Recent progress in molecular medicine has identified the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1) as the central regulator of protein and lipid synthesis, cell growth, proliferation, energy metabolism and autophagy. Age-related diseases of Western civilization such as obesity, diabetes mellitus, neurodegenerative diseases, and cancer are associated with enhanced mTORC1 signaling. According to the current opinion, metformin’s primary mode of action is the alteration of cellular energy metabolism stimulating 5-AMP-Activated Protein Kinase (AMPK). However, the notion that AMPK primarily mediates metformin´s anti-hyperglycemic action has recently been challenged, thrusting AMPK-independent effects into the focus of interest. We provide a new viewpoint on metformin´s mode of action as an inhibitor of mTORC1. Metformin´s insulin-lowering and AMPK-activating effects decrease RHEB-mediated stimulation of mTORC1. Independent of AMPK metformin inhibits mTORC1 in a RAG GTPase-dependent manner. Thus, metformin interferes with the two major pathways required for mTORC1 activation: 1) energy- and cell stress-mediated activation of AMPK attenuating the activity of the GTPase RHEB and 2) suppression of amino acid signaling down-regulating the activity of lysosomal RAG GTPases. Both RHEB- and RAG GTPase activation, which are required for mTORC1 activation at the lysosomal membrane, are thus suppressed by metformin. Metformin-induced suppression of mTORC1 subsequently decreases S6K1 activity and S6K1-mediated insulin resistance as well as AKT-FoxO1-mediated hepatic gluconeogenesis. Metformin represents an ideal, save and cheap drug targeting the pathogenesis of mTORC1-driven anabolic and hyperproliferative diseases of civilization.

Melnik BC, Schmitz G (2014) Metformin: an Inhibitor of mTORC1 Signaling. J Endocrinol Diabetes Obes 2(2): 1029.

•    AMPK
•    Diseases of civilization
•    Metformin; mTORC1
•    RHEB; RAG GT Pase

AA: Amino Acid; AAT: Amino Acid Transporter; AD: Alzheimer’s Disease; AKT: Akt Kinase (protein kinase B); AMPK: Adenosine Monophosphate-activated Protein Kinase; AS160: AKT Substrate 160kD (TBC1 domain family member 4); ATF4: Activating Transcription Factor 4; AMP: Adenosine Monophosphate; ATM: Ataxia Teleangiectasia Mutated; ATP: Adenosine Triphosphate; BCAA: Branched-Chain Amino Acid; DDIT4: DNA Damage-Inducible Transcript 4; DDR: DNA Damage Response; 4-EBP-1: Eukaryotic initiation factor (eIF) 4E-Binding Protein 1; ER: Endoplasmic Reticulum; ERK: Extracellular signal Regulated Kinase; FGF21: Fibroblast Growth Factor 21; FoxO1: Forkhead Box O1 Transcription Factor; GAP: GTPase-activating protein; GCN2: General amino acid Control-non-derepressible 2; GEF: Guanine nucleotide Exchange Factor; GDH: Glutamate Dehydrogenase; GLUT4: Glucose Transporter 4; GDP: Guanosine Diphosphate; G6Pase: Glucose-6-Phosphatase; GTP: Guanosine Triphosphate; IGF-1: Insulin-Like Growth Factor-1; IRS: Insulin Receptor Substrate; αKG: α-Ketoglutarate; LAT1: L-Type Amino Acid Transporter 1; LEL: Late Endosomes and Lysosomes; LKB1: Liver Kinase B1; Leu: Leucine; LeuRS: Leucyl-tRNA Synthase; MDM2: Mouse Double Minute 2 Homolog; MDMX: Mouse Double Minute 4 Homolog (p53-binding protein MDM4); mTORC1: Mechanistic (Mammalian) Target Of Rapamycin Complex 1; mTORC2: Mechanistic (Mammalian) Target Of Rapamycin Complex 2; OCT-1: Organic Cation Transporter-1; PAT: Proton-Assisted Amino Acid Transporter; PEPCK: Phosphoenolpyruvate Carboxykinase; PI3K: Phosphoinositol-3 Kinase; PPARγ: Peroxisome Proliferator-Activated Receptor-γ; PGC-1α: Peroxisome Proliferatator-Activated Receptor-γ Co-Activator 1α; PTEN: Phosphatase and Tensin Homolog; RAG: RAS-Related GTP-Binding Protein; RAPTOR: Regulatory-Associated Protein of mTOR; REDD1: Regulated in DNA Damage and Development 1; RHEB: RAS-Homolog Enriched in Brain; RICTOR: Rapamycin-Insensitive Companion of mTOR; ROS: Reactive Oxygen Species; RSK: Ribosomal S6 Kinase 90kD; SeP: Selenoprotein P; Ser: Serine; S6K: Ribosomal protein S6 kinase 70kD; SREBP: Sterol Response Element Binding Protein; SOCS3: Suppressor OF Cytokine Signaling 3; STAT3: Signal Transducer AND Activator of Transcription 3; TBC1D7: TBC (Tre2-Bib2-Cdc16)1 Domain family member 7; tRNA: Transfer-RNA; TSC1: Hamartin; TSC2: Tuberin; Tyr: Tyrosine; ULK-1: UNC51-Like Kinase 1; v-ATPase: Vacuolar H+ -ATPase

The Mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling pathway couples energy and nutrient abundance to anabolic metabolism by sensing and simultaneously orchestrating pivotal signals such as cell energy, cell stress, nutrient and especially amino acid availability and growth factors such as insulin and insulin-like growth factor-1 (IGF-1) (Figure 1) [1- 11]. It is the intention of this review to provide evidence that the major pharmacological action of metformin is the suppression of mTORC1.

In the past few years, the involvement of aberrant mTORC1 signaling in the onset and progression of ageing, obesity, type 2 diabetes mellitus, cancer and neurodegenerative diseases (type 3 diabetes) has been appreciated [12-18]. Enhanced mTORC1 signaling stimulates adipogenesis [19-22] and results in increased expression of the key adipogenic transcription factors peroxisome proliferator-activated receptor-γ (PPARγ) and sterol regulatory element binding transcription factor-1 (SREBP1) [10,23-27]. mTORC1 suppresses lipolysis, stimulates lipogenesis, and promotes fat storage [28], thus plays a pivotal role in adipogenesis [29]. The major mTORC1 downstream target, the ribosomal S6 kinase 1 (S6K1), plays a critical role in early adipocyte differentiation [30]. In fact, absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity [31].

Over-stimulation of mTORC1 signaling by excess food and high amino acid intake appears to be the crucial factor underlying the diabetes epidemics [12,32]. mTORC1 signaling is involved in pancreatic β-cell growth, β-cell mass regulation, insulin synthesis and secretion [33-37]. The mTORC1-S6K1 signaling axis controls, at least in part, glucose homeostasis, insulin sensitivity, adipocyte metabolism, body mass and energy balance, tissue and organ size, and aging [38,39]. Notably, S6K1-mediated phosphorylation of insulin receptor substrate-1 (IRS-1) plays a central role for the induction of insulin resistance [40-50]. Postnatal stimulation of mTORC1 by ablation of tuberosus sclerosis complex 2 (TSC2) in β-cells of mice resulted in a biphasic response with increased β-cell mass and insulin secretion in young but progressive β-cell apoptosis, insulin deficiency and hyperglycemia in adult mice [51].

mTORC1 is a master regulator of protein and lipid synthesis, nucleotide synthesis and cell cycle progression that couples nutrient availability to cell growth and cancer [52-59]. Dysregulation of multiple elements of the mTORC1 pathway (PI3K amplification/mutation, PTEN loss of function, AKT over expression, and S6K1-, 4EBP1- and eIF4E over expression) have all been reported in many types of cancer [60-66].

mTORC1 plays a role in amyloid-ß- and tau-induced neurodegeneration [67,68]. Increased phosphorylation of S6K and eIF4E in postmortem human Alzheimer´s disease (AD) brains compared to age-matched control patients suggests higher mTORC1 activity in AD brains [69-72]. Remarkably, inhibition of mTORC1 by rapamycin abolished cognitive deficits and reduced amyloid-β levels in a mouse model of AD [73]. Insufficient autophagy by increased mTORC1 activity may contribute to amyloid-β accumulation and formation of tau oligomers and insoluble aggregates, whereas appropriate autophagy enhances the clearance of soluble and aggregated forms of amyloid-β and tau proteins [74]. The frequently observed comorbidity of insulin resistance, type 2 diabetes mellitus and AD (type 3 diabetes) [75] appear to derive from a common underlying mechanism, i.e., increased mTORC1 signaling.

Aging is defined as an accumulation of cellular damage over time, promoting disease and death. Over-activated mTORC1 signaling, which suppresses autophagy [76,77], results in increased accumulation of damaged proteins and cell constituents, thereby promotes the process of aging. In fact, inhibition of mTORC1 signaling extends lifespan in yeast, worms, flies and mice [78-88].

Taken together, enhanced mTORC1 signaling appears to represent the molecular interface connecting metabolic stress with aging and age-related metabolic diseases [14,89].

According to the current opinion, attenuation of mTORC1 signaling either by dietary restriction and/or pharmacological intervention should be effective in the prevention of age-related diseases [90]. A well-tolerated and cheap drug for widespread use, which would effectively attenuate over-stimulated mTORC1 signaling, is highly desirable. In this review, we will provide evidence that the synthetic biguanide metformin (N,N-dimethylimido-dicarbonimidic diamide) perfectly corrects over-activated mTORC1 signaling. Naturally occurring biguanides originating from the French lilac (Galega officinalis) have been used for the treatment of diabetes in folk medicine for centuries. To understand the impact of metformin on mTORC1 pathways, a brief introduction into recent concepts of canonical mTORC1 signaling may be helpful.

Canonical mTORC1 Signaling

mTOR is a multi-domain protein of approximately 300 kDa exhibiting a serine/threonine protein kinase domain at its C-terminus related to phosphoinositol-3-kinases (PI3Ks). In mammalian cells two functionally different mTOR complexes exist: mTORC1 and mTORC2, respectively [1-5]. Among other functional proteins, mTORC1 contains the partner protein RAPTOR, which interacts with substrates for mTORC1-mediated phosphorylation. mTORC1 controls the G1 /S transition and G2 /M progression of the cell cycle [53]. In contrast to mTORC2, which contains the partner protein RICTOR, only mTORC1 plays a special role in sensing cellular nutrients, amino acids, energy (ATP) levels, and oxygen stress (ROS), which are all important signals for the regulation of cell growth and proliferation.

AMPK-Mediated Regulation of mTORC1

LKB1 and AMP-activated protein kinase (AMPK) are critical regulators of mTORC1 [91,92]. The serine/threonine kinase LKB1 represents the major kinase phosphorylating the AMPK activation loop (α-subunit of AMPK) under conditions of energy stress [93,94]. AMPK plays a key role in energy-dependent regulation of mTORC1. AMPK is activated during energy-deficient conditions, when AMP levels rise. AMPK phosphorylates TSC2 and RAPTOR, thereby suppressing mTORC1 [95]. AMPK-mediated inhibition of mTORC1 activates autophagy by initiating the kinase ULK-1 [96,97]. In response to glucose deprivation, hexokinase-II, which catalyzes the first step of glycolysis, binds to mTORC1 through its TOS motif, thereby decreasing mTORC1 activity [98,99].

The mTORC1 pathway is also regulated by oxidative stress that down-regulates mTORC1 signaling [1]. Ataxia Teleangiectasia Mutated (ATM) is another kinase of the PI3K family that responds to oxidative stress (ROS) and DNA damage by phosphorylating key substrates such as LKB1. This results in AMPK-mediated activation of TSC2 suppressing mTORC1 [100- 103]. ATM plays a central role in maintaining genomic stability [104,105].

Most functions of mTORC1 are inhibited by rapamycin, a triene macrolide antibiotic synthesized by Streptomyces hygroscopicus [2]. Growth factor signals (insulin and IGF-1) are integrated by the tuberous sclerosus protein TSC1 (hamartin) and TSC2 (tuberin) that regulate RHEB (RAS-homolog enriched in brain), one essential activator of mTORC1 [95,106-110]. In its GTP-bound form, RHEB directly activates mTORC1. The RHEB-specific GTPase-activating protein (GAP) is the TSC2 protein, which functions as a heterotrimer with its binding partners TSC1 and TBC1D7 [110]. Growth factor signaling via TSC2 phosphorylation reduces the inhibitory function of the TSC1/TSC2/TBC1D7 complex towards RHEB, which results in activation of RHEB and finally of mTORC1.

Amino Acid-Mediated Regulation of mTORC1

Amino acids, especially leucine, glutamine and arginine, play a most important role for the activation of mTORC1 [3-9]. Amino acids activate mTORC1 even in the absence of insulin but not vice versa [7,111,112]. The activation of mTORC1 depends on two major pathways: 1) the upstream activation of RHEB by signals derived from growth factors and 2) by the amino acid-dependent translocation of inactive mTORC1 to active RHEB localized in lysosome compartments [113-115]. Insulin and IGF-1 signaling, via activated AKT as well as other growth-related kinases such as ERK and RSK, phosphorylate TSC2 and thereby suppress the inhibitory function of the TSC1/TSC2/TBC1D7 complex towards RHEB. The TSC complex associates with the lysosome in a RHEB-dependent manner, and its dissociation in response to insulin requires AKT-mediated TSC2 phosphorylation. Loss of the PTEN tumor suppressor results in constitutive activation of mTORC1 through the AKT-dependent dissociation of the TSC complex from the lysosome [116]. These recent findings provide a unifying mechanism by which independent pathways affecting the spatial recruitment of mTORC1 and the TSC complex to RHEB at the lysosomal surface serve to integrate diverse growth signals [116]. The inhibition of either TSC1 or TSC2 leads to activation of RHEB and ultimately of mTORC1 [109,116-117].

Amino acid uptake into the cell is crucial for mTORC1 signaling. Nicklin [118] suggested that cellular export of glutamine is required for cellular leucine uptake and subsequent leucine-mediated mTORC1 activation. Intracellular glutamine is required for preloading the SLC7A5/SLC3A2 bidirectional amino acid transporter, which drives the efflux of glutamine and influx of leucine for leucine-mediated mTORC1 activation [118,119]. Remarkably, in response to amino acid depletion, mTORC1 activity is rapidly abolished [19]. Amino acid starvation even impairs binding of mTORC1 to RHEB [120].Of all essential amino acids, leucine exerts the greatest effects on mTORC1 signaling [3,4,19,111]. Notably, from all animal proteins, milk proteins provide the highest amounts of the essential branched-chain amino acids (BCAAs) leucine, isoleucine and valine [121].

Amino acids play a pivotal role in the translocation of inactive mTORC1 to lysosomal compartments enriched in activated RHEB [113,114]. The spatial regulation of inactive mTORC1 by amino acids is mediated by an active RAG heterodimer and is of crucial importance for amino acid sensing and activation of mTORC1 [122]. The pentameric Ragulator complex acts as a scaffold for the RAG GTPases and mTORC1 at the lysosomal membrane. According to the recent opinion, RHEB and RAGs come together at the lysosome to activate mTORC1 [116,123].

Amino acid accumulation in the lysosomal lumen generates an activating signal that is transmitted in a vacuolar H+ -ATPase (v-ATPase)-dependent fashion to activate the guanine nucleotide exchange factor (GEF) activity of Ragulator towards RAGA. Upon RAGA-GTP loading, mTORC1 is recruited to the lysosomal surface where it interacts with RHEB and becomes activated [122]. Thus, mTORC1 integrates insulin, IGF-1, energy-, and ROS-derived signals to RHEB. In parallel mTORC1 activation requires sufficient amino acid signals for complex assembly allowing efficient activation of mTORC1 [124].

Proton-assisted amino acid transporters (PATs) localized on late endosomes and lysosomes (LEL) interact with RAGs and are required for mTORC1 activation [125]. PAT1 (SLC36A1) is expressed at the luminal surface of the small intestine and is commonly found in lysosomes of many cell types [126]. PAT1 has a relative low affinity (Km 1-10 mM) for its substrates, which include zwitterionoic amino and imino acids, heterocyclic amino acids, and amino acid-based drugs and derivatives [126].

The v-ATPase interacts with the activated RAG/Ragulator complex to control amino acid-dependent mTORC1 activation, which is regulated by the rapid accumulation of extracellular amino acids in LELs [12,127]. Thus, in response to amino acids these molecules form a signaling complex that has been called the ‘nutrisome’ [12,125]. Cycling of protons through this nutrisomal engine induces conformational changes that may activate mTORC1. Importantly, signaling from the insulin receptor and subsequent activation of the PI3K/AKT/RHEB cascade promotes shuttling of PATs from the cell surface to LEL membranes, hence increasing PAT-dependent mTORC1 activation [125,127]. In addition, the accumulation of amino acids in the LEL lumen presumably involves transport into intracellular endosomal compartments via currently unknown amino acid transporters (AATs) or potentially via endocytosis. Cytoplasmic leucine, which is brought into cells via the heterodimeric amino acid transporter CD98 [128], has been shown to play a key role in activating mTORC1 in some cultured cells and may be important in this process. Influx of leucine or other amino acids into the LEL system may ultimately allow the amino acid substrates of PAT1 to accumulate in the LELs through amino acid exchange mechanisms, leading to PAT1-mediated activation of the nutrisome [125].

Leucyl-tRNA synthetase (LeuRS) acts as a cytosolic amino acid sensor [129,130]. LeuRS also plays a critical role in amino acid-induced mTORC1 activation by sensing intracellular leucine concentration and initiating mTORC1 activation by binding to and activating RAG GTPase [129,130]. LeuRS acts as a GTPase-activating protein (GAP) for RAGD, enhancing the GTP-bound form of RAGA/B crucial for amino acid-mediated mTORC1 activation [130]. Furthermore, Duran [131] suggested that leucine stimulates mTORC1 indirectly through its effects on glutaminolysis. Glutamine in combination with leucine increased GTP charging of exogeneously expressed RAGB, promoting mTORC1 activation by enhancing glutaminolysis and the production of α-ketoglutarate [131]. In contrast, the eIF2α (eukaryotic initiation factor 2α) kinase GCN2 (general amino acid control-non-derepressible 2) senses the absence of one or more amino acids by virtue of direct binding to uncharged cognate tRNAs [132].

These recent insights into regulation of mTORC1 underline that the activation status of AMPK and the availability of amino acids determines the magnitude of mTORC1 signaling.

Metformin by Activation of LKB1-AMPK Inhibits mTORC1

The primary target of metformin action is the liver. Hepatocytes abundantly express organic cation transporter-1 (OCT-1), the predominant transporter for cellular metformin uptake [133-136]. OCT-1 expression plays also an important role for the antiproliferative action of metformin in cancer cells [137]. Once taken up by the cell, the major action of metformin is believed to alterate cellular energy metabolism associated with direct and indirect stimulation of AMP-Activated Protein kinase (AMPK) [138]. AMPK is a serine/threonine protein kinase that acts as a sensor of cellular energy status [139]. AMPK represents a heterotrimeric complex composed of a catalytic subunit (AMPK-α) and two regulatory subunits (AMPK-β and AMPK-γ) [139]. Metformin-induced inhibition of complex I of the mitochondrial electron transport chain reduces ATP production and increases AMP levels [140-144]. This results in 5´-AMP-mediated activation of Liver Kinase B1 (LKB1) that phosphoylates and activates the catalytic α-subunit of AMPK [145-147]. Loss of LKB1 in intestinal tumors from LKB1+/- mice is accompanied by an increase in mTORC1 signaling, as detected by the phosphorylation of its major downstream targets p70 ribosomal S6 kinase (S6K) and Eukaryotic translation Initiation Factor (eIF) 4B binding protein 1 (4E-BP1) [148]. LKB1 is thus the major upstream activating kinase of AMPK in the liver [149]. It has been demonstrated that metformin requires LKB1 in the liver to lower blood glucose levels [149]. Recently, Zhang [150] reported that metformin has a stronger binding ability to the γ subunit of AMPK than to α subunit. AMPK-mediated down regulation of mTORC1 results from phosphorylation and activation of TSC2 [95,151], the negative regulator of RHEB [95,106-110]. Furthermore, AMPK phosphorylates and inhibits RAPTOR, a positive regulator and substrate processor of mTORC1 [95].

Metformin-Induced ATF4/REDD1-Mediated mTORC1- Inhibition

Inhibition of mitochondrial complex I activity by metformin enhanced the expression of fibroblast growth factor 21 (FGF21), an endocrine hepatic hormone that exhibits anti-obesity and anti-diabetes effects [152]. A strong dose-dependent increase in FGF21 expression has been observed in rat and human hepatocytes treated with metformin [153]. Both increased FGF21-expression as well as mTORC1 inhibition has been associated with increased lifespan [154-156]. The starvation hormone FGF21 induces hepatic fatty acid oxidation and ketogenesis and increases insulin sensitivity. Metformin induced expression of FGF21 is mediated through activating transcription factor 4 (ATF4) [152]. It has recently been demonstrated in hepatocytes that tetracyclines also induce ATF4, which was associated with mTORC1 inhibition [157]. Regulated in DNA damage and development 1 (REDD1) also known as DNA damage-inducible transcript 4 (DDIT4) functions to repress signaling through mTORC1 in response to diverse stress conditions such as increased endoplasmic reticulum stress. Notably, ATF4 facilitates the transcription of the REDD1 gene [158,159]. REDD1 inhibits mTORC1 by stabilizing the TSC1-TSC2-TBC1D7 inhibitory complex [160].

Metformin via p53/REDD1-Induction Inhibits mTORC1

The tumor suppressor protein p53, which induces Sestrin1/2 [161], and p63, required for the induction of REDD1 and activation of the TSC complex [162,163], inhibit mTORC1 signaling [164]. REDD1 is another target of p53 and mirrors the tissue specific pattern of the p53 family member p63 [162]. REDD1 suppresses mTORC1 activity by releasing TSC2 from AKT-mediated association with inhibitory 14-3-3 proteins [163], which leads to inhibition of mTORC1, a physiological mechanism in response to hypoxia [164].

Recent evidence underlines that p53 suppresses carcinogenesis by inhibiting mTORC1 [165]. Importantly, p53 is another target of AMPK. AMPK induces phosphorylation of p53 on Ser15, which induces AMPK-dependent cell-cycle arrest [166,167]. Critical for the control of p53 function are its main negative regulators Mdm2 and Mdmx [168]. Recently, He [169] demonstratd that metformin-activated AMPK results in phosphorylation and inactivation of Mdmx, leading to p53 stabilization and activation. AMPK-mediated phosphorylation of Mdmx on Ser342 enhanced the association between Mdmx and 14-3-3. This inhibits p53 ubiquitylation stabilizing and activating p53 [169]. p53-induced Sestrin1 and Sestrin2 activate AMPK, which phosphorylate TCS2 thereby inhibiting mTORC1 [161]. Thus, metformin-activated AMPK and AMPK-mediated p53 activation and Sestrin1/2 expression functions as a feed forward loop inhibiting mTORC1 signaling [170]. It has recenly been demonstrated that REDD1 expression is increased by metformin even when AMPK was down-regulated [171]. Remarkably, in LNCaP prostate cancer cells, REDD1 induced mTORC1 inhibition in response to metformin [171].

Metformin-Induction of ATM Suppresses mTORC1

The ATM gene is mutated in the human genetic disorder ataxia-telangiectasia [172]. ATM is a 350 kDa protein is a member of the PI3K super family. At the G1/S interface ATM plays a central role in radiation-induced activation of p53. ATM binds to p53 in a complex fashion and activates p53 in response to breaks in DNA by Ser15-phosphorylation [172]. AMPK, the major pharmacologic target of metformin, is one of the downstream targets of ATM [173-175]. In rat hepatoma cells, inhibition of ATM reduced metformin-stimulated phosphorylation of AMPK, suggesting that ATM plays a therapeutic role for the action of metformin [176,177].

By activating the ATM-mediated DNA Damage Response (DDR), metformin might sensitize cells against further damage, thus mimicking the precancerous stimulus that induces an intrinsic barrier against carcinogenesis. It has been proposed that metformin might function as a tissue sweeper of pre-malignant cells before they gain stem cell/tumor initiating properties [178]. Recent evidence indicates that variations of ATM alter the glycemic response to metformin in patients with type 2 diabetes [179]. The ATM rs11212617 variant has been associated with successful glycemic response to metformin in patients with type 2 diabetes and insulin resistent HIV-infected patients [179,180].

Metformin Inhibits Amino Acid-Mediated mTORC1 Activation

In addition to metformin´s inhibitory effects on mTORC1 by AMPK-TSC2-mediated suppression of RHEB, metformin inhibits mTORC1 in a RAG GTPase-dependent manner [181]. The mechanism by which metformin inhibits RAG GTPases resulting in suppressed mTORC1 signaling has not yet been clarified. Similar to amino acid withdrawal of cells treatment with the biguanide phenformin caused mTOR to disperse from perinuclear aggregates and to diffuse throughout the cytoplasm [181]. Proton-assisted amino acid transporters (PATs) localized on late endosomes and lysosomes (LEL) interact with RAGs and are required for mTORC1 activation [125,182,183]. PAT1 has been identified as an essential mediator of amino acid-dependent mTORC1 activation involved in the function of the PAT1/RAG/ Ragulator complex [125]. The mTORC1-regulatory role of the PATs is conserved in humans [184]. Activation of mTORC1 in starved HEK-293 cells stimulated by amino acids requires PAT1 and PAT4, and is elevated in PAT1-overexpressing cells. Importantly, in HEK-293 cells, PAT1 is highly concentrated in intracellular compartments, including endosomes, wherein mTOR shuttles upon amino-acid stimulation [184]. PAT1 and mTOR co-localize at the surface of the same intracellular compartments [184]. Therefore it has been proposed that PATs modulate the activity of mTORC1 not by transporting amino acids into the cell but by modulating the intracellular response to amino acids [184]. PAT1 and RAGC form part of a putative amino acid-sensing complex [184]. Knockdown of PAT1, PAT4, or mTOR in serum- and nutrient-starved cells reduced amino acid-dependent mTORC1 signaling following refeeding [184]. Conversely, over-expression of PAT1 in starved cells enhanced the sensitivity of the TORC1 response to amino acids during refeeding [184]. Notably, siRNA knock down of PAT1 inhibits mTORC1 activation [184]. Thus, suppression of PAT1 attenuates mTORC1 signaling. Intriguingly, Metzner [185] demonstrated that PAT1 accepts guanidine derivatives such as the anti-diabetic compound β-guanidinopropionic acid, and its derivatives guanidinoacetic acid, and guanidinobutyric acid as substrates. However, metformin in excess amounts (10 mM) only exhibited a modest PAT1 inhibition of L-[3 H]proline uptake in Caco-2 cells. To our knowledge no study investigated the effect of biguanides on PAT4 activity, which is also involved in the regulation of mTORC1 [186]. Future studies using different metformin concentrations and various lysosomal PAT1- and PAT4 expressing cell systems might be promising to eluciate the potential inhibitory effect of metformin and related biguanides on the regulation of PAT/RAG/ Ragulator/v-ATPase nutrisome-mediated mTORC1 signaling.

Metformin and mTORC1/FoxO1-Regulated Gluconeo-genesis

For about a decade AMPK was assumed the prime mediator of metformin´s anti-hyperglycemic action [138,145,146]. However, the suggested role of metformin’s mode of action stimulating AMPK-mediated inhibition of hepatic gluconeogenesis has been challenged. Genetic loss of function studies demonstrated that metformin lowered glucose production in liver of transgenic mice that lacked either AMPK or its upstream activator LKB1 [187]. Recently, Li [188] identified mTORC1 as an essential component in the insulin regulated pathway of hepatic lipogenesis but not gluconeogenesis. mTORC1 activates SREBP-1c and uncouples lipogenesis from gluconeogenesis [188,189]. Gluconeogenesis is primarily controled by the Fork head box class O1 transcription factor (FoxO1), which after insulin-induced AKT-mediated phosphorylation is extruded from the nucleus [190]. Nuclear FoxO1 stimulates the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase (PEPCK) involved in the net glucose output from the liver [191]. Glucose-6- phosphatase (G6Pase), the enzyme that catalyzes the final step of gluconeogenesis, also plays a key role in the control of blood glucose levels. Onuma [192] observed a correlation between FoxO1a and FoxO3a binding and the inhibition of basal G6Pase catalytic subunit gene transcription by insulin. FoxO1a and FoxO3a share an identical binding specificity for G6Pase insulin response sequences 1 (IRS1) and IRS2 [192]. Notably, AMPK directly phosphorylates FoxO transcription factors at six regulatory sites that enhance FoxO transcriptional activity [193]. FoxO1 is the transcription factor of starvation and is upregulated by nutrient restriction [190,194]. Metformin exerts comparable effects to that of nutrient restriction [195]. In adipocytes, metformin increased nuclear FoxO1 expression and induced FoxO1-dependent lysosomal acid lipase as well as lipid droplet degradation through lipophagy [195]. Hepatic gluconeogenesis is absolutely required for survival during prolonged fasting or starvation, whereas during phases of nutrient abundance insulin suppresses hepatic gluconeogenesis. Peroxisome proliferatator-activated receptor-γ co-activator 1α (PGC-1α), a transcriptional co-activator, binds and co-activates FoxO1 in a manner inhibited by AKT-mediated phosphorylation [196]. FoxO1 function is required for the robust activation of gluconeogenic gene expression in hepatic cells and in mouse liver by PGC-1α demonstrating that FoxO1 and PGC-1α interact in the execution of a program of insulin-regulated gluconeogenesis [196].

Thus, there appears to be a contradiction as metformin on the one hand increases FoxO1 expression in adipocytes but on the other hand apparently suppresses FoxO1-mediated hepatic gluconeogenesis. As metformin´s suppressive effect on hepatic gluconeogenesis is independent of LKB1 and AMPK [187], but strongly dependent on the degree of AKT-mediated FoxO1- phosphorylation, metformin´s primary target in the regulation of hepatic gluconeogenesis appears to be mTORC1-S6K1 signaling. Nutrient and amino acid excess results in hepatic mTORC1- overactivation enhancing the activity of S6K1. S6K1-mediated phosphorylation of IRS-1, the adaptor protein of key downstream effects of the insulin receptor, dampens the activity of AKT thereby increases nuclear FoxO1-levels that promote hepatic gluconeogenesis. Thus, S6K1-mediated phosphorylation of S6K1 leads to insulin desensensitization [31]. Notably, hyperactive mTORC1 signaling is an essential event in the development of hepatic insulin resistance in the presence of excess amino acids [197]. Exposure of HepG2 cells to excess amino acids reduced AMPK phosphorylation and impaired the insulin-stimulated phosphorylation of AKT Ser473 and IRS-1 Tyr612 [197]. Metformin inhibited mTORC1 signaling, thereby prevented hepatic insulin resistance induced by excess amino acid intake [197].

Metformin´s major mode of action thus appears to be the inhibition of mTORC1 decreasing the activity of S6K1 resulting in enhanced insulin sensitivity. Suppression of S6K1 reduces FoxO1-mediated hepatic gluconeogenesis and insulin resistance of peripheral tissues by increased AKT-mediated translocation of glucose transporter-4 (GLUT4) to the plasma membrane [198,199]. This mechanism explains the beneficial clinical effects of metformin treatment on glucose homeostasis (Figure 2).

This concept is supported by recent studies exploring the expression of the liver-derived secretory protein Selenoprotein P (SeP). SeP is regulated similarly to G6Pase. G6Pase-expression is stimulated by PGC-1α and FoxO1a [200]. SeP encoded by SEPP1 in humans induces insulin resistance in type 2-diabetes [201]. Remarkably, metformin suppresses SEPP1 expression by activating AMPK and subsequently inactivating FoxO3a in hepatocytes [202]. Treatment with metformin reduced SEPP1 promoter activity in a concentration- and time-dependent manner. Computational analysis of transcription factor binding sites conserved among the species resulted in identification of the FoxO-binding site in the metformin-response element of the SEPP1 promoter. Metformin did not affect FoxO3a expression, but it increased its phosphorylation and thereby decreased its nuclear localization. This effect is compatible with the proposed mode of metformin action centering on inhibition of mTORC1- S6K1-signaling, which explains increased AKT-mediated FoxO-phosphorylation and nuclear extrusion by metformin-mediated suppression of mTORC1 (Figure 2 ).

Metformin Modifies mRNA Translation and mTORC1 Signaling

Recently, Larsson [203] presented data of a genome-wide analysis of translational targets of canonical mTOR inhibitors compared with metformin. Metformin exerted profound effects on the translatome and perturbed the translatome to an extent comparable to canonical mTOR inhibitors such as rapamycin and PP242 [203]. Metformin suppressed translation of limited subsets of functionally related mRNAs that encode proteins involved in cell-cycle control, metabolism, mRNA translation, and RNA processing. Importantly, meformin´s antiproliferative activity could be explained by selective translational suppression of mRNAs encoding cell-cycle regulators via the mTORC1/4E-BP pathway [203].

Metormin interferes with cellular pathways that sense ROS signals and DNA damage responses, cellular energy homeostasis and amino acid availability finally converging in restrained mTORC1 signaling. In comparison to allosterical, natural or synthetic mTORC1 inhibitors such as rapamycin [204], resveratrol, curcumin, caffeine, epigallocatechin gallate, silymarin and others plant-derived polyphenols [205] and synthetic active-site mTORC1 inhibitors (TORkinibs) [206,207], metformin is a unique drug that attenuates the two major independent pathways required for efficient activation mTORC1. Metformin 1) suppresses RHEB-mediated signals integrating growth factors (insulin, IGF-1), cellular energy status (AMPK), ROS-status and ER stress (ATM, REDD1), and 2) suppresses amino acid-mediated PAT1/Ragulator-RAG-signaling (Table 1).

Is is thus conceivable that metformin functions as a unique mTORC1 inhibitor that attenuates the progression of mTORC1- driven diseases of civilization associated with increased food intake [208,209], obesity [210-217], insulin resistance and type 2 diabetes [32,138,145,218,219], hepatic steatosis [220], polycystic ovary syndrome [221,222], acne [223-225], dyslipoproteinemia [226], atherosclerosis and cardiovascular diseases [227,228], cancer [203,229-248], and neurodegenerative diseases [249,250]. Metformin in a pharmacological way attenuates both exaggerated nutrient signaling of Western diet associated with enhanced insulin/IGF-1 signaling (the RHEB-axis of mTORC1 activation) and nutritional overload of essential BCAAs (the RAG-axis of mTORC1 activation) [251-253]. Notably, the mTORC1 inhibitor metformin counteracts the anabolic signaling of milk [254], which has been identified as an endocrine postnatal signaling system promoting mTORC1-mediated postnatal growth [255]. From all animal proteins, milk proteins transport the highest amounts of essential BCAAs [121]. Elevated plasma levels of essential BCAAs significantly correlate with insulin resistance and the risk of type 2-diabetes [50,256,257]. Not only carbohydrate-and lipid overload but also amino acid excess induces mTORC1 hyper-activation resulting in S6K1/IRS-1- and STAT3/SOCS3- mediated insulin resistance [44,45,258-266] (Figure 2). In this regard, metformin by down-regulating mTORC1-S6K1 signaling compensates the adverse effects of persistent hyperalimentation, high milk, milk protein and milk fat consumption associated with anabolic nutrient signaling.

Metformin has been used for decades to improve glucose homeostasis and its major mode of action had focused on its role to activate AMPK. Now, we begin to understand that metformin´s pivotal mode of action is the attenuation of mTORC1-S6K1 signaling, a regulatory key node of cellular metabolism that in response to excess nutrients orchestrates the signaling pathways of anabolism. Accumulating evidence underlines that persistently increased mTORC1 signaling is the common promoter of diseases of civilization [12-18].

Suppression of mTORC1 explains metformin´s effectiveness in diabetes and cancer. Down-regulation of S6K1 explains IRS-1/PI3K/AKT/FoxO1-mediated suppression of hepatic gluconeogenesis as well as AKT-AS160-mediated translocation of GLUT4 that reduces insulin resistance.

Metformin compensates the adverse metabolic effects of Western sedentary lifestyle with overnutrion that promotes mTOR-driven age-related diseases. Thus, metformin exerts preventive and therapeutic effects for common metabolic diseases of civilization for which „less TOR is more“[90].

Metformin apparently normalizes the magnitude of mTORC1 signaling in cancer cells with genetic aberrations of signaling components of the mTORC1 pathway such as over-activated AKT of PTEN loss of function mutations. Metformin treatment appears to lower mTORC1 signaling to a level of a vegan low-protein diet [234]. Taken together, metformin by targeting mTORC1 signaling acts at the molecular interface connecting metabolic stress, aging, obesity, cardiovascular and neurodegenerative diseases and cancer.

Table 1: Meformin-mediated effects that inhibit mTORC1 signaling.

Metformin targets	Pathways suppressing mTORC1 signaling	Reference
Inhibition of complex I of the mitochondrial electron transport chain	Decrease in ATP, increase in AMP, activation of LKB1, LKB1-mediated activation of AMPK, activation of TSC2 → mTORC1 inhibition; AMPK-mediated inhibition of RAPTOR → mTORC1 inhibtion; AMPK-mediated inactivation of Mdmx, p53 stabilization, p53-induced expression of Sestrin1 and Sestrin 2, AMPK activation → mTORC1 inhibition	[95,140-149, 151] [95]   [169]
Induction of cellular stress transcription factor ATF4	ATF4-mediated expression of REDD1, TSC2 stabilization → mTORC1 inhibition	[157-160, 164]
Activation of ATM	ATM-mediated activation of AMPK → mTORC1 inhibition	[176,177]
Inhibition of RAG GTPase	Possible interference with PAT1 function (?), disintegration of the nutrisome complex (PAT1/RAG/Ragulator/v-ATPase)→mTORC1 inhibition	[184-186]
Modification of the translatome	Suppression of components of the mTORC1/ 4E-BP pathway→mTORC1 inhibition	[203]

Abbreviations: mTORC1: Mechanistic Target of Rapamycin Complex 1; AMP: Adenosine Monophosphate; ATP: Adenosine Triphosphate; LKB1: Liver kinase B1; AMPK: AMP-activated protein Kinase; TSC2: Tuberin; RAPTOR: Regulatory-Associated Protein of mTOR; ATF4: Activating Transcription Factor 4; REDD1: Regulated in DNA Damage and Development 1; ATM: Ataxia Teleangiectasia Mutated; PAT1: Proton-Assisted amino acid Transporter 1; RAG: RAS-related GTP-binding protein; v-ATPase: Vacuolar H+ -ATPase; 4-EBP: Eukaryotic initiation factor (eIF) 4E-Binding Protein.

BCM searched the literature and wrote the manuscript. GS provided further scientific informations, discussions and literature references. Both authors read and approved the final manuscript.

1. Foster KG, Fingar DC. Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem. 2010; 285: 14071-14077.

2. Inoki K, Ouyang H, Li Y, Guan KL. Signaling by target of rapamycin proteins in cell growth control. Microbiol Mol Biol Rev. 2005; 69: 79- 100.

3. Avruch J, Long X, Ortiz-Vega S, Rapley J, Papageorgiou A, Dai N. Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab. 2009; 296: E592-602.

4. Sengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell. 2010; 40: 310-322.

5. Laplante M, Sabatini DM. mTOR Signaling. Cold Spring Harb Perspect Biol. 2012; 4. pii: a011593.

6. Kim J, Guan KL. Amino acid signaling in TOR activation. Annu Rev Biochem. 2011; 80: 1001-1032.

7. Kim SG, Buel GR, Blenis J. Nutrient regulation of the mTOR complex 1 signaling pathway. Mol Cells. 2013; 35: 463-473.

8. Jewell JL, Guan KL. Nutrient signaling to mTOR and cell growth. Trends Biochem Sci. 2013; 38: 233-242.

9. Efeyan A, Sabatini DM. Nutrients and growth factors in mTORC1 activation. Biochem Soc Trans. 2013; 41: 902-905.

10. Laplante M, Sabatini DM. Regulation of mTORC1 and its impact on gene expression at a glance. J Cell Sci. 2013; 126: 1713-1719.

11. Catania C, Binder E, Cota D. mTORC1 signaling in energy balance and metabolic disease. Int J Obes (Lond). 2011; 35: 751-761.

12. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol. 2011; 12: 21-35.

13. Dazert E, Hall MN. mTOR signaling in disease. Curr Opin Cell Biol. 2011; 23: 744-755.

14. Cornu M, Albert V, Hall MN. mTOR in aging, metabolism, and cancer. Curr Opin Genet Dev. 2013; 23: 53-62.

15. Dann SG, Selvaraj A, Thomas G. mTOR Complex1-S6K1 signaling: at the crossroads of obesity, diabetes and cancer. Trends Mol Med. 2007; 13: 252-259.

16. Mieulet V, Lamb RF. Tuberous sclerosis complex: linking cancer to metabolism. Trends Mol Med. 2010; 16: 329-335.

17. Proud CG. mTOR Signalling in heath and disease. Biochem Soc Trans. 2011; 39: 431-436.

18. Takahara T, Maeda T. Evolutionarily conserved regulation of TOR signalling. J Biochem. 2013; 154: 1-10.

19. Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem. 1998; 273: 14484-14494.

20. Lynch CJ, Fox HL, Vary TC, Jefferson LS, Kimball SR. Regulation of amino acid-sensitive TOR signaling by leucine analogues in adipocytes. J Cell Biochem. 2000; 77: 234-251.

21. Lynch CJ. Role of leucine in the regulation of mTOR by amino acids: revelations from structure-activity studies. J Nutr. 2001; 131: 861S-865S.

22. Pham PT, Heydrick SJ, Fox HL, Kimball SR, Jefferson LS Jr, Lynch CJ. Assessment of cell-signaling pathways in the regulation of mammalian target of rapamycin (mTOR) by amino acids in rat adipocytes. J Cell Biochem. 2000; 79: 427-441.

23. Kim JE, Chen J. regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes. 2004; 53: 2748-2756.

24. Blanchard PG, Festuccia WT, Houde VP, St-Pierre P, Brûlé S, Turcotte V, Côté M. Major involvement of mTOR in the PPARγ-induced stimulation of adipose tissue lipid uptake and fat accretion. J Lipid Res. 2012; 53: 1117-1125.

25. Porstmann T, Santos CR, Lewis C, Griffiths B, Schulze A. A new player in the orchestra of cell growth: SREBP activity is regulated by mTORC1 and contributes to the regulation of cell and organ size. Biochem Soc Trans. 2009; 37: 278-283.

26. Peterson TR, Sengupta SS, Harris TE, Carmack AE, Kang SA, Balderas E, et al. mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell. 2011; 146: 408-420.

27. Bakan I, Laplante M. Connecting mTORC1 signaling to SREBP-1 activation. Curr Opin Lipidol. 2012; 23: 226-234.

28. Chakrabarti P, English T, Shi J, Smas CM, Kandror KV. Mammalian target of rapamycin complex 1 suppresses lipolysis, stimulates lipogenesis, and promotes fat storage. Diabetes. 2010; 59: 775-781.

29. Yoon MS, Zhang C, Sun Y, Schoenherr CJ, Chen J. Mechanistic target of rapamycin controls homeostasis of adipogenesis. J Lipid Res. 2013; 54: 2166-2173.

30. Carnevalli LS, Masuda K, Frigerio F, Le Bacquer O, Um SH, Gandin V, et al. S6K1 plays a critical role in early adipocyte differentiation. Dev Cell. 2010; 18: 763-774.

31. Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, et al. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature. 2004; 431: 200-205.

32. Melnik BC. Leucine signaling in the pathogenesis of type 2 diabetes and obesity. World J Diabetes. 2012; 3: 38-53.

33. McDaniel ML, Marshall CA, Pappan KL, Kwon G. Metabolic and autocrine regulation of the mammalian target of rapamycin by pancreatic beta-cells. Diabetes. 2002; 51: 2877-2885.

34. Kwon G, Marshall CA, Pappan KL, Remedi MS, McDaniel ML. Signaling elements involved in the metabolic regulation of mTOR by nutrients, incretins, and growth factors in islets. Diabetes. 2004; 53 Suppl 3: S225-232.

35. Yang J, Chi Y, Burkhardt BR, Guan Y, Wolf BA. Leucine metabolism in regulation of insulin secretion from pancreatic beta cells. Nutr Rev. 2010; 68: 270-279.

36. Blandino-Rosano M, Chen AY, Scheys JO, Alejandro EU, Gould AP, Taranukha T, et al. mTORC1 signaling and regulation of pancreatic β-cell mass. Cell Cycle. 2012; 11: 1892-1902.

37. Bartolome A, Guillén C. Role of the mammalian target of rapamycin (mTOR) complexes in pancreatic β-cell mass regulation. Vitam Horm. 2014; 95: 425-469.

38. Düvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell. 2010; 39: 171-183.

39. Magnuson B, Ekim B, Fingar DC. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J. 2012; 441: 1-21.

40. Zick Y. Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance. Sci STKE. 2005; 2005: pe4.

41. Boura-Halfon S, Zick Y. Phosphorylation of IRS proteins, insulin action, and insulin resistance. Am J Physiol Endocrinol Metab. 2009; 296: E581-591.

42. Carlson CJ, White MF, Rondinone CM. Mammalian target of rapamycin regulates IRS-1 serine 307 phosphorylation. Biochem Biophys Res Commun. 2004; 316: 533-539.

43. Krebs M, Roden M. Nutrient-induced insulin resistance in human skeletal muscle. Curr Med Chem. 2004; 11: 901-908.

44. Um SH, D’Alessio D, Thomas G. Nutrient overload, insulin resistance, and ribosomal protein S6 kinase , S6K1. Cell Metab. 2006; 3: 393-402.

45. Tremblay F, Brûlé S, Hee Um S, Li Y, Masuda K, Roden M, et al. Identification of IRS-1 Ser-1101 as a target of S6K1 in nutrient- and obesity-induced insulin resistance. Proc Natl Acad Sci U S A. 2007; 104: 14056-14061.

46. Barbour LA, McCurdy CE, Hernandez TL, Friedman JE. Chronically increased S6K1 is associated with impaired IRS1 signaling in skeletal muscle of GDM women with impaired glucose tolerance postpartum. J Clin Endocrinol Metab. 2011; 96: 1431-1441.

47. Krebs M, Brunmair B, Brehm A, Artwohl M, Szendroedi J, Nowotny P, et al. The mammalian target of rapamycin pathway regulates nutrient-sensitive glucose uptake in man. Diabetes. 2007; 56: 1600-1607.

48. Tremblay F, Krebs M, Dombrowski L, Brehm A, Bernroider E, Roth E, et al. Overactivation of S6 kinase 1 as a cause of human insulin resistance during increased amino acid availability. Diabetes. 2005; 54: 2674-2684.

49. Saha AK, Xu XJ, Lawson E, Deoliveira R, Brandon AE, Kraegen EW, et al. Downregulation of AMPK accompanies leucine- and glucose-induced increases in protein synthesis and insulin resistance in rat skeletal muscle. Diabetes. 2010; 59: 2426-2434.

50. Newgard CB, An J, Bain JR, Muehlbauer MJ, Stevens RD, Lien LF, et al. A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance. Cell Metab. 2009; 9: 311-326.

51. Shigeyama Y, Kobayashi T, Kido Y, Hashimoto N, Asahara S, Matsuda T, et al. Biphasic response of pancreatic beta-cell mass to ablation of tuberous sclerosis complex 2 in mice. Mol Cell Biol. 2008; 28: 2971- 2979.

52. Hsieh AC, Liu Y, Edlind MP, Ingolia NT, Janes MR, Sher A, et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature. 2012; 485: 55-61.

53. Wang X, Proud CG. Nutrient control of TORC, a cell-cycle regulator. Trends Cell Biol. 2009; 19: 260-267.

54. Efeyan A, Sabatini DM. mTOR and cancer: many loops in one pathway. Curr Opin Cell Biol. 2010; 22: 169-176.

55. Ekim B, Magnuson B, Acosta-Jaquez HA, Keller JA, Feener EP, Fingar DC. mTOR kinase domain phosphorylation promotes mTORC1 signaling, cell growth, and cell cycle progression. Mol Cell Biol. 2011; 31: 2787-2801.

56. Robitaille AM, Christen S, Shimobayashi M, Cornu M, Fava LL, Moes S, et al. Quantitative phosphoproteomics reveal mTORC1 activates de novo pyrimidine synthesis. Science. 2013; 339: 1320-1323.

57. Ben-Sahra I, Howell JJ, Asara JM, Manning BD. Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science. 2013; 339: 1323-1328.

58. Thoreen CC, Chantranupong L, Keys HR, Wang T, Gray NS, Sabatini DM. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature. 2012; 485: 109-113.

59. Clohessy JG, Reschke M, Pandolfi PP. Found in translation of mTOR signaling. Cell Res. 2012; 22: 1315-1318.

60. Pópulo H, Lopes JM, Soares P. The mTOR Signalling Pathway in Human Cancer. Int J Mol Sci. 2012; 13: 1886-1918.

61. Nardella C, Carracedo A, Alimonti A, Hobbs RM, Clohessy JG, Chen Z, et al. Differential requirement of mTOR in postmitotic tissues and tumorigenesis. Sci Signal. 2009; 2: ra2.

62. Vadlakonda L, Pasupuleti M, Pallu R. Role of PI3K-AKT-mTOR and Wnt Signaling Pathways in Transition of G1-S Phase of Cell Cycle in Cancer Cells. Front Oncol. 2013; 3: 85.

63. Mita MM, Mita A, Rowinsky EK. Mammalian target of rapamycin: a new molecular target for breast cancer. Clin Breast Cancer. 2003; 4: 126-137.

64. Barrett D, Brown VI, Grupp SA, Teachey DT. Targeting the PI3K/ AKT/mTOR signaling axis in children with hematologic malignancies. Paediatr Drugs. 2012; 14: 299-316.

65. Cho DC. Targeting the PI3K/Akt/mTOR pathway in malignancy: Rationale and clinical outlook.

66. Beauchamp EM, Platanias LC. The evolution of the TOR pathway and its role in cancer. Oncogene. 2013; 32: 3923-3932.

67. Oddo S. The role of mTOR signaling in Alzheimer disease. Front Biosci (Schol Ed). 2012; 4: 941-952.

68. Wong M. Mammalian target of rapamycin (mTOR) pathways in neurological diseases. Biomed J. 2013; 36: 40-50.

69. Li X, An WL, Alafuzoff I, Soininen H, Winblad B, Pei JJ. Phosphorylated eukaryotic translation factor 4E is elevated in Alzheimer brain. Neuroreport. 2004; 15: 2237-2240.

70. Li X, Alafuzoff I, Soininen H, Winblad B, Pei JJ. Levels of mTOR and its downstream targets 4E-BP, eEF2, and eEF2 kinase in relationships with tau in Alzheimer’s disease brain. FEBS J. 2005; 272: 4211-4220.

71. Pei JJ, Björkdahl C, Zhang H, Zhou X, Winblad B. p70 S6 kinase and tau in Alzheimer’s disease. J Alzheimers Dis. 2008; 14: 385-392.

72. Pei JJ, Hugon J. mTOR-dependent signalling in Alzheimer’s disease. J Cell Mol Med. 2008; 12: 2525-2532.

73. Spilman P, Podlutskaya N, Hart MJ, Debnath J, Gorostiza O, Bredesen D, et al. Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer’s disease. PLoS One. 2010; 5: e9979.

74. Cai Z, Zhao B, Li K, Zhang L, Li C, Quazi SH, et al. Mammalian target of rapamycin: a valid therapeutic target through the autophagy pathway for Alzheimer’s disease? J Neurosci Res. 2012; 90: 1105-1118.

75. Zhao WQ, Townsend M. Insulin resistance and amyloidogenesis as common molecular foundation for type 2 diabetes and Alzheimer’s disease. Biochim Biophys Acta. 2009; 1792: 482-496.

76. Kundu M. ULK, mammalian target of rapamycin, and mitochondria: linking nutrient availability and autophagy. Antioxid Redox Signal. 2011; 14: 1953-1958.

77. Yang J, Carra S, Zhu WG, Kampinga HH. The regulation of the autophagic network and its implications for human disease. Int J Biol Sci. 2013; 9: 1121-1133.

78. Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, Müller F. Genetics: influence of TOR kinase on lifespan in C. elegans. Nature. 2003; 426: 620.

79. Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr Biol. 2004; 14: 885-890.

80. Kaeberlein M, Powers RW 3rd, Steffen KK, Westman EA, Hu D, Dang N, et al. Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science. 2005; 310: 1193-1196.

81. Powers RW 3rd, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 2006; 20: 174-184.

82. Chen D, Thomas EL, Kapahi P. HIF-1 modulates dietary restriction-mediated lifespan extension via IRE-1 in Caenorhabditis elegans. PLoS Genet. 2009; 5: e1000486.

83. Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. 2009; 460: 392-395.

84. Bjedov I, Toivonen JM, Kerr F, Slack C, Jacobson J, Foley A, et al. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metab. 2010; 11: 35-46.

85. Anisimov VN, Zabezhinski MA, Popovich IG, Piskunova TS, Semenchenko AV, Tyndyk ML, et al. Rapamycin extends maximal lifespan in cancer-prone mice. Am J Pathol. 2010; 176: 2092-2097.

86. Miller RA, Harrison DE, Astle CM, Baur JA, Boyd AR, de Cabo R, et al. Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice. J Gerontol A Biol Sci Med Sci. 2011; 66: 191-201.

87. Robida-Stubbs S, Glover-Cutter K, Lamming DW, Mizunuma M, Narasimhan SD, Neumann-Haefelin E, et al. TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/ FoxO. Cell Metab. 2012; 15: 713-724.

88. Wilkinson JE, Burmeister L, Brooks SV, Chan CC, Friedline S, Harrison DE, et al. Rapamycin slows aging in mice. Aging Cell. 2012; 11: 675- 682.

89. Yang Z, Ming XF. mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases. Obes Rev. 2012; 13 Suppl 2: 58-68.

90. Kapahi P, Chen D, Rogers AN, Katewa SD, Li PW, Thomas EL, et al. With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging. Cell Metab. 2010; 11: 453-465.

91. Shaw RJ. LKB1 and AMP-activated protein kinase control of mTOR signalling and growth. Acta Physiol (Oxf). 2009; 196: 65-80.

92. Xu J, Ji J, Yan XH. Cross-talk between AMPK and mTOR in regulating energy balance. Crit Rev Food Sci Nutr. 2012; 52: 373-381.

93. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A. 2004; 101: 3329-3335.

94. Hardie DG. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat Rev Mol Cell Biol. 2007; 8: 774-785.

95. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell. 2008; 30: 214-226.

96. Egan D, Kim J, Shaw RJ, Guan KL. The autophagy initiating kinase ULK1 is regulated via opposing phosphorylation by AMPK and mTOR. Autophagy. 2011; 7: 643-644.

97. Shang L, Chen S, Du F, Li S, Zhao L, Wang X. Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK. Proc Natl Acad Sci U S A. 2011; 108: 4788-4793.

98. Roberts DJ, Tan-Sah VP, Ding EY, Smith JM, Miyamoto S. Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol Cell. 2014; 53: 521-533.

99. Rabinowitz JD, White E. Autophagy and metabolism. Science. 2010; 330: 1344-1348.

100. Lee JH, Paull TT. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks. Oncogene. 2007; 26: 7741- 7748.

101. Lavin MF, Khanna KK. ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia. Int J Radiat Biol. 1999; 75: 1201-1214.

102. Guo Z, Kozlov S, Lavin MF, Person MD, Paull TT. ATM activation by oxidative stress. Science. 2010; 330: 517-521.

103. Alexander A, Kim J, Walker CL. ATM engages the TSC2/mTORC1 signaling node to regulate autophagy. Autophagy. 2010; 6: 672-673.

104. Maréchal A, Zou L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harb Perspect Biol. 2013; 5.

105. Masai H. ATM in prevention of genomic instability. Cell Cycle. 2014; 13: 882-883.

106. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002; 4: 648-657.

107. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell. 2003; 115: 577-590.

108. Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell. 2002; 10: 151-162.

109. Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, Blenis J. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)- mediated downstream signaling. Proc Natl Acad Sci U S A. 2002; 99: 13571-13576.

110. Dibble CC, Elis W, Menon S, Qin W, Klekota J, Asara JM, et al. TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1. Mol Cell. 2012; 47: 535-546.

111. Dodd KM, Tee AR. Leucine and mTORC1: a complex relationship. Am J Physiol Endocrinol Metab. 2012; 302: E1329-1342.

112. Thedieck K, Hall MN. Translational control by amino acids and energy. Handbook of Cell Signaling. Three-Volume Set, 2nd edn. Elsevier Inc. chapter 274. 2285-2293, 2010.

113. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science. 2008; 320: 1496-1501.

114. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell. 2010; 141: 290-303.

115. Goberdhan DC. Intracellular amino acid sensing and mTORC1- regulated growth: new ways to block an old target? Curr Opin Investig Drugs. 2010; 11: 1360-1367.

116. Menon S, Dibble CC, Talbott G, Hoxhaj G, Valvezan AJ, Takahashi H, et al. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell. 2014; 156: 771-785.

117. Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell. 2002; 10: 151-162.

118. Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell. 2009; 136: 521-534.

119. Cohen A, Hall MN. An amino acid shuffle activates mTORC1. Cell. 2009; 136: 399-400.

120. Long X, Ortiz-Vega S, Lin Y, Avruch J. Rheb binding to mammalian target of rapamycin (mTOR) is regulated by amino acid sufficiency. J Biol Chem. 2005; 280: 23433-23436.

121. Millward DJ, Layman DK, Tomé D, Schaafsma G. Protein quality assessment: impact of expanding understanding of protein and amino acid needs for optimal health. Am J Clin Nutr. 2008; 87: 1576S-1581S.

122. Bar-Peled L, Schweitzer LD, Zoncu R, Sabatini DM. Ragulator is a GEF RAG GTPases that signal amino acid levels to mTORC1. Cell. 2012; 150: 1196-1208.

123. Groenewoud MJ, Zwartkruis FJ. Rheb and Rags come together at the lysosome to activate mTORC1. Biochem Soc Trans. 2013; 41: 951- 955.

124. Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, Gulati P, et al. Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc Natl Acad Sci U S A. 2005; 102: 14238-14243.

125. Ögmundsdóttir MH, Heublein S, Kazi S, Reynolds B, Visvalingam SM, Shaw MK, et al. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes. PLoS One. 2012; 7: e36616.

126. Thwaites DT, Anderson CM. The SLC36 family of proton-coupled amino acid transporters and their potential role in drug transport. Br J Pharmacol. 2011; 164: 1802-1816.

127. Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science. 2011; 334: 678-683.

128. Reynolds B, Laynes R, Ogmundsdóttir MH, Boyd CA, Goberdhan DC. Amino acid transporters and nutrient-sensing mechanisms: new targets for treating insulin-linked disorders? Biochem Soc Trans. 2007; 35: 1215-1217.

129. Bonfils G, Jaquenoud M, Bontron S, Ostrowicz C, Ungermann C, De Virgilio C. Leucyl-tRNA synthetase controls TORC1 via the EGO complex. Mol Cell. 2012; 46: 105-110.

130. Han JM, Jeong SJ, Park MC, Kim G, Kwon NH, Kim HK, et al. Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1- signaling pathway. Cell. 2012; 149: 410-424.

131. Durán RV, Oppliger W, Robitaille AM, Heiserich L, Skendaj R, Gottlieb E, et al. Glutaminolysis activates Rag-mTORC1 signaling. Mol Cell. 2012; 47: 349-358.

132. Gallinetti J, Harputlugil E, Mitchell JR. Amino acid sensing in dietary-restriction-mediated longevity: roles of signal-transducing kinases GCN2 and TOR. Biochem J. 2013; 449: 1-10.

133. Wang DS, Jonker JW, Kato Y, Kusuhara H, Schinkel AH, Sugiyama Y. Involvement of organic cation transporter 1 in hepatic and intestinal distribution of metformin. J Pharmacol Exp Ther. 2002; 302: 510- 515.

134. Shu Y, Sheardown SA, Brown C, Owen RP, Zhang S, Castro RA, et al. Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action. J Clin Invest. 2007; 117: 1422-1431.

135. Lozano E, Herraez E, Briz O, Robledo VS, Hernandez-Iglesias J, Gonzalez-Hernandez A, et al. Role of the plasma membrane transporter of organic cations OCT1 and its genetic variants in modern liver pharmacology. Biomed Res Int. 2013; 2013: 692071.

136. Graham GG, Punt J, Arora M, Day RO, Doogue MP, Duong JK, et al. Clinical pharmacokinetics of metformin. Clin Pharmacokinet. 2011; 50: 81-98.

137. Segal ED, Yasmeen A, Beauchamp MC, Rosenblatt J, Pollak M, Gotlieb WH. Relevance of the OCT1 transporter to the antineoplastic effect of biguanides. Biochem Biophys Res Commun. 2011; 414: 694-699.

138. Pernicova I, Korbonits M. Metformin--mode of action and clinical implications for diabetes and cancer. Nat Rev Endocrinol. 2014; 10: 143-156.

139. Sanz P. AMP-activated protein kinase: structure and regulation. Curr Protein Pept Sci. 2008; 9: 478-492.

140. El-Mir MY, Nogueira V, Fontaine E, Avéret N, Rigoulet M, Leverve X. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J Biol Chem. 2000; 275: 223-228.

141. Owen MR, Doran E, Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J. 2000; 348 Pt 3: 607- 614.

142. Detaille D, Guigas B, Leverve X, Wiernsperger N, Devos P. Obligatory role of membrane events in the regulatory effect of metformin on the respiratory chain function. Biochem Pharmacol. 2002; 63: 1259- 1272.

143. Miller RA, Birnbaum MJ. An energetic tale of AMPK-independent effects of metformin. J Clin Invest. 2010; 120: 2267-2270.

144. Kim KH, Jeong YT, Kim SH, Jung HS, Park KS, Lee HY, et al. Metformin-induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation. Biochem Biophys Res Commun. 2013; 440: 76-81.

145. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001; 108: 1167-1174.

146. Rena G, Pearson ER, Sakamoto K. Molecular mechanism of action of metformin: old or new insights? Diabetologia. 2013; 56: 1898-1906.

147. Shaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, Depinho RA, et al. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science. 2005; 310: 1642-1646.

148. Shaw RJ, Bardeesy N, Manning BD, Lopez L, Kosmatka M, DePinho RA, et al. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell. 2004; 6: 91-99.

149. Green AS, Chapuis N, Lacombe C, Mayeux P, Bouscary D, Tamburini J. LKB1/AMPK/mTOR signaling pathway in hematological malignancies: from metabolism to cancer cell biology. Cell Cycle. 2011; 10: 2115-2120.

150. Zhang Y, Wang Y, Bao C, Xu Y, Shen H, Chen J, et al. Metformin interacts with AMPK through binding to g subunit. Mol Cell Biochem. 2012; 368: 69-76.

151. Pikiou O, Vasilaki A, Leondaritis G, Vamvakopoulos N, Messinis IE. Effects of metformin on fertilisation of bovine oocytes and early embryo development: possible involvement of AMPK3-mediated TSC2 activation. Zygote. 2013.

152. Zhang M, Liu Y, Xiong ZY, Deng ZY, Song HL, An ZM. Changes of plasma fibroblast growth factor-21 (FGF-21) in oral glucose tolerance test and effects of metformin on FGF-21 levels in type 2 diabetes mellitus. Endokrynol Pol. 2013; 64: 220-224.

153. Nygaard EB, Vienberg SG, Ørskov C, Hansen HS, Andersen B. Metformin stimulates FGF21 expression in primary hepatocytes. Exp Diabetes Res. 2012; 2012: 465282.

154. Zhang Y, Xie Y, Berglund ED, Coate KC, He TT, Katafuchi T, et al. The starvation hormone, fibroblast growth factor-2, extends lifespan in mice. Elife. 2012; 1: e00065.

155. Rallis C, Codlin S, Bähler J. TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast. Aging Cell. 2013; 12: 563-573.

156. Mendelsohn AR, Larrick JW. Dissecting mammalian target of rapamycin to promote longevity. Rejuvenation Res. 2012; 15: 334- 337.

157. Brüning A, Brem GJ, Vogel M, Mylonas I. Tetracyclines cause cell stress-dependent ATF4 activation and mTOR inhibition. Exp Cell Res. 2014; 320: 281-289.

158. Dennis MD, McGhee NK, Jefferson LS, Kimball SR. Regulated in DNA damage and development 1 (REDD1) promotes cell survival during serum deprivation by sustaining repression of signaling through the mechanistic target of rapamycin in complex 1 (mTORC1). Cell Signal. 2013; 25: 2709-2716.

159. Kimball SR, Jefferson LS. Induction of REDD1 gene expression in the liver in response to endoplasmic reticulum stress is mediated through a PERK, eIF2α phosphorylation, ATF4-dependent cascade. Biochem Biophys Res Commun. 2012; 427: 485-489.

160. Yoshida T, Mett I, Bhunia AK, Bowman J, Perez M, Zhang L, et al. Rtp80, a suppressor of mTOR signaling, is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema. Nat Med. 2010; 16: 767-773.

161. Budanov AV, Karin M. p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell. 2008; 134: 451-460.

162. Ellisen LW, Ramsayer KD, Johannessen CM, Yang A, Beppu H, Minda K, et al. REDD, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species. Mol Cell. 2002; 10: 995-1005.

163. DeYoung MP, Horak P, Sofer A, Sgroi D, Ellisen LW. Hypoxia regulates TSC1/2-mTOR signaling and tumor suppression through REDD1- mediated 14-3-3 shuttling. Genes Dev. 2008; 22: 239-251.

164. Cam M, Bid HK, Xiao L, Zambetti GP, Houghton PJ, Cam H. p53/ TAp63 and AKT regulate mammalian target of rapamycin complex 1 (mTORC1) signaling through two independent parallel pathways in the presence of DNA damage. J Biol Chem. 2014; 289: 4083-4094.

165. Akeno N, Miller AL, Ma X, Wikenheiser-Brokamp KA. p53 suppresses carcinoma progression by inhibiting mTOR pathway activation. Oncogene. 2014; (Epub ahead of print).

166. Zhang XD, Qin ZH, Wang J. The role of p53 in cell metabolism. Acta Pharmacol Sin. 2010; 31: 1208-1212.

167. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, et al. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell. 2005; 18: 283-293.

168. Wade M, Wang YV, Wahl GM. The p53 orchestra: Mdm2 and Mdmx set the tone. Trends Cell Biol. 2010; 20: 299-309.

169. He G, Zhang YW, Lee JH, Zeng SX, Wang YV, Luo Z, et al. AMP-activated protein kinase induces p53 by phosphorylating MDMX and inhibiting its activity. Mol Cell Biol. 2014; 34: 148-157.

170. Hay N. Interplay between FOXO, TOR, and Akt. Biochim Biophys Acta. 2011; 1813: 1965-1970.

171. Ben Sahra I, Regazzetti C, Robert G, Laurent K, Le Marchand-Brustel Y, Auberger P, et al. Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1. Cancer Res. 2011; 71: 4366-4372.

172. Lavin MF, Khanna KK. ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia. Int J Radiat Biol. 1999; 75: 1201-1214.

173. Sanli T, Rashid A, Liu C, Harding S, Bristow RG, Cutz JC, et al. Ionizing radiation activates AMP-activated kinase (AMPK): a target for radiosensitization of human cancer cells. Int J Radiat Oncol Biol Phys. 2010; 78: 221-229.

174. Sun Y, Connors KE, Yang DQ. AICAR induces phosphorylation of AMPK in an ATM-dependent, LKB1-independent manner. Mol Cell Biochem. 2007; 306: 239-245.

175. Fu X, Wan S, Lyu YL, Liu LF, Qi H. Etoposide induces ATM-dependent mitochondrial biogenesis through AMPK activation. PLoS One. 2008; 3: e2009.

176. Vazquez-Martin A, Oliveras-Ferraros C, Cufí S, Martin-Castillo B, Menendez JA. Metformin activates an ataxia telangiectasia mutated (ATM)/Chk2-regulated DNA damage-like response. Cell Cycle. 2011; 10: 1499-1501.

177. Yee SW, Chen L, Giacomini KM. The role of ATM in response to metformin treatment and activation of AMPK. Nat Genet. 2012; 44: 359-360.

178. Menendez JA, Cufí S, Oliveras-Ferraros C, Martin-Castillo B, Joven J, Vellon L, et al. Metformin and the ATM DNA damage response (DDR): accelerating the onset of stress-induced senescence to boost protection against cancer. Aging (Albany NY). 2011; 3: 1063-1077.

179. GoDARTS and UKPDS Diabetes Pharmacogenetics Study Group, Wellcome Trust Case Control Consortium, Zhou K, Bellenguez C, Spencer CC, Bennett AJ, et al. Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes. Nat Genet. 2011; 43: 117-120.

180. Joven J, Menéndez JA, Fernandez-Sender L, Espinel E, Rull A, Beltrán-Debón R, et al. Metformin: a cheap and well-tolerated drug that provides benefits for viral infections. HIV Med. 2013; 14: 233-240.

181. Kalender A, Selvaraj A, Kim SY, Gulati P, Brûlé S, Viollet B, et al. Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPasedependent manner. Cell Metab. 2010; 11: 390-401.

182. Goberdhan DC, Meredith D, Boyd CA, Wilson C. PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids. Development. 2005; 132: 2365-2375.

183. Goberdhan DC. Intracellular amino acid sensing and mTORC1- regulated growth: new ways to block an old target? Curr Opin Investig Drugs. 2010; 11: 1360-1367.

184. Heublein S, Kazi S, Ogmundsdóttir MH, Attwood EV, Kala S, Boyd CA, et al. Proton-assisted amino-acid transporters are conserved regulators of proliferation and amino-acid-dependent mTORC1 activation. Oncogene. 2010; 29: 4068-4079.

185. Metzner L, Dorn M, Markwardt F, Brandsch M. The orally active antihyperglycemic drug beta-guanidinopropionic acid is transported by the human proton-coupled amino acid transporter hPAT1. Mol Pharm. 2009; 6: 1006-1011.

186. Matsui T, Fukuda M. Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino-acid transporter PAT4. EMBO Rep. 2013; 14: 450-457.

187. Foretz M, Hébrard S, Leclerc J, Zarrinpashneh E, Soty M, Mithieux G, et al. Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state. J Clin Invest. 2010; 120: 2355-2369.

188. Li S, Brown MS, Goldstein JL. Bifurcation of insulin signaling pathway in rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of gluconeogenesis. Proc Natl Acad Sci U S A. 2010; 107: 3441-3446.

189. Laplante M, Sabatini DM. mTORC1 activates SREBP-1c and uncouples lipogenesis from gluconeogenesis. Proc Natl Acad Sci U S A. 2010; 107: 3281-3282.

190. Gross DN, van den Heuvel AP, Birnbaum MJ. The role of FoxO in the regulation of metabolism. Oncogene. 2008; 27: 2320-2336.

191. Sekine K, Chen YR, Kojima N, Ogata K, Fukamizu A, Miyajima A. Foxo1 links insulin signaling to C/EBPalpha and regulates gluconeogenesis during liver development. EMBO J. 2007; 26: 3607-3615.

192. Onuma H, Vander Kooi BT, Boustead JN, Oeser JK, O’Brien RM. Correlation between FOXO1a (FKHR) and FOXO3a (FKHRL1) binding and the inhibition of basal glucose-6-phosphatase catalytic subunit gene transcription by insulin. Mol Endocrinol. 2006; 20: 2831-2847.

193. Greer EL, Banko MR, Brunet A. AMP-activated protein kinase and FoxO transcription factors in dietary restriction-induced longevity. Ann N Y Acad Sci. 2009; 1170: 688-692.

194. Kramer JM, Davidge JT, Lockyer JM, Staveley BE. Expression of Drosophila FOXO regulates growth and can phenocopy starvation. BMC Dev Biol. 2003; 3: 5.

195. Lettieri Barbato D, Tatulli G, Aquilano K, Ciriolo MR. FoxO1 controls lysosomal acid lipase in adipocytes: implication of lipophagy during nutrient restriction and metformin treatment. Cell Death Dis. 2013; 4: e861.

196. Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, et al. Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1alpha interaction. Nature. 2003; 423: 550-555.

197. Li H, Lee J, He C, Zou MH, Xie Z. Suppression of the mTORC1/STAT3/ Notch1 pathway by activated AMPK prevents hepatic insulin resistance induced by excess amino acids. Am J Physiol Endocrinol Metab. 2014; 306: E197-209.

198. Kane S, Sano H, Liu SC, Asara JM, Lane WS, Garner CC, et al. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J Biol Chem. 2002; 277: 22115-22118.

199. Thong FS, Bilan PJ, Klip A. The Rab GTPase-activating protein AS160 integrates Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4 traffic. Diabetes. 2007; 56: 414-423.

200. Speckmann B, Walter PL, Alili L, Reinehr R, Sies H, Klotz LO, et al. Selenoprotein P expression is controlled through interaction of the coactivator PGC-1alpha with FoxO1a and hepatocyte nuclear factor 4alpha transcription factors. Hepatology. 2008; 48: 1998-2006.

201. Misu H, Takamura T, Takayama H, Hayashi H, Matsuzawa-Nagata N, Kurita S, et al. A liver-derived secretory protein, selenoprotein P, causes insulin resistance. Cell Metab. 2010; 12: 483-495.

202. Takayama H, Misu H, Iwama H, Chikamoto K, Saito Y, Murao K, et al. Metformin suppresses expression of the selenoprotein P gene via an AMP-activated kinase (AMPK)/FoxO3a pathway in H4IIEC3 hepatocytes. J Biol Chem. 2014; 289: 335-345.

203. Larsson O, Morita M, Topisirovic I, Alain T, Blouin MJ, Pollak M, et al. Distinct perturbation of the translatome by the antidiabetic drug metformin. Proc Natl Acad Sci U S A. 2012; 109: 8977-8982.

204. Zhou H, Luo Y, Huang S. Updates of mTOR inhibitors. Anticancer Agents Med Chem. 2010; 10: 571-581.

205. Melnik BC. Western diet-mediated mTORC1-signaling in acne, psoriasis, atopic dermatitis, and related diseases of civilization: Therapeutic role of plant-derived natural mTORC1 inhibitors. Watson RR, Zibadi S, editors. In: Bioactive Dietary Factors and Plant Extracts in Dermatology. Nutrition and Health, Springer Science + Business Media New York. 2013. 397-419.

206. Wander SA, Hennessy BT, Slingerland JM. Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy. J Clin Invest. 2011; 121: 1231-1241.

207. Feldman ME, Apsel B, Uotila A, Loewith R, Knight ZA, Ruggero D, et al. Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol. 2009; 7: e38.

208. Kim HJ, Zhang XH, Park EY, Shin KH, Choi SH, Chun BG, et al. Metformin decreases meal size and number and increases c-Fos expression in the nucleus tractus solitarius of obese mice. Physiol Behav. 2013; 110-111: 213-20.

209. Glueck CJ, Aregawi D, Agloria M, Winiarska M, Sieve L, Wang P. Sustainability of 8% weight loss, reduction of insulin resistance, and amelioration of atherogenic-metabolic risk factors over 4 years by metformin-diet in women with polycystic ovary syndrome. Metabolism. 2006; 55: 1582-1589. 

210. Freemark M, Bursey D. The effects of metformin on body mass index and glucose tolerance in obese adolescents with fasting hyperinsulinemia and a family history of type 2 diabetes. Pediatrics. 2001; 107: E55.

211. Glueck CJ, Fontaine RN, Wang P, Subbiah MT, Weber K, Illig E, et al. Metformin reduces weight, centripetal obesity, insulin, leptin, and low-density lipoprotein cholesterol in nondiabetic, morbidly obese subjects with body mass index greater than 30. Metabolism. 2001; 50: 856-861.

212. Matsui Y, Hirasawa Y, Sugiura T, Toyoshi T, Kyuki K, Ito M. Metformin reduces body weight gain and improves glucose intolerance in high-fat diet-fed C57BL/6J mice. Biol Pharm Bull. 2010; 33: 963-970.

213. Ibáñez L, Lopez-Bermejo A, Diaz M, Marcos MV, de Zegher F. Pubertal metformin therapy to reduce total, visceral, and hepatic adiposity. J Pediatr. 2010; 156: 98-102.

214. Lee CG, Boyko EJ, Barrett-Connor E, Miljkovic I, Hoffman AR, Everson-Rose SA, et al. Insulin sensitizers may attenuate lean mass loss in older men with diabetes. Diabetes Care. 2011; 34: 2381-2386.

215. Brufani C, Crinò A, Fintini D, Patera PI, Cappa M, Manco M. Systematic review of metformin use in obese nondiabetic children and adolescents. Horm Res Paediatr. 2013; 80: 78-85.

216. Wang H, Ni Y, Yang S, Li H, Li X, Feng B. The effects of gliclazide, metformin, and acarbose on body composition in patients with newly diagnosed type 2 diabetes mellitus. Curr Ther Res Clin Exp. 2013; 75: 88-92.

217. McDonagh MS, Selph S, Ozpinar A, Foley C. Systematic review of the benefits and risks of metformin in treating obesity in children aged 18 years and younger. JAMA Pediatr. 2014; 168: 178-184.

218. Cicero AF, Tartagni E, Ertek S. Metformin and its clinical use: new insights for an old drug in clinical practice. Arch Med Sci. 2012; 8: 907-917.

219. Salpeter SR, Buckley NS, Kahn JA, Salpeter EE. Meta-analysis: metformin treatment in persons at risk for diabetes mellitus. Am J Med. 2008; 121: 149-157.

220. Woo SL, Xu H, Li H, Zhao Y, Hu X, Zhao J, et al. Metformin ameliorates hepatic steatosis and inflammation without altering adipose phenotype in diet-induced obesity. PLoS One. 2014; 9: e91111.

221. Teede HJ, Meyer C, Norman RJ. Insulin-sensitisers in the treatment of polycystic ovary syndrome. Expert Opin Pharmacother. 2005; 6: 2419-2427.

222. Pasquali R, Gambineri A. Insulin sensitizers in polycystic ovary syndrome. Front Horm Res. 2013; 40: 83-102.

223. Melnik B. Dietary intervention in acne: Attenuation of increased mTORC1 signaling promoted by Western diet. Dermatoendocrinol. 2012; 4: 20-32.

224. Melnik BC, Schmitz G. Are therapeutic effects of antiacne agents mediated by activation of FoxO1 and inhibition of mTORC1? Exp Dermatol. 2013; 22: 502-504.

225. Melnik BC, John SM, Plewig G. Acne: risk indicator for increased body mass index and insulin resistance. Acta Derm Venereol. 2013; 93: 644-649.

226. Goldberg R, Temprosa M, Otvos J, Brunzell J, Marcovina S, Mather K, et al. Lifestyle and metformin treatment favorably influence lipoprotein subfraction distribution in the Diabetes Prevention Program. J Clin Endocrinol Metab. 2013; 98: 3989-3998.

227. Martinet W, De Loof H, De Meyer GR. mTOR inhibition: a promising strategy for stabilization of atherosclerotic plaques. Atherosclerosis. 2014; 233: 601-607.

228. Preiss D, Lloyd SM, Ford I, McMurray JJ, Holman RR, Welsh P,et al. Metformin for non-diabetic patients with coronary heart disease (the CAMERA study): a randomised controlled trial. Lancet Diabetes Endocrinol. 2014; 2: 116-124.

229. Erdemoglu E, Güney M, Giray SG, Take G, Mungan T. Effects of metformin on mammalian target of rapamycin in a mouse model of endometrial hyperplasia. Eur J Obstet Gynecol Reprod Biol. 2009; 145: 195-199.

230. Ben Sahra I, Le Marchand-Brustel Y, Tanti JF, Bost F. Metformin in cancer therapy: a new perspective for an old antidiabetic drug? Mol Cancer Ther. 2010; 9: 1092-1099.

231. Jalving M, Gietema JA, Lefrandt JD, de Jong S, Reyners AK, Gans RO, et al. Metformin: taking away the candy for cancer? Eur J Cancer. 2010; 46: 2369-2380.

232. Micic D, Cvijovic G, Trajkovic V, Duntas LH, Polovina S. Metformin: its emerging role in oncology. Hormones (Athens). 2011; 10: 5-15.

233. Algire C, Amrein L, Bazile M, David S, Zakikhani M, Pollak M. Diet and tumor LKB1 expression interact to determine sensitivity to anti-neoplastic effects of metformin in vivo. Oncogene. 2011; 30: 1174- 1182.

234. McCarty MF. mTORC1 activity as a determinant of cancer risk-- rationalizing the cancer-preventive effects of adiponectin, metformin, rapamycin, and low-protein vegan diets. Med Hypotheses. 2011; 77: 642-648.

235. Vitale-Cross L, Molinolo AA, Martin D, Younis RH, Maruyama T, Patel V, et al. Metformin prevents the development of oral squamous cell carcinomas from carcinogen-induced premalignant lesions. Cancer Prev Res (Phila). 2012; 5: 562-573.

236. Grimaldi C, Chiarini F, Tabellini G, Ricci F, Tazzari PL, Battistelli M, et al. AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications. Leukemia. 2012; 26: 91-100.

237. Melnik BC, John SM, Carrera-Bastos P, Cordain L. The impact of cow’s milk-mediated mTORC1-signaling in the initiation and progression of prostate cancer. Nutr Metab (Lond). 2012; 9: 74.

238. Risk of cancer in diabetes: the effect of metformin. ISRN Endocrinol. 2013; 2013: 636927.

239. Hardie DG. AMPK: a target for drugs and natural products with effects on both diabetes and cancer. Diabetes. 2013; 62: 2164-2172.

240. Yin M, Zhou J, Gorak EJ, Quddus F. Metformin is associated with survival benefit in cancer patients with concurrent type 2 diabetes: a systematic review and meta-analysis. Oncologist. 2013; 18: 1248- 1255.

241. Ko EM, Walter P, Jackson A, Clark L, Franasiak J, Bolac C, et al. Metformin is associated with improved survival in endometrial cancer. Gynecol Oncol. 2014; 132: 438-442.

242. Cerezo M, Tichet M, Abbe P, Ohanna M, Lehraiki A, Rouaud F, et al. Metformin blocks melanoma invasion and metastasis development in AMPK/p53-dependent manner. Mol Cancer Ther. 2013; 12: 1605- 1615.

243. Russo GL, Russo M, Ungaro P. AMP-activated protein kinase: a target for old drugs against diabetes and cancer. Biochem Pharmacol. 2013; 86: 339-350.

244. Soares HP, Ni Y, Kisfalvi K, Sinnett-Smith J, Rozengurt E. Different patterns of Akt and ERK feedback activation in response to rapamycin, active-site mTOR inhibitors and metformin in pancreatic cancer cells. PLoS One. 2013; 8: e57289. 

245. Sinnett-Smith J, Kisfalvi K, Kui R, Rozengurt E. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: dependence on glucose concentration and role of AMPK. Biochem Biophys Res Commun. 2013; 430: 352-357.

246. Tseng CH. Metformin may reduce bladder cancer risk in Taiwanese patients with type 2 diabetes. Acta Diabetol. 2014; 51: 295-303.

247. Rosilio C, Ben-Sahra I, Bost F, Peyron JF. Metformin: a metabolic disruptor and anti-diabetic drug to target human leukemia. Cancer Lett. 2014; 346: 188-196.

248. Checkley LA, Rho O, Angel JM, Cho J, Blando J, Beltran L, et al. Metformin inhibits skin tumor promotion in overweight and obese mice. Cancer Prev Res (Phila). 2014; 7: 54-64.

249. Gupta A, Bisht B, Dey CS. Peripheral insulin-sensitizer drug metformin ameliorates neuronal insulin resistance and Alzheimer’s-like changes. Neuropharmacology. 2011; 60: 910-920.

250. Patrone C, Eriksson O, Lindholm D. Diabetes drugs and neurological disorders: new views and therapeutic possibilities. Lancet Diabetes Endocrinol. 2014; 2: 256-262.

251. Melnik BC, John SM, Schmitz G. Over-stimulation of insulin/IGF-1 signaling by western diet may promote diseases of civilization: lessons learnt from laron syndrome. Nutr Metab (Lond). 2011; 8: 41.

252. Excessive leucine-mTORC1-signalling of cow milk-based infant formula: the missing link to understand early childhood obesity. J Obes. 2012; 2012: 197653.

253. Melnik BC, Schmitz G, John S, Carrera-Bastos P, Lindeberg S, Cordain L. Metabolic effects of milk protein intake strongly depend on pre-existing metabolic and exercise status. Nutr Metab (Lond). 2013; 10: 60.

254. Yamin HB, Barnea M, Genzer Y, Chapnik N, Froy O. Long-term commercial cow’s milk consumption and its effects on metabolic parameters associated with obesity in young mice. Mol Nutr Food Res. 2014; 58: 1061-1068.

255. Melnik BC, John SM, Schmitz G. Milk is not just food but most likely a genetic transfection system activating mTORC1 signaling for postnatal growth. Nutr J. 2013; 12: 103.

256. McCormack SE, Shaham O, McCarthy MA, Deik AA, Wang TJ, Gerszten RE, et al. Circulating branched-chain amino acid concentrations are associated with obesity and future insulin resistance in children and adolescents. Pediatr Obes. 2013; 8: 52-61.

257. Morris C, O’Grada C, Ryan M, Roche HM, Gibney MJ, Gibney ER, et al. The relationship between BMI and metabolomic profiles: a focus on amino acids. Proc Nutr Soc. 2012; 71: 634-638.

258. Lu J, Xie G, Jia W, Jia W. Insulin resistance and the metabolism of branched-chain amino acids. Front Med. 2013; 7: 53-59.

259. Hoppe C, Mølgaard C, Vaag A, Barkholt V, Michaelsen KF. High intakes of milk, but not meat, increase s-insulin and insulin resistance in 8-year-old boys. Eur J Clin Nutr. 2005; 59: 393-398.

260. Patti ME, Brambilla E, Luzi L, Landaker EJ, Kahn CR. Bidirectional modulation of insulin action by amino acids. J Clin Invest. 1998; 101: 1519-1529.

261. Krebs M, Krssak M, Bernroider E, Anderwald C, Brehm A, Meyerspeer M, et al. Mechanism of amino acid-induced skeletal muscle insulin resistance in humans. Diabetes. 2002; 51: 599-605.

262. Wang TJ, Larson MG, Vasan RS, Cheng S, Rhee EP, McCabe E, et al. Metabolite profiles and the risk of developing diabetes. Nat Med. 2011; 17: 448-453.

263. Adams SH. Emerging perspectives on essential amino acid metabolism in obesity and the insulin-resistant state. Adv Nutr. 2011; 2: 445-456.

264. Kim JH, Kim JE, Liu HY, Cao W, Chen J. Regulation of interleukin-6-induced hepatic insulin resistance by mammalian target of rapamycin through the STAT3-SOCS3 pathway. J Biol Chem. 2008; 283: 708-715.

265. Kim JH, Yoon MS, Chen J. Signal transducer and activator of transcription 3 (STAT3) mediates amino acid inhibition of insulin signaling through serine 727 phosphorylation. J Biol Chem. 2009; 284: 35425-35432.

266. Yasuda M, Tanaka Y, Kume S, Morita Y, Chin-Kanasaki M, Araki H, et al. Fatty acids are novel nutrient factors to regulate mTORC1 lysosomal localization and apoptosis in podocytes. Biochim Biophys Acta 2014; 1842:1097-1108.