Brain amino acid infusion failed to block this LP-induced increase in food intake, except for a transient effect on day 2 with the highest dose (20×AA). Left: daily food intake. Right: average intake from days 2–6. *P < 0.05 vs. NP+Saline.