Oral Leucine Supplementation Is Sensed by the Brain but neither Reduces Food Intake nor Induces an Anorectic Pattern of Gene Expression in the Hypothalamus

Leucine activates the intracellular mammalian target of the rapamycin (mTOR) pathway, and hypothalamic mTOR signaling regulates food intake. Although central infusion of leucine reduces food intake, it is still uncertain whether oral leucine supplementation is able to affect the hypothalamic circuits that control energy balance. We observed increased phosphorylation of p70s6k in the mouse hypothalamus after an acute oral gavage of leucine. We then assessed whether acute oral gavage of leucine induces the activation of neurons in several hypothalamic nuclei and in the brainstem. Leucine did not induce the expression of Fos in hypothalamic nuclei, but it increased the number of Fos-immunoreactive neurons in the area postrema. In addition, oral gavage of leucine acutely increased the 24 h food intake of mice. Nonetheless, chronic leucine supplementation in the drinking water did not change the food intake and the weight gain of ob/ob mice and of wild-type mice consuming a low- or a high-fat diet. We assessed the hypothalamic gene expression and observed that leucine supplementation increased the expression of enzymes (BCAT1, BCAT2 and BCKDK) that metabolize branched-chain amino acids. Despite these effects, leucine supplementation did not induce an anorectic pattern of gene expression in the hypothalamus. In conclusion, our data show that the brain is able to sense oral leucine intake. However, the food intake is not modified by chronic oral leucine supplementation. These results question the possible efficacy of leucine supplementation as an appetite suppressant to treat obesity.     

Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling

Phosphatidylinositol 3-OH-kinase (PI3K) and STAT3 are signal transduction molecules activated by leptin in brain areas controlling food intake. To investigate their role in leptin-mediated inhibition of hypothalamic neuropeptide Y (Npy) and agouti-related peptide (Agrp) gene expression, male Sprague-Dawley rats (n = 5/group) were either fed ad libitum or subjected to a 52-h fast. At 12-h intervals, the PI3K inhibitor LY-294002 (LY, 1 nmol) or vehicle was injected intracerebroventricularly (ICV) as a pretreatment, followed 1 h later by leptin (3 microg icv) or vehicle. Fasting increased hypothalamic Npy and Agrp mRNA levels (P < 0.05), and ICV leptin administration prevented this increase. As predicted, LY pretreatment blocked this inhibitory effect of leptin, such that Npy and Agrp levels in LY-leptin-treated animals were similar to fasted controls. By comparison, leptin-mediated activation of hypothalamic STAT3 signaling, as measured by induction of both phospho-STAT3 immunohistochemistry and suppressor of cytokine signaling-3 (Socs3) mRNA, was not significantly attenuated by ICV LY pretreatment. Because NPY/AgRP neurons project to the hypothalamic paraventricular nucleus (PVN), we next investigated whether leptin activation of PVN neurons is similarly PI3K dependent. Compared with vehicle, leptin increased the number of c-Fos positive cells within the parvocellular PVN (P = 0.001), and LY pretreatment attenuated this effect by 35% (P = 0.043). We conclude that leptin requires intact PI3K signaling both to inhibit hypothalamic Npy and Agrp gene expression and activate neurons within the PVN. In addition, these data suggest that leptin activation of STAT3 is insufficient to inhibit expression of Npy or Agrp in the absence of PI3K signaling.     

The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes

Background

The branched-chain amino acid (BCAA) leucine has been identified to be a key regulator of skeletal muscle anabolism. Activation of anabolic signalling occurs via the mammalian target of rapamycin (mTOR) through an undefined mechanism. System A and L solute carriers transport essential amino acids across plasma membranes; however it remains unknown whether an exogenous supply of leucine regulates their gene expression. The aim of the present study was to investigate the effects of acute and chronic leucine stimulation of anabolic signalling and specific amino acid transporters, using cultured primary human skeletal muscle cells.

Results

Human myotubes were treated with leucine, insulin or co-treated with leucine and insulin for 30 min, 3 h or 24 h. Activation of mTOR signalling kinases were examined, together with putative nutrient sensor human vacuolar protein sorting 34 (hVps34) and gene expression of selected amino acid transporters. Phosphorylation of mTOR and p70S6K was transiently increased following leucine exposure, independently to insulin. hVps34 protein expression was also significantly increased. However, genes encoding amino acid transporters were differentially regulated by insulin and not leucine.

Conclusions

mTOR signalling is transiently activated by leucine within human myotubes independently of insulin stimulation. While this occurred in the absence of changes in gene expression of amino acid transporters, protein expression of hVps34 increased.     

Increased hypothalamic melanin concentrating hormone gene expression during energy restriction involves a melanocortin-independent, estrogen-sensitive mechanism

Increased expression of melanin concentrating hormone (MCH), an orexigenic neuropeptide produced by neurons in the lateral hypothalamic area (LHA), is implicated in the effect of energy restriction to increase food intake. Since melanocortins inhibit Mch gene expression, this effect of energy restriction to increase Mch signaling may involve reduced hypothalamic melanocortin signaling. Consistent with this hypothesis, we detected increased hypothalamic Mch mRNA levels in agouti (Ay) mice (by 102%; P < 0.05), a model of genetic obesity resulting from impaired melanocortin signaling, compared to wild-type controls. If reduced melanocortin signaling mediates the effect of energy restriction, hypothalamic Mch gene expression in Ay mice should not be increased further by energy restriction, since melanocortin signaling is impaired in these animals regardless of nutritional state. We therefore investigated the effects of energy restriction on hypothalamic Mch gene expression in both Ay mice and in wild-type mice with diet-induced obesity (DIO). Responses in these mice were compared to those induced by administration of 17beta-estradiol (E2) at a dose previously shown to reduce food intake and Mch expression in rats. In both Ay and DIO mice, energy restriction increased hypothalamic Mch mRNA levels (P < 0.05 for each) via a mechanism that was fully blocked by E2. However, E2 did not lower levels of Mch mRNA below basal values in Ay mice, whereas it did so in DIO mice. Thus, the effect of energy restriction to increase hypothalamic Mch gene expression involves an E2-sensitive mechanism that is not altered by impaired melanocortin signaling. By comparison, impaired melanocortin signaling increases hypothalamic Mch gene expression via a mechanism that is insensitive to E2. These findings suggest that while both energy restriction and reduced melanocortin signaling stimulate hypothalamic Mch gene expression, they do so via distinct mechanisms.     

Requirement for lysosomal localization of mTOR for its activation differs between leucine and other amino acids

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.     

Insulin-like growth factor-1 (IGF-1) and leucine activate pig myogenic satellite cells through mammalian target of rapamycin (mTOR) pathway

Myogenic satellite cells are adult stem cells and have important roles in skeletal muscle growth, repair, and regeneration. Both insulin-like growth factor-1 (IGF-1) and leucine stimulate skeletal muscle growth, which link to the activation and proliferation of myogenic satellite cells in skeletal muscle. Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis and cell proliferation. Thus, IGF-1 and leucine may stimulate activation of myogenic satellite cells through mTOR signaling. In this study, myogenic satellite cells were isolated from 6-month-old pigs and subjected to IGF-1 and leucine treatments. Both IGF-1 and leucine upregulated mTOR signaling in myogenic satellite cells. The phosphorylation of mTOR at Ser(2448) increased 83.8 +/- 7.7% by IGF-1 (P < 0.05) and 83.4 +/- 5.7% by leucine (P < 0.05). The downstream targets of mTOR, S6 kinase, and 4E-binding protein 1 (4EBP1) were also phosphorylated due to IGF-1 and leucine treatments. Treatment with IGF-1 and leucine induced the phosphorylation of tuburin (TSC2), a key mediator upstream of mTOR signaling, by 272.8 +/- 26.4% and 94.2 +/- 28.7%, respectively. Treatment of cells with both IGF-1 and leucine did not show synergistic effect on mTOR signaling. Inhibition of mTOR by rapamycin abolished the protein synthesis and cell proliferation stimulated by both IGF-1 and leucine. In summary, our data showed that in preliminary cultured myogenic satellite cells mTOR signaling was activated due to IGF-1 and leucine treatments, and this mTOR activation is necessary for the activation of myogenic satellite cells.     

Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs

Leucine is unique among the amino acids in its ability to promote protein synthesis by activating translation initiation via the mammalian target of rapamycin (mTOR) pathway. Previously, we showed that leucine infusion acutely stimulates protein synthesis in fast-twitch glycolytic muscle of neonatal pigs but this response cannot be maintained unless the leucine-induced fall in amino acids is prevented. To determine whether leucine can stimulate protein synthesis in muscles of different fiber types and in visceral tissues of the neonate in the long-term if baseline amino acid concentrations are maintained, overnight fasted neonatal pigs were infused for 24 h with saline, leucine (400 μmol kg⁻¹ h⁻¹), or leucine with replacement amino acids to prevent the leucine-induced hypoaminoacidemia. Changes in the fractional rate of protein synthesis and activation of mTOR, as determined by eukaryotic initiation factor 4E binding protein (4E-BP1) and S6 kinase 1 (S6K1) phosphorylation, in the gastrocnemius and masseter muscles, heart, liver, jejunum, kidney, and pancreas were measured. Leucine increased mTOR activation in the gastrocnemius and masseter muscles, liver, and pancreas, in both the absence and presence of amino acid replacement. However, protein synthesis in these tissues was increased only when amino acids were infused to maintain baseline levels. There were no changes in mTOR signaling or protein synthesis in the other tissues we examined. Thus, long-term infusion of leucine stimulates mTOR signaling in skeletal muscle and some visceral tissues but the leucine-induced stimulation of protein synthesis in these tissues requires sustained amino acid availability.     

Nutrient-sensing mTOR-mediated pathway regulates leptin production in isolated rat adipocytes

Leptin biosynthesis in adipose cells in vivo is increased by food intake and decreased by food deprivation. However, the mechanism that couples leptin production to food intake remains unknown. We found that addition of leucine to isolated rat adipocytes significantly increased leptin production by these cells, suggesting that postprandial leptin levels may be directly regulated by dietary leucine. The effect of leucine was inhibited by rapamycin and not by actinomycin D. Besides, leucine administration did not increase the amount of leptin mRNA in adipocytes. Therefore, we concluded that leucine activates leptin expression in adipose cells at the level of translation via a mammalian target of rapamycin (mTOR)-mediated pathway. Because leptin is a secreted protein, its biosynthesis is compartmentalized on the endoplasmic reticulum. To analyze mTOR signaling in this subcellular fraction, we separated adipose cells by centrifugation into a heavy membrane fraction that includes virtually all endoplasmic reticulum and the cytosolic extract. Phosphorylation of the major mTOR targets, the ribosomal protein S6 and the translational inhibitor 4E-binding protein (BP)/phosphorylated heat- and acid-stable protein (PHAS)-1, was stimulated by leucine in the cytosolic extract, whereas, in the heavy fraction, S6 was constitutively phosphorylated and leucine only induced phosphorylation of 4E-BP/PHAS-1. We also found that 60-70% of leptin mRNA was stably associated with the heavy fraction, and leucine administration did not change the ratio between compartmentalized and free cytoplasmic leptin mRNA. We suggest that, in adipose cells, a predominant part of leptin mRNA is compartmentalized on the endoplasmic reticulum, and leucine activates translation of these messages via the mTOR/4E-BP/PHAS-1-mediated signaling pathway.     

Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4

Obese women have an increased risk to deliver large babies. However, the mechanisms underlying fetal overgrowth in these pregnancies are not well understood. Obese pregnant women typically have elevated circulating lipid levels. We tested the hypothesis that fatty acids stimulate placental amino acid transport, mediated via toll-like receptor 4 (TLR4) and mammalian target of rapamycin (mTOR) signaling pathways. Circulating NEFA levels and placental TLR4 expression were assessed in women with varying prepregnancy body mass index (BMI). The effects of oleic acid on system A and system L amino acid transport, and on the activation of the mTOR (4EBP1, S6K1, rpS6), TLR4 (IĸBɑ, JNK, p38 MAPK), and STAT3 signaling pathways were determined in cultured primary human trophoblast cells. Maternal circulating NEFAs (n = 33), but not placental TLR4 mRNA expression (n = 16), correlated positively with BMI (P < 0.05). Oleic acid increased trophoblast JNK and STAT3 phosphorylation (P < 0.05), whereas mTOR activity was unaffected. Furthermore, oleic acid doubled trophoblast system A activity (P < 0.05), without affecting system L activity. siRNA-mediated silencing of TLR4 expression prevented the stimulatory effect of oleic acid on system A activity. Our data suggest that maternal fatty acids can increase placental nutrient transport via TLR4, thereby potentially affecting fetal growth.     

A role for hypothalamic malonyl-CoA in the control of food intake

The cellular level of malonyl-CoA, an intermediate in fatty acid biosynthesis, depends on its rate of synthesis catalyzed by acetyl-CoA carboxylase relative to its rate of utilization and degradation catalyzed by fatty acid synthase and malonyl-CoA decarboxylase, respectively. Recent evidence suggests that hypothalamic malonyl-CoA functions in the regulation of feeding behavior by altering the expression of key orexigenic and anorexigenic neuropeptides. Here we report that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a 5'-AMP kinase activator, rapidly lowers malonyl-CoA both in GT1-7 hypothalamic neurons and in the hypothalami of mice. These effects correlate closely with the phosphorylation of acetyl-CoA carboxylase, an established target of AMP kinase. Intracerebroventricular (i.c.v.) administration of AICAR rapidly lowers hypothalamic [malonyl-CoA] and increases food intake. Expression of an adenoviral cytosolic malonyl-CoA decarboxylase vector (Ad-cMCD) in hypothalamic GT1-7 cells decreases malonyl-CoA. When delivered by bilateral stereotaxic injection into the ventral hypothalamus (encompassing the arcuate nucleus) of mice, Ad-cMCD increases food intake and body weight. Ad-MCD delivered into the ventral hypothalamus also reverses the rapid suppression of food intake caused by i.c.v.-administered C75, a fatty acid synthase inhibitor that increases hypothalamic [malonyl-CoA]. Taken together these findings implicate malonyl-CoA in the hypothalamic regulation of feeding behavior.     

Effect of leptin on hypothalamic GLP-1 peptide and brain-stem pre-proglucagon mRNA

Leptin, the adipocyte-derived plasma hormone, and CNS GLP-1 neurons reduce food intake and body weight. GLP-1 is produced in the CNS by post-translational processing of pre-proglucagon. ICV leptin administration prevented the reduction in hypothalamic GLP-1 peptide content seen in pair-fed food-restricted rats (P < 0.05). There was a significant overall positive correlation between pre-proglucagon mRNA expression in the NTS and hypothalamic GLP-1 peptide content (r = +0.34, P < 0.05). Intraperitoneal leptin administration also increased hypothalamic GLP-1 peptide in food-restricted mice (P < 0. 05). This supports the hypothesis that the anorectic actions of leptin are in part due to stimulation of GLP-1 neurons. Reduced CNS GLP-1 neuronal activity during food deprivation may act to stimulate feeding behaviour, and perhaps also inhibit hypothalamic LHRH neurons, as part of the neuroendocrine response to starvation.     

Nutritionally essential amino acids and metabolic signaling in aging

Aging is associated with a gradual decline in skeletal muscle mass and strength leading to increased risk for functional impairments. Although basal rates of protein synthesis and degradation are largely unaffected with age, the sensitivity of older muscle cells to the anabolic actions of essential amino acids appears to decline. The major pathway through which essential amino acids induce anabolic responses involves the mammalian target of rapamycin (mTOR) Complex 1, a signaling pathway that is especially sensitive to regulation by the branched chain amino acid leucine. Recent evidence suggests that muscle of older individuals require increasing concentrations of leucine to maintain robust anabolic responses through the mTOR pathway. While the exact mechanisms for the age-related alterations in nutritional signaling through the mTOR pathway remain elusive, there is increasing evidence that decreased sensitivity to insulin action, reductions in endothelial function, and increased oxidative stress may be underlying factors in this decrease in anabolic sensitivity. Ensuring adequate nutrition, including sources of high quality protein, and promoting regular physical activity will remain among the frontline defenses against the onset of sarcopenia in older individuals.     

Glutamine is a key regulator for amino acid-controlled cell growth through the mTOR signaling pathway in rat intestinal epithelial cells

Amino acids, especially branched-chain amino acids such as l-leucine, have been shown to regulate activation of p70 S6 kinase and phosphorylation of 4E-BP1 through the mTOR signaling pathway. In our recent study, l-arginine was also shown to activate the mTOR signaling pathway in rat intestinal epithelial cells. l-Glutamine is an amino acid that is required for culturing of numerous cell types, including rat intestinal epithelial cells. In this study, we showed that l-glutamine inhibited the activation of p70 S6 kinase and phosphorylation of 4E-BP1 induced by arginine or leucine in rat intestinal epithelial cells. Although the molecular mechanism of l-glutamine-induced inhibition of the mTOR signaling pathway is still unknown, the presence of this novel signal pathway may indicate that individual amino acids play specific roles for cellular proliferation and growth.     

FoxO1对下丘脑摄食中枢的影响研究进展

下丘脑是人体的摄食中枢,它通过抑制食欲的阿黑皮素原(POMC)神经元和促进食欲的神经肽相关蛋白(AgRP)神经元调节摄食及能量代谢。叉头转录因子O亚族1(FoxO1)是胰岛素信号通路和瘦素信号通路中重要的调节蛋白,FoxO1的生理作用是促进下丘脑Agrp基因表达、抑制Pomc基因表达,抑制瘦素信号通路的转录激活因子3(STAT3)蛋白对Pomc基因转录的促进作用,从而促进食欲。瘦素和胰岛素均可激活经典的IRS/PI(3)K/Akt信号通路,使FoxO1磷酸化失去活性,抑制食欲。此外,沉默信息调节因子Sirt1也可以通过去乙酰化,影响FoxO1的转录活性。本文综述了胰岛素、瘦素、Sirt1通过FoxO1调节下丘脑摄食中枢的作用机制。