Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine’s action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) complex-1 (mTORC1). Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine (400 μmol·kg−1·h−1) for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) phosphorylation in gastrocnemius and masseter muscles (P < 0.05), but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTORC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of eIF2α and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation.
leucine; mTORC1; neonatal pigs; rapamycin; skeletal muscle
Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine's action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) complex-1 (mTORC1). Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine (400 μmol·kg-1·h-1) for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) phosphorylation in gastrocnemius and masseter muscles (P < 0.05), but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTORC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of eIF2α and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation.
leucine; mTORC1; neonatal pigs; rapamycin; skeletal muscle
Branched-chain amino acids, such as leucine and glucose, stimulate protein synthesis and increase the phosphorylation and activity of the mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase (p70S6K). We examined in skeletal muscle whether the effects of leucine and glucose on these parameters and on insulin resistance are mediated by the fuel-sensing enzyme AMP-activated protein kinase (AMPK).
RESEARCH DESIGN AND METHODS
Rat extensor digitorum longus (EDL) muscle was incubated with different concentrations of leucine and glucose with or without AMPK activators. Muscle obtained from glucose-infused rats was also used as a model.
In the EDL, incubation with 100 or 200 μmol/l leucine versus no added leucine suppressed the activity of the α2 isoform of AMPK by 50 and 70%, respectively, and caused concentration-dependent increases in protein synthesis and mTOR and p70S6K phosphorylation. Very similar changes were observed in EDL incubated with 5.5 or 25 mmol/l versus no added glucose and in muscle of rats infused with glucose in vivo. Incubation of the EDL with the higher concentrations of both leucine and glucose also caused insulin resistance, reflected by a decrease in insulin-stimulated Akt phosphorylation. Coincubation with the AMPK activators AICAR and α-lipoic acid substantially prevented all of those changes and increased the phosphorylation of specific sites of mTOR inhibitors raptor and tuberous sclerosis complex 2 (TSC2). In contrast, decreases in AMPK activity induced by leucine and glucose were not associated with a decrease in raptor or TSC2 phosphorylation.
The results indicate that both leucine and glucose modulate protein synthesis and mTOR/p70S6 and insulin signaling in skeletal muscle by a common mechanism. They also suggest that the effects of both molecules are associated with a decrease in AMPK activity and that AMPK activation prevents them.
The postprandial rise in amino acids and insulin independently stimulate protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to amino acids. We have shown that the postprandial rise in leucine, but not isoleucine or valine, acutely stimulates muscle protein synthesis in piglets. Leucine increases muscle protein synthesis by modulating the activation of mammalian target of rapamycin complex 1 (mTORC1) and signaling components of translation initiation. Leucine increases the phosphorylation of mTOR, 70-kDa ribosomal protein S6 kinase-1 (S6K1), eukaryotic initiation factor (eIF) 4E-binding protein-1 (4EBP1), and eIF4G, decreases eIF2α phosphorylation, and increases the association of eIF4E with eIF4G. However, leucine does not affect the upstream activators of mTOR, that is, protein kinase B (PKB), adenosine monophosphate (AMP)-activated protein kinase (AMPK), and tuberous sclerosis complex 1/2 (TSC1/2), or the activation translation of elongation regulator, eukaryotic elongation factor 2 (eEF2). Leucine’s action can be replicated by α-ketoisocaproate (KIC) but not by norleucine. Interference by rapamycin with the raptor-mTOR interaction blocks leucine-induced muscle protein synthesis. The acute leucine-induced stimulation of muscle protein synthesis is not maintained for prolonged periods, despite continued activation of mTOR signaling, because circulating amino acids fall as they are utilized for protein synthesis. However, when circulating amino acid levels are maintained, the leucine-induced stimulation of muscle protein synthesis is maintained for prolonged periods. Thus, leucine acts as a nutrient signal to stimulate translation initiation but whether this translates into a prolonged increase in protein synthesis depends on the sustained availability of all amino acids.
amino acids; leucine; muscle; newborn; pig; translation initiation
It has long been known that excesses of glucose and branched chain amino acids, such as leucine, lead to insulin resistance in skeletal muscle. A recent study in incubated rat muscle suggests that both molecules may do so by virtue of their ability to downregulate the fuel sensing and signaling enzyme AMP-activated protein kinase (AMPK) and activate mTOR/p70S6 kinase (p70S6K) signaling. The results also demonstrated that inhibition of mTOR/p70S6K with rapamycin prevented the development of insulin resistance but had no effect on AMPK activity (Thr172 phosphorylation of its catalytic subunit). In contrast, activation of AMPK by both AICAR and α-lipoic acid led to the phosphorylation of specific molecules that diminished both mTOR/p70S6K signaling and insulin resistance. These findings suggest that downregulation of AMPK precedes mTOR/p70S6K activation in mediating glucose and leucine-induced insulin resistance, although the mechanism by which it does so remains to be determined. Also requiring study is how an excess of the two nutrients leads to AMPK downregulation.
insulin resistance; AMP-activated protein kinase; mTOR/p70S6K; protein synthesis; branched chain amino acids
We recently showed that resistance exercise and ingestion of essential amino acids with carbohydrate (EAA+CHO) can independently stimulate mammalian target of rapamycin (mTOR) signaling and muscle protein synthesis in humans. Providing an EAA+CHO solution postexercise can further increase muscle protein synthesis. Therefore, we hypothesized that enhanced mTOR signaling might be responsible for the greater muscle protein synthesis when leucine-enriched EAA+CHOs are ingested during postexercise recovery. Sixteen male subjects were randomized to one of two groups (control or EAA+CHO). The EAA+CHO group ingested the nutrient solution 1 h after resistance exercise. mTOR signaling was assessed by immunoblotting from repeated muscle biopsy samples. Mixed muscle fractional synthetic rate (FSR) was measured using stable isotope techniques. Muscle protein synthesis and 4E-BP1 phosphorylation during exercise were significantly reduced (P < 0.05). Postexercise FSR was elevated above baseline in both groups at 1 h but was even further elevated in the EAA+CHO group at 2 h postexercise (P < 0.05). Increased FSR was associated with enhanced phosphorylation of mTOR and S6K1 (P < 0.05). Akt phosphorylation was elevated at 1 h and returned to baseline by 2 h in the control group, but it remained elevated in the EAA+CHO group (P < 0.05). 4E-BP1 phosphorylation returned to baseline during recovery in control but became elevated when EAA+CHO was ingested (P < 0.05). eEF2 phosphorylation decreased at 1 and 2 h postexercise to a similar extent in both groups (P < 0.05). Our data suggest that enhanced activation of the mTOR signaling pathway is playing a role in the greater synthesis of muscle proteins when resistance exercise is followed by EAA+CHO ingestion.
muscle protein synthesis; mammalian target of rapamycin; essential amino acids
Insulin like growth factor-1 (IGF-1) is established as an anabolic factor that can induce skeletal muscle growth through activating the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. While this signaling pathway has been heavily studied, the molecular mechanisms linking IGF-1 binding to mTOR activation are still poorly defined in muscle. The purpose of this study was to test the hypothesis that IGF-1 activation of mTOR in C2C12 myotubes requires a phosphorylation dependent, altered distribution of the tuberous sclerosis complex (TSC)1/TSC2 complex from the membrane to the cytosol. We found that IGF-1 treatment does not affect complex formation between TSC1 and TSC2, but rather IGF-1 induces an altered distribution of the TSC1/TSC2 complex in C2C12 myotubes. In response to IGF-1 treatment, there was a relative re-distribution of the TSC1/TSC2 complex, composed of TSC1 and phosphorylated TSC2, from the membrane to the cytosol. IGF-1 stimulated TSC1/TSC2 phosphorylation and re-distribution were completely prevented by the PI3K inhibitor wortmannin, but were not with the downstream mTOR inhibitor, rapamycin. When a non-phosphorylatable form of TSC2 (S939A) was overexpressed, phosphorylation-dependent binding of the scaffold protein 14-3-3 to TSC2 was diminished and no re-distribution of the TSC1/TSC2 complex was observed following IGF-1 stimulation. These results indicate that TSC2 phosphorylation in response to IGF-1 treatment is necessary for the altered distribution of the TSC1/TSC2 complex to the cytosol and we suggest that this translocation is likely critical for mTOR activation by dissociating the interaction between the GAP activity of the TSC1/TSC2 complex and its downstream target Rheb.
mTOR; TSC1; TSC2; wortmannin; rapamycin
Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.
Leucine, a the branched-chain amino acids that must be supplied in daily diet, plays an important role in controlling protein synthesis and regulating cell metabolism in various cell types. In pancreatic β cells, leucine acutely stimulates insulin secretion by serving as both metabolic fuel and allosteric activator of glutamate dehydrogenase to enhance glutaminolysis. Leucine has also been shown to regulate gene transcription and protein synthesis in pancreatic islet β cells via both mTOR-dependent and -independent pathways at physiological concentrations. Long-term treatment of leucine has been shown to improve insulin secretory dysfunction of human diabetic islets via upregulation of certain key metabolic genes. In vivo, leucine administration improves glycemic control in humans and rodents with type 2 diabetes. This review aims to summarize and discuss the recent findings regarding the effects of leucine metabolism on pancreatic β cell function.
leucine; glutamate dehydrogenase; mTOR; ATP synthase
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.
Growth; Muscle; Translation initiation; Mammalian target of rapamycin; Eukaryotic initiation factor 4E binding protein; Ribosome protein S6 kinase
Leucine acts as a signal nutrient in promoting protein synthesis in skeletal muscle and adipose tissue via mTOR pathway activation, and may be of interest in age-related sarcopenia. However, hyper-activation of mTOR/S6K1 has been suggested to inhibit the first steps of insulin signaling and finally promote insulin resistance. The impact of long-term dietary leucine supplementation on insulin signaling and sensitivity was investigated in old rats (18 months old) fed a 15% protein diet supplemented (LEU group) or not (C group) with 4.5% leucine for 6 months. The resulting effects on muscle and fat were examined. mTOR/S6K1 signaling pathway was not significantly altered in muscle from old rats subjected to long-term dietary leucine excess, whereas it was increased in adipose tissue. Overall glucose tolerance was not changed but insulin-stimulated glucose transport was improved in muscles from leucine-supplemented rats related to improvement in Akt expression and phosphorylation in response to food intake. No change in skeletal muscle mass was observed, whereas perirenal adipose tissue mass accumulated (+45%) in leucine-supplemented rats. A prolonged leucine supplementation in old rats differently modulates mTOR/S6K pathways in muscle and adipose tissue. It does not increase muscle mass but seems to promote hypertrophy and hyperplasia of adipose tissue that did not result in insulin resistance.
Leucine supplementation; Sarcopenia; Muscle; Adipose tissue; Insulin signaling
mTOR is a major actor of skeletal muscle mass regulation in situations of atrophy or hypertrophy. It is established that Phospholipase D (PLD) activates mTOR signaling, through the binding of its product phosphatidic acid (PA) to mTOR protein. An influence of PLD on muscle cell size could thus be suspected. We explored the consequences of altered expression and activity of PLD isoforms in differentiated L6 myotubes. Inhibition or down-regulation of the PLD1 isoform markedly decreased myotube size and muscle specific protein content. Conversely, PLD1 overexpression induced muscle cell hypertrophy, both in vitro in myotubes and in vivo in mouse gastrocnemius. In the presence of atrophy-promoting dexamethasone, PLD1 overexpression or addition of exogenous PA protected myotubes against atrophy. Similarly, exogenous PA protected myotubes against TNFα-induced atrophy. Moreover, the modulation of PLD expression or activity in myotubes showed that PLD1 negatively regulates the expression of factors involved in muscle protein degradation, such as the E3-ubiquitin ligases Murf1 and Atrogin-1, and the Foxo3 transcription factor. Inhibition of mTOR by PP242 abolished the positive effects of PLD1 on myotubes, whereas modulating PLD influenced the phosphorylation of both S6K1 and Akt, which are respectively substrates of mTORC1 and mTORC2 complexes. These observations suggest that PLD1 acts through the activation of both mTORC1 and mTORC2 to induce positive trophic effects on muscle cells. This pathway may offer interesting therapeutic potentialities in the treatment of muscle wasting.
Phospholipase D; Phosphatidic acid; Muscle wasting; Muscle hypertrophy; Myotubes; mTOR
The insulin/IGF signalling (IIS) cascade has an important role in regulating normal development and physiology, as evidenced by its effects in a host of major human diseases including cancer, Type II diabetes and neurodegeneration. Recently it has become clear that multiple types of local nutrient sensing mechanism impact on cellular insulin sensitivity through the downstream kinase TOR (target of rapamycin). In vivo analysis in flies has surprisingly highlighted proton-assisted amino acid transporters as having a uniquely potent role in regulating IIS/TOR activity and growth, potentially via a novel signalling mechanism. Other molecules such as the heterodimeric amino acid transporter, CD98, which provides the principal route for cellular uptake of leucine, an amino acid implicated in regulating TOR, also appear to have important effects. As our understanding of how nutrient sensing impacts on IIS/TOR increases, novel targets to modulate aberrant IIS in disease are likely to emerge, which could complement current strategies designed to block kinases in this pathway.
cancer; nutrient sensing; transceptor; transporters; Drosophila
Chlamydiaceae are obligate intracellular bacterial pathogens that strictly depend on host metabolites, such as nucleotides, lipids, and amino acids. Depletion of amino acids in cell culture media results in abnormal chlamydial development in vitro. Surprisingly, enrichment of certain amino acids also retards chlamydial growth. Our experiments revealed that the antichlamydial effects are largely independent of changes in the host cell transcriptome or proteome and in the major signal transduction pathway modulated by amino acids, the mTOR (mammalian target of rapamycin) pathway. Furthermore, the chlamydial growth inhibition induced by leucine, isoleucine, methionine, or phenylalanine was completely reversed by concomitant addition of valine. In contrast, the growth inhibition induced by serine, glycine, or threonine was not reversed by valine addition. Functional characterization of the only predicted chlamydial transporter for branched-chain amino acids, BrnQ, revealed that it can be blocked by leucine, isoleucine, methionine, or phenylalanine but not by serine, glycine, or threonine. This chlamydial transporter is the only known BrnQ homolog possessing specificity for methionine, suggesting a unique strategy for methionine uptake among gram-negative bacteria. The antichlamydial effects of leucine, isoleucine, methionine, and phenylalanine could be explained as competitive inhibition of the BrnQ transporter and subsequent valine starvation.
Elevations in branched chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, the first two enzymatic steps catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm), branched chain α-keto acid dehydrogenase (BCKD, E1α subunit) complex were assessed in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293 or total BCKD E1α or BCKD kinase, in liver, BCKD E1-α was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1α. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue, but not liver. BCAAs were also reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1α in omental and subcutaneous fat. Our results are consistent with the idea that tissue specific alterations in BCAA metabolism, in liver and adipose tissue but not muscle, may contribute to the rise in plasma BCAAs in obesity.
Obesity; branched chain amino acids; mitochondrial branched chain amino acid transaminase (BCATm); branched chain keto acid dehydrogenase; branched chain keto acid dehydrogenase kinase; ob/ob mice; diet induced obesity; Zucker rats; bariatric surgery; humans
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that exquisitely regulates protein metabolism in skeletal muscle. mTOR integrates input from amino acids, growth factors and intracellular cues to make or break muscle protein. mTOR accomplishes this task by stimulating the phosphorylation of substrates that control protein translation while simultaneously inhibiting proteasomal and autophagic protein degradation. In a metabolic twist of fate, sepsis induces muscle atrophy in part by the aberrant regulation of mTOR. In this review we track the steps of normal mTOR signaling in muscle and examine where they go astray in sepsis and inflammation.
Insulin regulates both glucose uptake and postnatal cardiac growth. The anabolic effects of insulin are mediated by the mammalian target of rapamycin (mTOR), an evolutionarily conserved kinase which is also a convergence point between nutrient sensing and cell growth. We postulated that mTOR signalling in the heart requires the metabolism of glucose.
We interrogated the insulin-mediated mTOR signalling pathway in response to different metabolic interventions regulating substrate metabolism in the isolated working rat heart and in isolated cardiomyocytes.
Although insulin enhanced Akt activity, phosphorylation of mTOR and its downstream targets (p70S6K and 4EBP1) required the addition of glucose. Glucose-dependent p70S6K phosphorylation was independent of the hexosamine biosynthetic pathway, the AMP kinase pathway, and the pentose phosphate pathway. However, inhibition of glycolysis downstream of hexokinase markedly enhanced p70S6K phosphorylation. Furthermore, 2-deoxyglucose activated p70S6K suggesting that phosphorylation of glucose is required for carbohydrate-mediated mTOR signalling in the heart. Lastly, we also found enhanced p70S6K phosphorylation in the hearts of diabetic rats.
Phosphorylation of glucose is necessary for insulin-dependent mTOR activity in the heart, suggesting a link between intermediary metabolism and cardiac growth.
Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.
ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and muscle ring finger protein 1 (MuRF1); and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1), FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2 = 0.36, p<0.05) between insulin infusion dose and phosphorylated Akt was demonstrated.
We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.
mTOR Complex 1 (mTORC1; mammalian target of rapamycin (mTOR) in complex with raptor) is a key regulator of protein synthesis and cell growth in response to nutrient amino acids. Here we report that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid kinase activities, regulates amino acid signaling to mTORC1. This regulation is independent of IPMK's catalytic function, instead reflecting its binding with mTOR and raptor, which maintains the mTOR-raptor association. Thus, IPMK appears to be a physiologic mTOR cofactor, serving as a determinant of mTORC1 stability and amino acid-induced mTOR signaling. Substances that block IPMK-mTORC1 binding may afford therapeutic benefit in nutrient amino acid-regulated conditions such as obesity and diabetes.
The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, acts as a “master switch” for cellular anabolic and catabolic processes, regulating the rate of cell growth and proliferation. Dysregulation of the mTOR signaling pathway occurs frequently in a variety of human tumors, and thus, mTOR has emerged as an important target for the design of anticancer agents. mTOR is found in two distinct multiprotein complexes within cells, mTORC1 and mTORC2. These two complexes consist of unique mTOR-in teracting proteins and are regulated by different mechanisms. Enormous advances have been made in the development of drugs known as mTOR inhibitors. Rapamycin, the first defined inhibitor of mTOR, showed effectiveness as an anticancer agent in various preclinical models. Rapamycin analogues (rapalogs) with better pharmacologic properties have been developed. However, the clinical success of rapalogs has been limited to a few types of cancer. The discovery that mTORC2 directly phosphorylates Akt, an important survival kinase, adds new insight into the role of mTORC2 in cancer. This novel finding prompted efforts to develop the second generation of mTOR inhibitors that are able to target both mTORC1 and mTORC2. Here, we review the recent advances in the mTOR field and focus specifically on the current development of the second generation of mTOR inhibitors as anticancer agents.
mTOR; inhibitor; rapamycin; rapalogs; cancer
The loss of muscle mass and strength with aging (i.e., sarcopenia) has a negative effect on functional independence and overall quality of life. One main contributing factor to sarcopenia is the reduced ability to increase skeletal muscle protein synthesis in response to habitual feeding, possibly due to a reduction in postprandial insulin release and an increase in insulin resistance. Branched-chain amino acids (BCAA), primarily leucine, increases the activation of pathways involved in muscle protein synthesis through insulin-dependent and independent mechanisms, which may help counteract the “anabolic resistance” to feeding in older adults. Leucine exhibits strong insulinotropic characteristics, which may increase amino acid availability for muscle protein synthesis, reduce muscle protein breakdown, and enhance glucose disposal to help maintain blood glucose homeostasis.
amino acids; leucine; skeletal muscle; protein synthesis; insulin secretion
Insulin-like growth factor-I (IGF-I) is a major anabolic hormone for skeletal muscle and a potent stimulus for protein synthesis and translation initiation. Recent studies suggest that translation can be inhibited by over expression of the mammalian target of rapamycin (mTOR) repressor REDD1. The purpose of the present study was to determine whether IGF-I alters the expression of REDD1 and whether this is associated with a concomitant change in protein synthesis in vitro. Subcutaneous injection of IGF-I or intravenous delivery of insulin for 3–4 h increased REDD1 mRNA in skeletal muscle 7- to10-fold. A 3-fold increase in REDD1 was observed when C2C12 myotubes were treated with IGF-I. REDD1 protein continued to be expressed for up to 24 h after addition of IGF-I to cells. Withdrawal of IGF-I from myotubes lead to a rapid loss of REDD1 protein content. IGF-I-induced REDD1 mRNA and protein expression were prevented by inhibitors of transcription and translation. IGF-I had an additive effect with dexamethasone (Dex) on REDD1 protein content in myotubes. The PI3K inhibitor LY294002 blocked IGF-I but not Dex induced REDD1. IGF-I also stimulated REDD1 promoter activity. Although REDD1 protein was elevated 5–6 h after addition of IGF-I to myotubes, protein synthesis measured during this 1 h window was paradoxically greater in myotubes expressing more REDD1. In contrast to the IGF-I induced increase in REDD1 mRNA, REDD2 mRNA was decreased by IGF-I. We conclude that IGF-I stimulates REDD1 expression in skeletal muscle and myotubes but under these conditions the REDD1 response is not sufficient to repress protein synthesis.
Skeletal Muscle; IGF-I; C2C12 Myotubes; Protein Synthesis
Amino acids (AA) activate the mammalian target of rapamycin (mTOR) signaling pathway but overactivation has a negative feedback effect on insulin signaling which may lead to insulin resistance and type 2 diabetes (T2DM). Purpose: To determine the effect of reduced AA concentrations on mTOR and insulin signaling during increased nutrient and insulin availability.
Six control and six T2DM subjects were studied at baseline and following a 5h AA lowering high energy and insulin clamp. Stable isotopic techniques in combination with femoral catheterizations were used to measure AA kinetics across the leg while muscle biopsies were used to measure mTOR and insulin signaling proteins using immunoblotting techniques.
AA concentrations decreased by ~30–60% in both groups (P<0.05). Phospho-mTOR, S6K1, eEF2, and eIF2α were unchanged in both groups following the clamp (P>0.05). In T2DM subjects, IRS-1 serine phosphorylation was unchanged while phospho-AMPKα decreased and phospho-Akt, phospho-AS160 and glucose uptake increased following the clamp (P<0.05). In comparison, AA concentrations were maintained in a separate group during an insulin infusion. In this group, phospho-Akt, mTOR and S6K1 (n=4) increased.
Amino acids are necessary for insulin-induced activation of mTOR signaling and protein synthesis in both healthy and insulin resistant skeletal muscle.
mammalian target of rapamycin; muscle protein synthesis; amino acid availability; adenosine 5′-monophosphate-activated protein kinase
Since in vitro study of Buse and Reid in 1975 showing a stimulatory effect of leucine upon rat muscle protein synthesis and reduction in proteolysis, a similar effect has been sought in humans. Sherwin demonstrated in humans in 1978 an improvement in N balance with infusion of leucine in obese subjects fasting to lose weight. A variety of subsequent studies have been performed in humans where leucine alone or the branched-chain amino acids (BCAAs) have been administered in varying amounts and durations and the effect upon protein metabolism has been measured. Measurement of changes in muscle amino acid metabolism were made by arterio-venous difference measurements and by biopsies. An anabolic effect of leucine and the BCAAs on reduction of muscle protein breakdown was found in these studies with no measured effect upon muscle protein synthesis. Later studies using stable isotope tracers to define both whole body protein turnover and leg or arm protein metabolism have similarly concluded that leucine administration specifically induces a reduction in protein breakdown without increasing protein synthesis. This anabolic effect produced through a reduction of protein breakdown in vivo in humans by leucine is contrary to in vitro studies of rat muscle where stimulation of protein synthesis has been demonstrated by leucine. Likewise an increase in protein synthesis has also been demonstrated by insulin in rat muscle that is not seen in humans. Of the various studies administering BCAAs or leucine to humans for varying periods of time and amount, the results have been consistent. In addition no untoward effects have been reported in any of these studies from infusion of the BCAAs at upwards three-times basal flux or six-times normal dietary intake during the fed portion of the day.
protein synthesis; protein breakdown; leucine
RAFT1/FRAP/mTOR is a key regulator of cell growth and division and the mammalian target of rapamycin, an immunosuppressive and anticancer drug. Rapamycin deprivation and nutrient deprivation have similar effects on the activity of S6 kinase 1 (S6K1) and 4E-BP1, two downstream effectors of RAFT1, but the relationship between nutrient- and rapamycin-sensitive pathways is unknown. Using transcriptional profiling, we show that, in human BJAB B-lymphoma cells and murine CTLL-2 T lymphocytes, rapamycin treatment affects the expression of many genes involved in nutrient and protein metabolism. The rapamycin-induced transcriptional profile is distinct from those induced by glucose, glutamine, or leucine deprivation but is most similar to that induced by amino acid deprivation. In particular, rapamycin treatment and amino acid deprivation up-regulate genes involved in nutrient catabolism and energy production and down-regulate genes participating in lipid and nucleotide synthesis and in protein synthesis, turnover, and folding. Surprisingly, however, rapamycin had effects opposite from those of amino acid starvation on the expression of a large group of genes involved in the synthesis, transport, and use of amino acids. Supported by measurements of nutrient use, the data suggest that RAFT1 is an energy and nutrient sensor and that rapamycin mimics a signal generated by the starvation of amino acids but that the signal is unlikely to be the absence of amino acids themselves. These observations underscore the importance of metabolism in controlling lymphocyte proliferation and offer a novel explanation for immunosuppression by rapamycin.