OBJECTIVE—A high-protein diet (HPD) is known to promote the reduction of body fat, but the mechanisms underlying this change are unclear. AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) function as majors regulators of cellular metabolism that respond to changes in energy status, and recent data demonstrated that they also play a critical role in systemic energy balance. Here, we sought to determine whether the response of the AMPK and mTOR pathways could contribute to the molecular effects of an HPD.

RESEARCH DESIGN AND METHODS—Western blotting, confocal microscopy, chromatography, light microscopy, and RT-PCR assays were combined to explore the anorexigenic effects of an HPD.

RESULTS—An HPD reduced food intake and induced weight loss in both normal rats and ob/ob mice. The intracerebroventricular administration of leucine reduced food intake, and the magnitude of weight loss and reduction of food intake in a leucine-supplemented diet are similar to that achieved by HPD in normal rats and in ob/ob mice, suggesting that leucine is a major component of the effects of an HPD. Leucine and HPD decrease AMPK and increase mTOR activity in the hypothalamus, leading to inhibition of neuropeptide Y and stimulation of pro-opiomelanocortin expression. Consistent with a cross-regulation between AMPK and mTOR to control food intake, our data show that the activation of these enzymes occurs in the same specific neuronal subtypes.

CONCLUSIONS—These findings provide support for the hypothesis that AMPK and mTOR interact in the hypothalamus to regulate feeding during HPD in a leucine-dependent manner.

Low-carbohydrate, high-protein diets (HPDs) have become increasingly popular, and many bestselling diet books have promoted this approach (13). However, only in recent years have studies begun to examine the effects of HPD on energy expenditure, subsequent energy intake, and weight loss, as compared with lower-protein diets (46). Currently, there is convincing evidence that a higher protein intake increases thermogenesis and satiety, compared with diets of lower protein content (7). The weight of evidence also suggests that high-protein meals lead to a reduced subsequent energy intake (7,8).

Recently, hypothalamic AMP-activated protein kinase (AMPK) signaling has become an important focus of interest in the control of food intake (911). AMPK is the downstream component of a kinase cascade that acts as a sensor of cellular energy charge, being activated by rising AMP coupled with falling ATP. Once activated, AMPK phosphorylates acetyl-CoA carboxylase (ACC) and switches on energy-producing pathways at the expense of energy-depleting processes (12). Another target molecule for the control of food intake and energy homeostasis is the mammalian target of rapamycin (mTOR) catalytic activity, which has been suggested to be affected by the phosphatidylinositol 3-kinase/Akt pathway (13). Activated signaling through mTOR phosphorylates the serine/threonine kinase p70S6K and the translational repressor eukaryotic initiation factor (eIF) 4E binding protein (4EBP1) (14). mTOR signaling is inhibited under conditions of low nutrients, such as glucose and amino acids, and low intracellular ATP levels (15). Whereas mTOR was presumed to serve as the direct cellular sensor for ATP levels (16), mounting evidence has implicated AMPK in the regulation of mTOR activity (1719).

Leucine has been reported to more potently activate mTOR than other amino acids (20). Leucine may regulate mTOR signaling through the tuberous sclerosis complexes and Rheb (21). However, Xu et al. (22) recently proposed that leucine stimulates the mTOR pathway, in part, by serving both as a mitochondrial fuel through oxidative carboxylation and an allosteric activation of glutamate dehydrogenase. This hypothesis may support the idea that leucine modulates mTOR function, in part by regulating mitochondrial function and AMPK activity.

In this study, we sought to determine whether the response of the AMPK and mTOR pathways could contribute to the molecular effects of an HPD. We therefore examined the hypothalamic modulation of the AMPK/ACC and mTOR signaling pathways induced by HPD as well as the role of leucine in these signaling pathways.

2-Deoxy-d-glucose (2-DG) and l-leucine were from Sigma (St. Louis, MO). The amino acid mix consisted of all amino acids without leucine. Alanine, arginine, asparagine, aspartic, cysteine, glutamic, glutamine, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were also from Sigma. The mTOR inhibitor, rapamycin, was from LC Laboratories (Woburn, MA). mTOR antisense oligonucleotide (ASO) was obtained from Invitrogen (Gaithersburg, MD). The sequence was obtained from NCBI Entrez Nucleotide Bank based on the Mus musculus mTOR mRNA complete code (sense 5′ GAACCTCAGGGCAAGAT 3′ and antisense 5′ ATCTTGCCCTGAGGTTC 3′). Routine reagents were purchased from Sigma, unless otherwise specified.

Animals and diets.

Male 8-week-old Wistar-Hannover rats from the University of Campinas Breeding Center and male 10-week-old ob/ob and db/db mice (C57BL/6J background), originally imported from The Jackson Laboratory (Bar Harbor, ME), were used in accordance with the guidelines of the Brazilian College for Animal Experimentation (COBEA), and the ethics committee at the State University of Campinas approved experiments.

Control animals were fed standard rodent chow (Table 1), given ad libitum. Diets were designed to maintain similar contributions of energy from different macronutrients. The HPD consisted of a 50% protein-enriched diet (Table 1). The leucine-supplemented diet was obtained with leucine addition in place of an equivalent amount of casein used in an HPD (Table 1).

Physiological and metabolic parameters.

After 6 h of fasting, rats were submitted to an insulin tolerance test (1 unit/kg body wt of insulin) as previously described (23). Plasma glucose was determined using a glucose meter (Roche Diagnostic, Rotkreuze, Switzerland), and radioimmunoassay was used to measure serum insulin, according to a previous description (24). Leptin concentrations were determined using an enzyme-linked immunosorbent assay kit (Crystal Chem, Chicago, IL). Cerebrospinal fluid (CSF) and plasma leucine were measured following the spectrophotometric methods described by Rosen (25).

Body composition determination.

The carcass (without the gastrointestinal tract) was weighed and stored at −20°C for analysis of body composition. Carcass water was determined as the difference between the dry and wet weights. Total fat was extracted with petroleum ether using a Soxhlet apparatus. The carcass without fat was dried to determine the lean mass.

Taste reactivity test.

The video cameras (JVC, Digital Still Camera GR-DVL557) were placed outside the chamber, 300 mm from the ring holding the food cup. The video signal was recorded on a conventional VHS tape at 50 frames/s using a recorder (Compact Super VHS, JVC: GR-SXM307). Videotapes were analyzed by slow-motion playback to count taste reactivity components. Taste reactivity components to HPD or leucine-supplemented diet were classified as hedonic, aversive, or neutral, as has previously been described (26).

Intracerebroventricular cannulation and cisterna magna puncture.

The rats were anesthetized with intraperitoneal injection of a mix of ketamin (10 mg) and diazepam (0.07 mg) (0.2 ml/100 g body wt), after overnight fasting and positioning on a Stoelting stereotaxic apparatus. The implantation of an intracerebroventricular catheter into the third ventricle (27) and the method for liquor sampling (28) have been previously described.

Treatments.

In acute treatments, rats were deprived of food for 6 h with free access to water and were injected intracerebroventricularly (3 μl bolus injection) with vehicle, rapamycin (15 μg), leucine (0.5, 2.0, or 4.0 mmol/l), mTOR ASO (1 mmol/μl), or amino acid mix (4.0 mmol/l for each amino acid). Chronic treatment with leucine (4.0 mmol/l) or mTOR ASO was performed by intracerebroventricular infusion every day between 5:00 and 6:00 p.m., during 1 or 3 weeks. Similar studies were carried out in rats that were initially pretreated with i.p. 2-DG, intracerebroventricular microinjection of rapamycin or either vehicles, and after 40 min with intracerebroventricular leucine (4.0 mmol/l) or vehicles, respectively.

Western blot analysis.

After the diets and intraperitoneal, intravenous, and/or intracerebroventricular treatments, animals were anesthetized with an intraperitoneal injection and brown adipose tissue, gastrocnemius muscle, and hypothalamus were quickly removed, minced coarsely, and homogenized immediately. Western blot was performed as previously described (29).

The antibodies used for Western blot were as follows: anti-Akt phosphoserine 473-specific, anti-p70S6K phosphothreonine 389-specific, anti-p70S6K, anti-eIF4E phosphoserine 209-specific, and anti-eIF4E antibodies from Cell Signaling Technology (Beverly, MA); anti-UCP-1, anti-IR, anti-Akt, and antiphosphotyrosine antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); and the anti-mTOR antibodies (mTAB1 and 2) from Upstate Biotechnology (Lake Placid, NY).

mRNA isolation and RT-PCR.

mRNA isolation and RT-PCR were performed as previously described (30). The primers used were as follows: RPS-29 (NCBI: NM012876), sense: 5′-AGGCAAGATGGGTCACCAGC-3′, antisense: 5′-AGTCGAATCATCCATTCAGGTCG-3′; neuropeptide Y (NPY) (NCBI: NM012614), sense: 5′-AGAGATC CAGCCCT GAGACA-3′, antisense: 5′-AACGACAACAAGGGAAATGG-3′; pro-opiomelanocortin (POMC) (NCBI: AF510391), sense: 5′-CTCCTGCTTCAGACCTCCAT-3′, antisense: 5′-TTGGGGTACACCTTCACAGG-3′.

Chromatography.

Chromatographic analyses were carried out on a Waters Alliance equipment series 2695 (Milford, MA) equipped with a quaternary pump, an autosampler, a degasser, and a Waters 2475 fluorescence detector model. The fluorescence of derivatized compounds (ATP, ADP, and AMP) was monitored with excitation and emission wavelengths set at 280 and 420 nm, respectively, as previously described (31).

Light microscopy.

Visceral (epididymal) white adipose tissue depots were dissected and assessed by light microscopy as previously described (32).

Confocal microscopy.

Paraformaldehyde-fixed hypothalami were sectioned (5 μm) and used in regular single- or double-immunofluorescence staining using DAPI, anti-AMPK (1:200; Santa Cruz Biotechnology), anti-phospho-p70SK (1:200; Santa Cruz Biotechnology), anti-mTOR (1:200; ABCAM, Cambridge, MA), and anti–phospho-ACC (1:200; Cell Signaling Technology) antibodies, according to a previously described protocol (33). Analysis and photodocumentation of results were performed using a LSM 510 laser confocal microscope (Zeiss, Jena, Germany). The anatomical correlations were made according to the landmarks given in a stereotaxic atlas (34).

Statistical analysis.

Where appropriate, the results are expressed as the mean ± SE accompanied by the indicated number of animals used in experiments. Comparisons among groups were made using parametric two-way ANOVA; where F ratios were significant, further comparisons were made using the Bonferroni test.

An HPD reduces food intake and body weight in rodents.

We gave male Wistar rats and ob/ob mice standard rodent chow or HPD for 3 weeks. HPD significantly reduced food intake and body weight in Wistar rats and ob/ob mice (Fig. 1A and B). Next, we assessed whether these differences in weight were related to alterations in adiposity. Weight gain and expansion of fat mass was significantly attenuated in HPD-fed rats without changes in total water and lean body mass (Fig. 1C). The fat pad weights of Wistar rats and ob/ob mice after 3 weeks on an HPD showed a 50 and 35% decreased, respectively, compared with the respective control groups (Fig. 1D). In accordance, sections of adipose tissue from rats and mice submitted to HPD exhibit decreases in adipocyte size relative to controls (Fig. 1E). To evaluate the possibility that the anorectic effect of HPD was caused by an aversive effect, we measured taste reactivity. As shown in Fig. 1F, the total numbers of hedonic and neutral reactions during the 15-min recording period remained the same; the aversive reactions were increased on the first day of HPD and returned to control levels thereafter.

Uncoupling protein (UCP-1) in brown adipose tissue is a chief regulator of energy expenditure in rodents (35). Normal rats and obese mice on HPD during 1 week demonstrated an increase of 2.2- and 1.9-fold, respectively, in UCP-1 protein expression in brown adipose tissue (Fig. 1G), indicating that weight loss on an HPD may also be associated with an increase in energy expenditure.

We also observed that 1 week of HPD did not alter plasma glucose in Wistar rats under fasting conditions, but increased insulin levels compared with standard rodent chow (1.82 ± 0.36 vs. 1.05 ± 0.17 ng/ml, respectively; P < 0.05) and reduced leptin levels (3.59 ± 1.43 vs. 5.43 ± 1.12 ng/ml, respectively; P < 0.05). In addition, 1 week of HPD promoted reduction in plasma glucose in ob/ob mice under fasting conditions when compared with the respective control group (166.7 ± 9.0 vs. 275.9 ± 8.8 mg/dl, respectively; P < 0.05) and increased insulin levels (13.1 ± 2.04 vs. 8.98 ± 2.56 ng/ml, respectively; P < 0.05). Despite the increased insulin levels, we did not observe changes in insulin tolerance test (Fig. 1H) and in insulin receptor (Fig. 1I) and Akt (Fig. 1J) phosphorylation in Wistar rats. The phosphorylated p70S6K was increased in HPD-fed rats at both basal and after insulin stimulation (Fig. 1K).

HPD modulates the hypothalamic AMPK/ACC signaling pathway.

To assess the effect of HPD on hypothalamic AMPK signaling, we gave an HPD to Wistar rats and obese mice (ob/ob) for 3 weeks. HPD suppressed AMPK and ACC phosphorylation in the hypothalamus of Wistar rats in a time-dependent manner (Fig. 2A). Similar results were observed in ob/ob mice (Fig. 2B).

To further delineate the mechanism by which the HPD reduced the AMPK/ACC pathway, we analyzed ATP content and the AMP-to-ATP ratio in the hypothalami tissues of HPD-fed rats. The chromatographic analysis shows that 3 days of HPD changed the hypothalamic ATP, ADP, and AMP concentrations in Wistar rats (Fig. 2C). HPD markedly increased ATP content by ∼35% (Fig. 2D) and reduced the AMP-to-ATP ratio by ∼60% in the hypothalamus of Wistar rats (Fig. 2E).

HPD activates mTOR in the hypothalamus.

We next examined whether HPD modulates mTOR activity in hypothalamus. HPD caused a significant increase in the phosphorylation of two downstream targets of mTOR—p70 ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E (eIF4E)—in a time-dependent manner in the hypothalamus of Wistar rats (Fig. 3A). Similar results were observed in ob/ob mice (Fig. 3B).

To determine whether mTOR is required for the HPD-dependent reduction of food intake, we chronically administered mTOR ASO in animals submitted to HPD. As shown in Fig. 3C, intracerebroventricularly administration of mTOR ASO reduced mTOR expression in a time-dependent manner and completely reversed the HPD-induced anorexia in Wistar rats (Fig. 3D). Moreover, while HPD-fed rats lost a significant amount of weight, the mTOR ASO treatment prevented the effect of HPD on body weight during the time frame observed (Fig. 3E). According to these data, treatment with mTOR ASO during 3 days prevents the increase in phosphorylation of p70S6K and eIF4E, both induced by HPD (Fig. 3F and G).

HPD modulates AMPK and mTOR in the same specific neuronal subset and modulates hypothalamic neuropeptides.

To investigate coexpression of AMPK with mTOR, we carried out double-staining confocal microscopy. Using this technique, most neurons expressing AMPK in arcuate and paraventricular nuclei were shown to possess mTOR (Fig. 4A). Consistent with results to detect AMPK, we found that hypothalamic phosphorylated ACC (pACC) immunoreactivity was highly localized in neurons of the arcuate and paraventricular nuclei in fasted rats (Fig. 4B). After 3 days of HPD in rats, low-intensity pACC immunofluorescence was detected in cells of arcuate and paraventricular nuclei of rats (Fig. 4D). Conversely, high-intensity pp70S6K immunofluorescence was detected in cells of arcuate and paraventricular nuclei of rats after HPD treatment (Fig. 4D). Similar results were observed in lateral nucleus (data not shown). The HPD-induced pp70S6K immunoreactivity was not increased in surrounding hypothalamic areas, including the ventromedial hypothalamic nucleus and zona incerta area (Fig. 4C and E).

To explore the mechanism(s) by which HPD regulates food intake, we examined the expression of hypothalamic neuropeptides involved in the control of energy homeostasis in Wistar rats. Under ad libitum fed and 12 h fasting, HPD decreased NPY mRNA levels to a greater extent than in controls (Fig. 4D, left panel). Consistent with these findings, HPD increased POMC mRNA levels to a greater extent than in controls (Fig. 4D, right panel). Similar results were observed in ob/ob mice (Fig. 4E).

Leucine reduces food intake and body weight in rodents.

To determine whether a major component of HPD is responsible for its effect in the control of food intake and body weight, we tested the possibility of a direct activation of this kinase by leucine. We measured plasma and CSF leucine levels after 1 week of treatment; leucine supplementation increased plasma and CSF leucine concentrations to a similar extent to HPD, compared with standard rodent chow (Fig. 5A). To determine whether leucine reduces food intake and body weight, we chronically administered leucine in Wistar rats and in two models of obese mice (ob/ob and db/db). The addition of leucine, in place of an equivalent amount of casein used in the HPD, to the diet of Wistar rats during 3 weeks led to a large decrease in food intake and body weight (Fig. 5B and C). Weight gain and expansion of fat mass were significantly attenuated in leucine-supplemented rats without changes in total water and lean body mass (Fig. 5D). To evaluate the possibility that the anorectic effects of leucine supplementation were caused by diet taste aversion, we carried out a taste reactivity test. As shown in Fig. 5E, the total numbers of hedonic and neutral reactions during the 15-min recording period remained the same; the aversive reactions were increased on the first day of HPD and returned to control levels thereafter. We also did not observe changes in insulin tolerance test (Fig. 5F) and on insulin receptor (Fig. 5G) and Akt (Fig. 5H) phosphorylation in Wistar rats. The phosphorylated p70S6K was increased in leucine-supplemented rats at both basal and after insulin stimulation (Fig. 5I). To explore the anorectic effects of leucine-supplemented diet in obese model animals, we gave this diet to ob/ob and db/db mice. Leucine supplementation promoted a reduction in cumulative food intake and induced weight loss in ob/ob (Fig. 5J and K) and db/db (Fig. 5L and M) mice.

Leucine decreases hypothalamic AMPK and activates mTOR signaling.

To determine whether leucine modulates hypothalamic AMPK/ACC signaling, we injected leucine into the third ventricle of rats and evaluated food intake and AMPK signaling. Leucine caused a significant reduction in food intake (Fig. 6A) and suppressed hypothalamic AMPK and ACC phosphorylation in a dose-dependent manner (Fig. 6B and C). We next investigated whether the microinfusion of leucine modulates the hypothalamic ATP concentration. Figure 6D shows that leucine (4.0 mmol/l) changed ATP, ADP, and AMP concentrations in the hypothalamus of rats, whereas 40 min after leucine injection, the ATP content increased by ∼32% (Fig. 6E) and reduced the AMP-to-ATP ratio by ∼55% in Wistar rats (Fig. 6F).

Interestingly, the activation of the AMPK/ACC pathway is inhibited by intracerebroventricular leucine in vivo; thus, we evaluated the effect of intraperitoneal 2-DG on the activities of these enzymes in male Wistar rats. We showed that the activation of AMPK results in an increase in food intake (Fig. 6G) and body weight gain (Fig. 6H); however, pretreatment with intracerebroventricular leucine with a dose that did not alter food intake (0.5 mmol/l) reversed the effects of 2-DG. In parallel, intraperitoneal injection of 2-DG induced hypothalamic AMPK (Fig. 6I) and ACC (Fig. 6J) phosphorylation, and these effects were blocked by leucine.

Consistent with previous work (13), leucine increased p70S6K and peIF4E phosphorylation in a dose-dependent manner (Fig. 6K and L). To determine whether the effects of leucine on food intake are mTOR dependent, we first identified a dose of rapamycin that did not alter food intake (15 μg) when administered at the onset of the dark cycle. We then evaluated the effect of intracerebroventricular pretreatment with this dose of the inhibitor, or its vehicle, on the anorectic response to intracerebroventricular leucine (4 mmol/l), and we observed that the anorectic response to intracerebroventricular leucine was reversed by rapamycin (Fig. 6M).

Leucine modulates AMPK and mTOR in the same specific neuronal subset and modulates hypothalamic neuropetides.

After the administration of leucine intracerebroventricularly into rat hypothalamus, low intensity of pACC immunofluorescence was detected in cells of arcuate and paraventricular nuclei of rats. Conversely, high intensity of pp70S6K immunofluorescence was detected in cells of arcuate and paraventricular nuclei of rats (Fig. 7A). Similar results were observed in the lateral nucleus (data not shown). The leucine-induced pp70S6K immunoreactivity was not increased in surrounding hypothalamic areas, including the ventromedial hypothalamic nucleus and zona incerta area (not shown).

To explore the mechanism(s) by which leucine regulates food intake, we examined the expression of hypothalamic neuropeptides involved in the control of energy homeostasis in Wistar rats. Under ad libitum fed and 12 h fasting, HPD decreased NPY mRNA levels to a greater extent than in controls (Fig. 7B). Consistent with these findings, leucine increased POMC mRNA levels to a greater extent than in controls (Fig. 7C), and rapamycin blocked the decrease in NPY and the increase in POMC caused by leucine (Fig. 7B and C).

The results of this study show that an HPD markedly reduces food intake and body weight in both normal rats and ob/ob mice. Our data indicate that an HPD, signaling through AMPK and mTOR, inhibits NPY and increases POMC expression in the hypothalamus. The intracerebroventricular administration of leucine, but not other amino acids (data not shown), reduced food intake in a dose-dependent manner, and the magnitude of weight loss and reduction of food intake in leucine-supplemented diet is similar to that achieved by an HPD in normal rats and in ob/ob mice, suggesting that leucine is a major component of the effects of HPD.

Another possibility that should be mentioned as a cause of the reduced food intake is that an HPD may induce conditioned taste aversion (36). Conversely, another study has shown that the behavioral effect of HPD is not caused by conditioned taste aversion, but is probably due to a lower initial palatability of HPD and its enhanced satiety effect (26). We cannot exclude the influence of the adverse reaction to the HPD or leucine-supplemented diet to reduce food intake, since our results showed an increased aversiveness to these diets on the first day.

Although leucine, when administered to rat brains, suppresses feeding and weight gain (13), it is critical to determine whether physiological changes in amino acid concentration act on the hypothalamus to influence energy balance. Our results show that, after 3 weeks of HPD, the body weights of rats and ob/ob mice fed on an HPD were much lower than these of rats fed on a standard diet. Subcutaneous fat pad measurements and expansion of fat mass revealed remarkable differences between the two groups. These results are in agreement with others (37,38) and can be explained, to some extent, by the reduction in caloric intake, but we also observed increased UCP-1 levels that may reflect an increase in energy expenditure.

Changes in hypothalamic AMPK activity regulates food intake; orexigenic factors (e.g., ghrelin) activate hypothalamic AMPK, whereas anorexigenic agents (e.g., leptin) suppresses AMPK activity in the hypothalamus. To gain further insight into the mechanism by which HPD produces weight loss, we examined the role of AMPK. Our results demonstrate that after HPD, hypothalamic ATP level was increased and the AMP-to-ATP ratio was reduced. In parallel, we observed a decrease in AMPK and ACC phosphorylation in rats submitted to HPD.

The mTOR, central to integrating similar signals to control food intake, has now emerged as a detector of hormonal and nutritional signals in the hypothalamus (13). In this study, we investigated whether HPD activates mTOR. HPD increased mTOR activity; moreover, knockdown of central mTOR reverses the anorectic effect of HPD. Consistent with a cross-regulation between AMPK and mTOR to control food intake, our data show that the activation of these enzymes occurs in the same specific neuronal subtypes.

Several factors make leucine an ideal direct nutrient signal to the brain. First, in contrast to plasma glucose and triglyceride concentrations, which do not change appreciably with feeding, leucine concentrations increased severalfold in circulation during a meal (39). Second, leucine regulates the mTOR-signaling pathway in neurons, in vivo (40). Third, leucine is more efficacious than other amino acids as a modulator of mTOR signaling. Indeed, in accordance with Cota et al. (13), we did not observe the other amino acids to be regulators of mTOR signaling in hypothalamus. Lastly, leucine is the most abundant amino acid in many dietary proteins. To further explore the potential nutrient regulation of food intake by leucine, we examined the effect of leucine supplementation on food intake. Animals eating the rat food supplemented with leucine had lower food intake and weight gain than the control animals.

Consistent with the hypothesis that leucine is a major regulator of the mTOR-sensitive response of food intake to HPD, HPD caused a similar reduction in food intake, fat mass, and increased CSF leucine compared with when leucine was supplemented in the diet. Furthermore, mTOR knockdown causes a similar change in the HPD-associated rise in food intake, compared with when leucine was supplemented in the diet. Similarly to an HPD, leucine enhances POMC and diminishes NPY expression in the hypothalamus in an AMPK- and mTOR-dependent manner; on the other hand, we could not detect alterations in food intake with other amino acids. However, the ability of HPD to effect contrasting changes in NPY and POMC neurons deserves further investigation.

How leucine activates mTOR in neurons is unknown. In other cells, it has been suggested that amino acids may activate the mTOR/Raptor/G-protein–β-subunit–like protein complex by promoting phosphorylation of the tumor suppressor complex and inhibition of the small Ras homolog enriched in brain GTPase (41,42). Recently, it has been shown that intracerebroventricular administration of leucine or leptin increases hypothalamic mTOR signaling and decreases food intake (13). Our data extend these findings by implicating AMPK in the anorectic actions of leucine and suggest an intricate relationship between these enzymes.

Nevertheless, this study shows that an HPD is associated with decreased AMPK and increased mTOR activity that results in a reduction in food intake and weight loss in rats and in ob/ob and db/db mice and suggests leucine to be the principal modulator of the AMPK and mTOR pathway present in HPD. These findings provide support for the hypothesis that AMPK and mTOR interact in the hypothalamus to regulate feeding during HPD in a leucine-dependent manner.

FIG. 1.

HPD causes weight loss and anorexia in Wistar rats and ob/ob mice. Cumulative food intake (A) and body weight (B) of animals on an HPD and chow diet (n = 6) are shown. C: Body composition of HPD-fed Wistar rats (n = 8). D: Epididymal fat pad weights (n = 6). E: Histological sections of epididymal fat pads (original magnification ×50, n = 6). F: Evaluation of taste reactivity in Wistar rats on an HPD and chow diet (n = 8). *P < 0.05, HPD 1 day vs. control and 2 and 21 days. G: Western blot analysis of UCP-1 expression in brown adipose tissue (n = 6). *P < 0.05, HPD vs. control. H: Insulin tolerance test (n = 8). Western blot analysis of insulin receptor (IR), tyrosine phosphorylation (I), phospho-AktSer473, serine-phosphorylated Akt (J), and phospho-p70S6Kthr389 and threonine-phosphorylated p70S6K (K) (n = 6). *P < 0.05 vs. vehicle-injected rats; #P < 0.05 vs. the respective control group. IB, immunoblotted; IP, immunoprecipitated; Py, phosphotyrosine.

FIG. 1.

HPD causes weight loss and anorexia in Wistar rats and ob/ob mice. Cumulative food intake (A) and body weight (B) of animals on an HPD and chow diet (n = 6) are shown. C: Body composition of HPD-fed Wistar rats (n = 8). D: Epididymal fat pad weights (n = 6). E: Histological sections of epididymal fat pads (original magnification ×50, n = 6). F: Evaluation of taste reactivity in Wistar rats on an HPD and chow diet (n = 8). *P < 0.05, HPD 1 day vs. control and 2 and 21 days. G: Western blot analysis of UCP-1 expression in brown adipose tissue (n = 6). *P < 0.05, HPD vs. control. H: Insulin tolerance test (n = 8). Western blot analysis of insulin receptor (IR), tyrosine phosphorylation (I), phospho-AktSer473, serine-phosphorylated Akt (J), and phospho-p70S6Kthr389 and threonine-phosphorylated p70S6K (K) (n = 6). *P < 0.05 vs. vehicle-injected rats; #P < 0.05 vs. the respective control group. IB, immunoblotted; IP, immunoprecipitated; Py, phosphotyrosine.

FIG. 2.

Effects of HPD on hypothalamic AMPK activity and ATP content. Representative Western blots of six independent experiments showing hypothalamic lysates from Wistar rats (A) and ob/ob mice (B) and phospho-AMPKthr172, threonine-phosphorylated AMPK, and phospho-ACCser79, serine phosphorylated ACC are shown (CE). Typical chromatographic run (C) depicting the ATP, ADP, and AMP fractions in control (black line) and HPD (dotted line) rats, as mean ATP content (D) and as AMP-to-ATP ratio (n = 6) (E), is represented. *P < 0.05, HPD vs. control.

FIG. 2.

Effects of HPD on hypothalamic AMPK activity and ATP content. Representative Western blots of six independent experiments showing hypothalamic lysates from Wistar rats (A) and ob/ob mice (B) and phospho-AMPKthr172, threonine-phosphorylated AMPK, and phospho-ACCser79, serine phosphorylated ACC are shown (CE). Typical chromatographic run (C) depicting the ATP, ADP, and AMP fractions in control (black line) and HPD (dotted line) rats, as mean ATP content (D) and as AMP-to-ATP ratio (n = 6) (E), is represented. *P < 0.05, HPD vs. control.

FIG. 3.

HPD increases hypothalamic mTOR activity. Representative Western blots of six independent experiments showing hypothalamic lysates from Wistar rats (A) and ob/ob mice (B) and phospho-p70S6Kthr389, threonine-phosphorylated p70S6k, and phospho-eIF4Eser209, serine phosphorylated eIF4E, are shown. C: Western blot of mTOR protein expression in the hypothalamus of rats treated with mTOR ASO at the time points indicated (n = 4). Cumulative food intake (D) and body weight (E) of rats on an HPD and chow diet treated with mTOR ASO or mTOR sense oligonucleotide (SO) (n = 6 per group) are represented. F and G: Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats on chow diet or HPD treated with mTOR ASO or SO. *P < 0.05 vs. other groups.

FIG. 3.

HPD increases hypothalamic mTOR activity. Representative Western blots of six independent experiments showing hypothalamic lysates from Wistar rats (A) and ob/ob mice (B) and phospho-p70S6Kthr389, threonine-phosphorylated p70S6k, and phospho-eIF4Eser209, serine phosphorylated eIF4E, are shown. C: Western blot of mTOR protein expression in the hypothalamus of rats treated with mTOR ASO at the time points indicated (n = 4). Cumulative food intake (D) and body weight (E) of rats on an HPD and chow diet treated with mTOR ASO or mTOR sense oligonucleotide (SO) (n = 6 per group) are represented. F and G: Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats on chow diet or HPD treated with mTOR ASO or SO. *P < 0.05 vs. other groups.

FIG. 4.

HPD enhances phosphorylated p70S6k (pp70S6K) immunoreactivity primarily in AMPK-expressing arcuate and paraventricular nuclei and modulates hypothalamic neuropetides. A: Confocal microscopy of AMPK and mTOR in the arcuate and paraventricular nucleus of Wistar rat. pACC and pp70S6K in the arcuate and paraventricular nucleus (B) and in ventromedial nucleus and zona incerta (C) of 12-h fasted rats is shown. Also represented is pACC and pp70S6K in the arcuate and paraventricular nucleus (D) and in ventromedial nucleus and zona incerta (E) of HPD-fed rats. Rats were submitted to chow diet, 12 h of fasting, or 3 days of HPD; sections (×200 magnification) are shown of arcuate, paraventricular, and ventromedial nucleus and zona incerta immunostained for 4′,6-diamidine-2-phenylindole (DAPI, blue), AMPK (green), mTOR (red), phospho-ACC (green), and phospho-p70S6K (red). HPD modulates mRNA levels of NPY and POMC in the ad libitum fed and fasting state in Wistar rats (F) and ob/ob mice (G) (n = 5 per group). *P < 0.05 HPD vs. chow diet. IB, immunoblotted; IP, immunoprecipitated; PY, phosphotyrosine.

FIG. 4.

HPD enhances phosphorylated p70S6k (pp70S6K) immunoreactivity primarily in AMPK-expressing arcuate and paraventricular nuclei and modulates hypothalamic neuropetides. A: Confocal microscopy of AMPK and mTOR in the arcuate and paraventricular nucleus of Wistar rat. pACC and pp70S6K in the arcuate and paraventricular nucleus (B) and in ventromedial nucleus and zona incerta (C) of 12-h fasted rats is shown. Also represented is pACC and pp70S6K in the arcuate and paraventricular nucleus (D) and in ventromedial nucleus and zona incerta (E) of HPD-fed rats. Rats were submitted to chow diet, 12 h of fasting, or 3 days of HPD; sections (×200 magnification) are shown of arcuate, paraventricular, and ventromedial nucleus and zona incerta immunostained for 4′,6-diamidine-2-phenylindole (DAPI, blue), AMPK (green), mTOR (red), phospho-ACC (green), and phospho-p70S6K (red). HPD modulates mRNA levels of NPY and POMC in the ad libitum fed and fasting state in Wistar rats (F) and ob/ob mice (G) (n = 5 per group). *P < 0.05 HPD vs. chow diet. IB, immunoblotted; IP, immunoprecipitated; PY, phosphotyrosine.

FIG. 5.

Leucine supplementation causes anorexia and weight loss. A: Leucine levels in the plasma and CSF of standard chow–fed, leucine-supplemented, or HFD-fed rats (n = 6). Cumulative food intake (B) and body weight (C) of rats on a leucine-supplemented and chow diet (n = 6) is shown. D: Body composition of standard chow–and leucine supplementation–fed rats (n = 8). E: Taste reactivity test (n = 8). *P < 0.05 vs. control group. F: Insulin tolerance test (n = 8). Western blot analysis of insulin receptor (IR) tyrosine phosphorylation (G), phospho-AktSer473, serine-phosphorylated Akt (H), and phospho-p70S6Kthr389, threonine-phosphorylated p70S6K (I) (n = 6) is shown. *P < 0.05 vs. vehicle-injected rats; #P < 0.05 vs. the respective control group. Cumulative food intake (J) and body weight (K) of ob/ob mice on a leucine-supplemented and chow diet (n = 5). Cumulative food intake (L) and body weight (M) of db/db mice on a leucine-supplemented and chow diet (n = 5) are shown. *P < 0.05 vs. control. IB, immunoblotted; IP, immunoprecipitated; PY, phosphotyrosine.

FIG. 5.

Leucine supplementation causes anorexia and weight loss. A: Leucine levels in the plasma and CSF of standard chow–fed, leucine-supplemented, or HFD-fed rats (n = 6). Cumulative food intake (B) and body weight (C) of rats on a leucine-supplemented and chow diet (n = 6) is shown. D: Body composition of standard chow–and leucine supplementation–fed rats (n = 8). E: Taste reactivity test (n = 8). *P < 0.05 vs. control group. F: Insulin tolerance test (n = 8). Western blot analysis of insulin receptor (IR) tyrosine phosphorylation (G), phospho-AktSer473, serine-phosphorylated Akt (H), and phospho-p70S6Kthr389, threonine-phosphorylated p70S6K (I) (n = 6) is shown. *P < 0.05 vs. vehicle-injected rats; #P < 0.05 vs. the respective control group. Cumulative food intake (J) and body weight (K) of ob/ob mice on a leucine-supplemented and chow diet (n = 5). Cumulative food intake (L) and body weight (M) of db/db mice on a leucine-supplemented and chow diet (n = 5) are shown. *P < 0.05 vs. control. IB, immunoblotted; IP, immunoprecipitated; PY, phosphotyrosine.

FIG. 6.

Effects of leucine on food intake, hypothalamic AMPK and mTOR activity, and ATP content. A: Effect of intracerebroventricular administration of leucine on food intake (n = 5 animals per group). B and C: Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK (B); phospho-ACCser79, serine phosphorylated ACC (C). Typical chromatographic run (D) depicting the ATP, ADP, and AMP fractions in control (black line) and in intracerebroventricular leucine-treated animals (dotted line), as mean ATP content (E) and as AMP-to-ATP ratio (n = 6) (F), is shown. Pretreatment with leucine blocks 2-DG–induced increases in food intake (G), body weight gain (H) (n = 8–10), and phosphorylation of AMPK (I) and ACC (J) in rat hypothalamus. *P < 0.05, leucine vs. vehicle; #P < 0.05, 2-DG vs. other groups. Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats are shown. K: Phospho-p70S6Kthr389, threonine-phosphorylated p70S6K. L: Phospho-eIF4Eser209, serine- phosphorylated eIF4E. Pretreatment with rapamycin blocks leucine-induced anorexia (M) (n = 5 animals per group). Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats are shown. Phospho-p70S6Kthr389, threonine-phosphorylated p70S6k; *P < 0.05 vs. vehicle-injected rats.

FIG. 6.

Effects of leucine on food intake, hypothalamic AMPK and mTOR activity, and ATP content. A: Effect of intracerebroventricular administration of leucine on food intake (n = 5 animals per group). B and C: Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK (B); phospho-ACCser79, serine phosphorylated ACC (C). Typical chromatographic run (D) depicting the ATP, ADP, and AMP fractions in control (black line) and in intracerebroventricular leucine-treated animals (dotted line), as mean ATP content (E) and as AMP-to-ATP ratio (n = 6) (F), is shown. Pretreatment with leucine blocks 2-DG–induced increases in food intake (G), body weight gain (H) (n = 8–10), and phosphorylation of AMPK (I) and ACC (J) in rat hypothalamus. *P < 0.05, leucine vs. vehicle; #P < 0.05, 2-DG vs. other groups. Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats are shown. K: Phospho-p70S6Kthr389, threonine-phosphorylated p70S6K. L: Phospho-eIF4Eser209, serine- phosphorylated eIF4E. Pretreatment with rapamycin blocks leucine-induced anorexia (M) (n = 5 animals per group). Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats are shown. Phospho-p70S6Kthr389, threonine-phosphorylated p70S6k; *P < 0.05 vs. vehicle-injected rats.

FIG. 7.

Leucine enhances phosphorylated p70S6k (pp70S6K) immunoreactivity primarily in arcuate and paraventricular nuclei and modulates hypothalamic neuropetides. pACC and pp70S6K in the arcuate and paraventricular nucleus (A) of the Wistar rat are shown. The animals were submitted to leucine-supplemented diet during 3 days, and the sections (×200 magnification) are shown of arcuate and paraventricular immunostained for 4′,6-diamidine-2-phenylindole (DAPI, blue), phospho-ACC (green), and phospho-p70S6K (red) with images merged. Rapamycin blocks leucine modulation of NPY (B) and POMC (C) mRNA levels in Wistar rats (n = 5 per group). *P < 0.05 vs. other groups.

FIG. 7.

Leucine enhances phosphorylated p70S6k (pp70S6K) immunoreactivity primarily in arcuate and paraventricular nuclei and modulates hypothalamic neuropetides. pACC and pp70S6K in the arcuate and paraventricular nucleus (A) of the Wistar rat are shown. The animals were submitted to leucine-supplemented diet during 3 days, and the sections (×200 magnification) are shown of arcuate and paraventricular immunostained for 4′,6-diamidine-2-phenylindole (DAPI, blue), phospho-ACC (green), and phospho-p70S6K (red) with images merged. Rapamycin blocks leucine modulation of NPY (B) and POMC (C) mRNA levels in Wistar rats (n = 5 per group). *P < 0.05 vs. other groups.

TABLE 1

Components of standard rodent chow, HPD, and leucine-supplemented diet

Standard chow (g/kg diet)HPD (g/kg diet)Leucine-supplemented diet (g/kg diet)
Casein 202 593.5 202 
Leucine — — 50 
Sucrose 100 39 100 
Cornstarch 397 150 347 
Dextrinated starch 130.5 47 130.5 
Soybean oil 70 70 70 
Cellulose 50 50 50 
Mineral mix AIN-93 35 35 35 
Vitamin mix AIN-93 10 10 10 
l-cystin 
Choline 2.5 2.5 2.5 
Total 1,000 1,000 1,000 
Energy (kj/g) 15.9 15.8 15.9 
Standard chow (g/kg diet)HPD (g/kg diet)Leucine-supplemented diet (g/kg diet)
Casein 202 593.5 202 
Leucine — — 50 
Sucrose 100 39 100 
Cornstarch 397 150 347 
Dextrinated starch 130.5 47 130.5 
Soybean oil 70 70 70 
Cellulose 50 50 50 
Mineral mix AIN-93 35 35 35 
Vitamin mix AIN-93 10 10 10 
l-cystin 
Choline 2.5 2.5 2.5 
Total 1,000 1,000 1,000 
Energy (kj/g) 15.9 15.8 15.9 

Published ahead of print at http://diabetes.diabetesjournals.org on 5 December 2007. DOI: 10.2337/db07-0573.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These studies were supported by grants from Fundacão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de desenvolvimento científico e tecnológico (CNPq).

We thank Dr. Nicola Conran for English language editing.

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