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Keywords:

  • AMPK;
  • mTOR;
  • adipogenesis;
  • white adipose tissue;
  • brown adipose tissue;
  • obesity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

Recent advances have demonstrated that the adipose tissue plays a central role in regulating overall energy balance. Obesity results from a chronic deregulation of energy balance, with energy intake exceeding energy expenditure. Recently, new mechanisms that control the obesity phenotype such as the equilibrium between white and brown adipose tissue function has been identified. In this context, it is becoming increasingly clear that in addition to cellular growth, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) also regulate lipid metabolism and adipogenesis. Here, we review recent advances in the understanding of the molecular mechanisms involved in white and brown differentiation programs focusing on AMPK and mTOR signaling pathways, which may play differential roles in white adipose tissue and brown adipose tissue development. In view of the worldwide epidemic of obesity and its associated metabolic disorders such as insulin resistance and type 2 diabetes, targeting these kinases may represent a potential approach for reducing adiposity and improving obesity-related diseases. © 2013 IUBMB Life, 65(7):572–583, 2013.


Abbreviations
ADSC

adipose-derived stem cells

AICAR

5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside

AMPK

AMP-activated protein kinase

BAT

brown adipose tissue

CaM

calmodulin

CaMKK2

Ca2 +/CaM-dependent protein kinase kinase 2

C/EBP

CCAAT enhancer-binding proteins

4E-BP1

eukaryotic initiation factor 4E-binding protein 1

FABP4

fatty acid binding protein

LKB1

liver kinase B1

MAPK

mitogen-activated protein kinases

MSC

mesenchymal stem cells

mTOR

mammalian target of rapamycin

mTORC1

mTOR complex 1

mTORC2

mTOR complex 2

PGC-1α

peroxisome proliferator-activated receptor-alpha coactivator 1

PI3K

phosphoinositide 3-kinase

PPAR

peroxisome proliferator-activated receptor

p70S6K

70-kDa ribosomal protein S6 kinase

TSC

tuberous sclerosis complex

TZDs

thiazolidinediones

T2D

type 2 diabetes

UCP

uncoupling protein

WAT

white adipose tissue

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

Adipose tissue plays an active role in energy balance because it is not only a lipid storing and mobilizing organ (white fat) but also consists of functionally specialized tissue able to produce heat (brown fat). Animals have highly integrated systems to regulate the proper balance between energy expenditure and storage capacity. However, nowadays there is excessive consumption of energy-rich foods as well as sedentary behavior, which have led to an increase in the prevalence of obesity and associated metabolic disorders such as type 2 diabetes (T2D) and cardiovascular diseases. White and brown adipocytes are organized to form a large organ with discrete anatomy, specific vascular and nerve supplies, and high physiological plasticity. Adipose tissues can thus be considered a multidepot organ that contributes to many of the crucial survival needs such as thermogenesis, immune responses, and fuel for metabolism [1]. In this context, our understanding of adipose tissue biology and the role of adipocytes in obesity-related diseases such as T2D has benefited greatly from the study of the molecular mechanisms controlling the differentiation process of white and brown adipocytes. Although adipogenesis is considered an active area of research and last years have brought to light some examples of potential factors differentially regulating white versus brown cell differentiation, the developmental relationship between white and brown adipose tissue (BAT) is far from clear. Emerging data reveal a divergence between white and brown precursor cells in early development but also a great plasticity of adipose tissue where several conditions associated with enhanced energy expenditure correlate with the appearance of a brown phenotype dispersed within white adipose tissue (WAT). It is well known that the complex protein network that senses and precisely reacts to environmental changes is thus mainly regulated by rapid and reversible post-translational modifications such as phosphorylation. This review focuses on the mechanisms by which preadipocytes differentiate into adipocytes and provide a functional and regulatory overview of the serine/threonine protein kinases AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), two interconnected major junctions which have been revealed to play key roles not only in growth control and cell proliferation but also in metabolism. We revise the impact of these two opposed pathways regulating catabolic versus anabolic routes in the control of white and brown adipogenesis processes.

The Adipose Organ

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

The adipose organ consists of several depots located at various anatomical sites that have different physiological functions and pathophysiological roles. Advances in the last two decades in our understanding of adipocyte biology have clarified their role as a key regulator of both energy balance and intermediary metabolism. WAT has long been recognized as the main site for the storage of energy excess derived from food intake [2]. Nevertheless, adipose tissue is not only an inert energy-storage depot, but an endocrine organ releasing a wide range of adipokines, which can regulate immune responses, blood pressure, angiogenesis, hemostasis, bone mass, and thyroid and reproductive function [3]. Moreover, it has also been described that adipose tissue secretes lipid factors (lipokines) such as C16:1n7-palmitoleate, to communicate with distant organs and regulate systemic metabolic homeostasis [4]. Thus, owing to its endocrine function and its classic role as lipid storage, the presence of functional adipose tissue in proper proportion to body size is essential to control whole-body metabolism. In contrast, BAT is specialized primarily for cold-induced nonshivering thermogenesis. The unique thermogenic capacity of BAT results from the expression of the uncoupling protein (UCP)1, which is located in the mitochondrial inner membrane. This protein allows the consumption of the energy derived from fatty acid (FA) oxidation for the generation of heat. Until quite recently, BAT was thought to be of metabolic importance only in small mammals and infant humans. However, recent studies using positron-emission tomography scanning suggest that adult humans have several discrete areas of metabolically active BAT [5, 6]. In this regard, BAT may be far more relevant in human metabolism than was previously appreciated, and loss of BAT function is linked to obesity and metabolic disease [7]. Most fat depots can be characterized as either brown or white although some brown fat cells, which have been recently named “brite adipocytes” (“brown-in-white”) [8], can also be found dispersed through white fat depots both in rodents and in humans, forming a multidepot organ with plastic properties (reviewed in ref. [9]).

Fat tissues contain several cell types, including stromal-vascular cells such as fibroblasts, macrophages, smooth muscle cells, pericytes, endothelial cells, and adipogenic progenitor cells. However, mature adipocytes constitute the majority of cells in both WAT and BAT. As adipose tissue plays a central role in whole-body energy metabolism, defects in adipose function can lead to severe metabolic abnormalities. Obesity, which has deleterious effects on metabolic balance and is an important risk factor for the development of insulin resistance and T2D, is characterized by increased adipose tissue mass that results from both increased fat-cell number (hyperplasia) and increased fat-cell size (hypertrophy) [10, 11]. The number of adipocytes present in an organism is determined to a large degree by the adipocyte differentiation process, which generates mature adipocytes from fibroblast-like preadipocytes and mesenchymal stem cells (MSCs) [12]. The knowledge of molecular pathways involved in adipogenic commitment and terminal differentiation as well as how these two stages of adipogenesis is physiologically integrated during development and is essential for the progress of future pharmacological interventions designed to prevent obesity.

Adipogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

Adipocytes are derived from MSCs, which are a self-renewing population of multipotent cells present in bone marrow and many other adult tissues that can differentiate into multiple lineage-specific cells that form bone, fat, cartilage, muscle, and tendon [13]. When MSCs are appropriately stimulated, they undergo a multistep process of commitment in which the progenitor cells become restricted to the adipocyte lineage. Recruitment to this lineage gives rise to preadipocytes, which, when induced, undergo multiple rounds of mitosis (mitotic clonal expansion) and then differentiate into adipocytes. Pluripotent stem cell lines such as CH310T1/2, murine preadipocyte cells (i.e., 3T3-L1, 3T3-F442A) as well as MSCs have been indispensable in identifying/characterizing the steps in the commitment and differentiation programs. Whether adipogenesis occurs in the adult adipose tissue remains controversial. Although white adipocyte numbers increase through puberty but are relatively steady in the mature fat pad, within human adult WAT, adipocytes seem to undergo an annual turnover of approximately 10% [10]. In addition, human studies correlating fat mass with cell number have yielded conflicting results [10, 14, 15]. Thus, further work must be carried out to conclude whether increased adipogenesis plays a crucial role in the development of obesity in humans.

Historically, white and brown adipocytes were thought to derive from the same precursor cell. However, recent studies have revealed that BAT is more closely related to skeletal muscle than WAT, as both muscle and BAT have progenitors that express the early muscle marker myogenic factor 5 (MYF5), contrasting to WAT [16]. These surprising findings indicate that there is a divergence between white and brown precursor cells in early development and it has been reported that transcription factors such as PRMD16 may control brown fat determination [16]. In addition, upon prolonged cold exposure or in response to β-adrenergic signaling, WAT can display characteristics of BAT, such as expression of UCP1. The brown-like adipocytes within WAT are developmentally distinct from brown adipocytes found in BAT [16]; thus, the plasticity of WAT in response to these stimuli may be owing to transdifferentiation of mature white adipocytes into brown adipocytes [17] or the result of de novo adipocyte formation.

Nevertheless, the process of differentiating into brown or white adipocytes appears to employ a similar canonical transcriptional pattern. Along these lines, several nuclear factors regulate some processes that are common to both cell types such as lipogenesis. Once the preadipocyte population has been defined (commitment), hormonal stimulation initiates a transcriptional regulatory cascade that results in a gene expression profile specific for adipocyte functions. Several critical transcription factors and the crosstalk among them have been well studied, including peroxisome proliferator-activated receptors (PPARs) and CCAAT enhancer-binding proteins (C/EBPs) [18]. Exogenous adipogenic stimuli induce the expression of C/EBPβ and δ in preadipocytes rapidly and transiently, which in turn mediate the induction of PPARγ and C/EBPα expression. Once activated upon initiation of differentiation, both transcription factors form a positive feedback loop to reinforce and maintain each other's expression. This cooperative interplay activates the essential adipogenic gene expression required for adipocyte functions and maintains the terminally differentiated state [18]. Although C/EBPα cannot promote adipogenesis in the absence of PPARγ, PPARγ is both necessary and sufficient for adipogenesis. However, PPARγ is only one of the many key modulators of adiposity. More specifically, some coactivators such as peroxisome proliferator-activated receptor-alpha coactivator 1 (PGC-1α) have been described as characteristic of BAT in opposition to WAT; and other nuclear receptors including PPARδ, liver X receptors or estrogen-related receptors have been proposed as playing a potential role in mediating brown fat differentiation (reviewed in ref. [19]).

Adipogenesis is also influenced by a variety of extrinsic factors and intracellular signaling pathways. In this regard, upstream signals regulating the expression and activation of the transcriptional events during adipocyte differentiation are far from clear. Insulin is a potent inducer of adipogenesis, and differentiation of white and brown adipocytes requires many components of the insulin signaling pathways. The phosphoinositide 3-kinase (PI3K) pathway appears to be required for the differentiation of 3T3-L1 adipocytes and several studies have also revealed an important role for the insulin/IGF-1 system in BAT development [19]. In contrast, mitogen-activated protein kinase (MAPK) pathways differentially regulate adipogenesis. Although ERK1/2 pathways display both positive and negative effects through the process, the role of p38MAPK seems to be related to UCP1 expression in brown adipocytes [20], but is still controversial in white (reviewed in ref. [21]). In addition, adipogenesis is regulated by calcium as increases of intracellular Ca2 + in preadipocytes during the early phase of differentiation can inhibit adipogenesis [22]. Although the mechanism by which Ca2 + represses adipogenesis is not fully understood, the calmodulin (CaM) kinase cascade through Ca2 +/CaM-dependent protein kinase kinase 2 (CaMKK2) activation seems to play an important role (see below).

The Energy-Sensing Kinase AMPK and Adipose Tissue

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

AMPK is a heterotrimeric serine/threonine kinase consisting of one catalytic (α) and two regulatory (β and γ) subunits. Multiple isoforms of each mammalian subunit exist (α1-2, β1-2, γ1-3) and are differentially expressed in various tissues enabling the potential formation of 12 heterotrimeric combinations that are thought to exhibit differences in subcellular localization and tissue-specific signaling functions [23]. In response to energy starvation or cellular stress, AMPK is allosterically activated by AMP through its binding to AMPKγ regulatory subunit, which also promotes phosphorylation by the upstream kinases such as liver kinase B1 (LKB1) and CaMKK2, and protects the enzyme against dephosphorylation [23]. Therefore, AMPK activation stimulates pathways, leading to ATP production such as FA oxidation, and blocking anabolic processes that consume ATP such as lipid and cholesterol synthesis (Fig. 1). Regarding glucose metabolism, it has been described that 3T3-L1 adipocytes treated with AMPK activators showed enhanced GLUT4 translocation [24]. Similarly, in human adipocytes TNF-α increases basal glucose uptake and GLUT4 in the plasma membrane by a mechanism dependent on AMPK activation [25]. However, these AMPK activators seem to inhibit insulin-stimulated glucose uptake [26]. It has been proposed that in contrast to skeletal muscle, in which AMPK stimulation might promote glucose transport to provide ATP as a fuel in response to contraction, AMPK stimulation might inhibit insulin-stimulated glucose transport in adipocytes, inhibiting triacylglycerol synthesis to conserve ATP. AKT substrate of 160 kDa (AS160) was revealed as a mediator of both insulin- and contraction-stimulated glucose uptake. However, their paralog TBC1D1 expression is higher in skeletal muscle compared with adipose tissue and it has been suggested that AMPK may play a greater role in phosphorylation of TBC1D1 than for AS160 [27].

image

Figure 1. AMPK activation leads to energy preservation for cell survival at the expense of growth and proliferation. AMPK is allosterically activated by AMP in response to energy-poor conditions such as energy starvation or cellular stress through its binding to AMPKγ regulatory subunit, which also promotes phosphorylation by the upstream kinases LKB1 and CaMKK2. AMPK activity is also controlled by the histone-modifying proteins acetyltransferases p300 ( + ) and deacetylase HDAC1 (−). Fasting, exercise, and cold exposure have been shown to activate AMPK through increasing cAMP levels in a β-adrenergic-dependent mechanism. AMPK activation by catecholamines such as epinephrine in adipose tissue seems to be secondary to the effects on lipolysis. The mechanism by which the adipokines leptin and adiponectin induce AMPK activation is not known. Antidiabetic drugs such as metformin and TZDs have also shown to induce AMPK activation in adipocytes. All these AMPK activation pathways lead to ATP production by stimulating catabolic processes such as FA oxidation, as well as inhibiting anabolic pathways such as lipid/cholesterol synthesis. White and brown fat development is differentially regulated by AMPK. Thus, white adipogenesis is negatively controlled by AMPK but this kinase is required for brown adipocyte differentiation, suggesting that this kinase differentially regulate white and brown fat development. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Overall, AMPK activation leads to energy preservation for cell survival at the expense of growth and proliferation. The AMP:ATP ratio within the cell is critical in regulating AMPK activity. Recent studies have revealed that AMPK activity is also controlled by histone-modifying proteins such as the acetyltransferases p300 and deacetylase HDAC1, which antagonize each other through modification of the side chains of lysine residues in histone proteins (Fig. 1). Emerging data reveal that deacetylation of AMPK enhances physical interaction with the upstream kinase LKB1, leading to AMPK phosphorylation and activation [28]. However, further studies are necessary to address the functional significance of nuclear acetylation/deacetylation and cytoplasmic phosphorylation with regard to the regulation of short- and long-term metabolic effects of AMPK activation in adipocytes.

The important role of AMPK in regulating adipocyte lipolysis, glucose uptake, and FA oxidation has been extensively reviewed [29]. Nevertheless and as we discuss below, AMPK also regulates the expression of several genes involved in the control of cellular differentiation and mitochondrial function in the adipocyte. AMPK activity in BAT is higher than in liver [30], and heterotrimeric complexes containing the α1 catalytic subunit account for most of the activities of AMPK in adipocytes. However, some studies have revealed that both AMPKα1 and α2 could potentially play an important role in the regulation of adiposity [31, 32].

Catecholamines activate AMPK both in white and in brown adipocytes. In WAT, fasting, exercise, and cold exposure have been shown to activate AMPK through increasing β-adrenergic stimulation and cAMP levels (Fig. 1) [30, 32-34]. Accordingly, decreasing the levels of GRK2, a kinase known to desensitize adrenergic receptors, also enhances AMPK phosphorylation, FA oxidation, and thermogenesis after acute cold exposure [35]. There is a good evidence that activation of AMPK in adipocytes by cAMP-inducing agents such as epinephrine is a consequence of lipolysis and not of PKA activation [36], suggesting that AMPK activation might be caused by an increase in the AMP:ATP ratio, as consequence to the acylation of FAs. Therefore, in this context, AMPK activation appears to control the energy depletion and oxidative stress caused by lipolysis.

More recently, it has been described that the adrenergic nervous system regulates AMPK in vivo in an adrenergic receptor-dependent manner by modulating AMPKα but not AMPKβ subunit levels [37]. On the other hand, adipokines such as leptin [38] and adiponectin [39] have also been shown to induce AMPK activation in WAT. Similarly, biguanides such as metformin and thiazolidinediones (TZDs), PPARγ agonists that have been classically used as antidiabetic drugs can also induce AMPK activity in adipocytes. Although the mechanism of AMPK activation by metformin and PPARγ agonists seems to be dependent on the inhibition of mitochondrial respiration and the increase in intracellular AMP:ATP ratio [40], TZDs may also act on AMPK by triggering the release of adiponectin [41]. However, it still remains unclear how signaling through the leptin and adiponectin receptors leads to AMPK activation (Fig. 1). Otherwise, pharmacological effects of PPARγ agonists exerted through the modulation of AMPK activity seem to be independent of receptor activation [42].

Role of AMPK in Adipogenesis

Compatible with the role of AMPK in shutting down anabolic pathways when activated, studies from several laboratories have also revealed that AMPK negatively regulates white adipogenesis. Thus, treatment of 3T3-L1 fibroblasts with AMPK agonists such as 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) has been shown to inhibit cell proliferation blocking clonal expansion, an essential step for the differentiation process in murine 3T3L1 but not in human primary cells [43, 44]. Similarly, hypoxic conditions biphasically activate AMPK and concomitantly block clonal expansion of preadipocytes attenuating adipocyte differentiation [45]. On the other hand, AMPK activation in the early phase of differentiation inhibits PPARγ and C/EBPα expression as well as late adipogenic markers such as fatty acid synthase (FAS) and acetyl-CoA carboxylase. Thus, it is well accepted that AMPK activity is inversely related to white adipogenesis. Recently, Lee et al. reported that inhibition of adipogenesis by AICAR-induced AMPK activation in 3T3-L1 cells is dependent on the WNT/β-catenin pathway. Thus, inhibition of adipogenesis by AICAR was accompanied by significantly enhanced expression and nuclear accumulation of β-catenin, and knocking down β-catenin expression by siRNA prevented the suppressive effects of AICAR on the expression of major genes involved in adipogenesis such as PPARγ, C/EBPα, fatty acid-binding protein (FABP4), and lipoprotein lipase [46]. It has been proposed that the Wnt/β-catenin signaling pathway might maintain the undifferentiated state of preadipocytes by inhibiting adipogenic gene expression. In fact, a reciprocal relationship between β-catenin and PPARγ has been described during adipogenesis. Thus, PPARγ activation resulted in a decrease in β-catenin by a mechanism that involves the proteasome [47]. Once adipocytes are differentiated, activation of AMPK may induce a decrease in adipocyte size through a reduced expression of proteins involved in triglyceride synthesis such as glycerol phosphate acyltransferase and acyl CoA:diacylglycerol acyltransferase [43]. Thus, nutritional and pharmacological approaches to manipulate AMPK activity such as antiobesity therapies are of great interest and subject of recent studies. On the other hand, upstream AMPK kinases, such as CaMKK2, which have been described to regulate AMPK activity in adipocytes, are also related to white adipogenesis. In this context, CaMKK2 null mice have shown increased adiposity and larger adipocytes than wild-type mice. This effect is a consequence of AMPK inhibition as CaMKKβ deletion enhances the differentiation of preadipocytes into mature adipocytes, an effect that was reversed by AICAR-induced AMPK activation [48]. Furthermore, it has recently been demonstrated that AMPK may also play an important role in fate cell decision of human adipose-derived stem cells (ADSCs) as a regulator between osteogenesis and adipogenesis. Thus, inhibition of AMPK reduced osteogenesis but induced adipogenesis of ADSC, even in osteogenic medium [49].

Contrary to the previous data reported on white adipocytes, a recent study carried out at our laboratory has shown that AMPK activation is required for brown adipocyte differentiation [50]. Our study reveals that inhibition of AMPK induced different effects, depending on the stage of differentiation. Thus, AMPK inhibition from the beginning of the process precluded brown adipocyte differentiation, whereas in the later stages AMPK inhibition increased lipid accumulation. No significant differences in cell number were detected, suggesting that AMPK activation is necessary for brown adipogenesis but is not involved in the proliferative step of this process. In addition, we observed that in vivo, AMPK activation with AICAR induced an accumulation of brown adipocytes in WAT detected by immunolocalization with anti UCP1 antibody, which partially correlated with the body weight reduction detected in response to the treatment with AICAR [50]. As no differences were detected in BAT from AICAR- compared to vehicle-treated animals, we hypothesize that only brown preadipocytes or MSCs within white fat depots, and not mature cells in BAT, might be sensitive to AICAR-induced UPC1 expression. Thus, our results support the hypothesis that brown adipocyte-like cells present in WAT and the brown adipocytes constituting BAT are subjected to different control systems. Along these lines, it had been previously reported that AMPK activation induced the expression of UCP1 and mitochondrial enzymes in WAT of obese mice [51]. Our studies support the hypothesis that AMPK may play a positive role in mitochondrial biogenesis and thermogenesis. The opposite effect of AMPK on brown and white adipocytes could be explained by the fact that brown adipocytes specialize in lipid catabolism rather than storage, much like oxidative skeletal muscle tissue. However, the molecular mechanisms by which AMPK is activated during brown adipocyte differentiation are currently under study. A change in energy status might be an appropriate candidate because inhibition of AMP synthesis with the adenosine kinase inhibitor iodotubercidin also precluded brown adipocyte differentiation [50]. Moreover, activation of AMPK by the acquisition of thermogenic properties should not be ruled out. In this regard, the inhibition of AMPK might preclude cell energetic adaptation, and thus impairing the brown adipocyte differentiation process.

The Nutrient-Sensing Kinase mTOR and Adipose Tissue

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

mTOR is a conserved serine–threonine kinase that controls protein synthesis, cell size, and proliferation, according to the availability of amino acids, growth factors, nutrients, and cell energy status. Consequently, mTOR signaling pathway has been described to be involved in several cellular processes including adipogenesis and deregulated in human diseases such as obesity and T2D (reviewed in ref. [52]). mTOR is activated downstream of Akt, and in turn it promotes enhanced translation through its targets eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), 70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic elongation factor 2. mTOR is the catalytic core of two distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), that have different downstream targets and biological functions. The adaptor proteins, Raptor and Rictor, determine the substrate specificity of mTORC1 and mTORC2, respectively. mTORC1 and mTORC2 have been traditionally characterized, depending on their sensitivity to inhibition by the bacterial macrolide rapamycin: mTORC1 as the rapamycin-sensitive and mTORC2 as the insensitive complex (Fig. 2). However, this paradigm might not be entirely precise, as chronic rapamycin, in some cases, inhibits mTORC2 activity [53] and important mTORC1 functions seem to be resistant to inhibition by rapamycin [54]. One of the most important sensors involved in the regulation of mTORC1 activity is the tuberous sclerosis complex (TSC), which is a heterodimer that comprises TSC1 (hamartin) and TSC2 (tuberin). TSC1/2 functions as a GTPase-activating protein for the small Ras-related GTPase Rheb. Although the TSC1–TSC2 complex was believed to function as a heterodimer, recently TBC1D7 has been revealed as a third subunit of the TSC1–TSC2 complex upstream of mTORC1 [55]. Growth factors stimulate mTORC1 through the activation of the canonical insulin and Ras signaling pathways. TSC2 phosphorylation is increased after stimulation through AKT, ERK1/2, and RSK1 [56-58], which leads to the inactivation of TSC1/2 and thus to the activation of mTORC1. Some studies, however, might suggest that the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation is unclear. In this regard, PRAS40 has been revealed as a key mediator of AKT signals to mTOR but also a dominant-negative effector of mTOR over TSC-Rheb signaling. In this context, PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. Thus, AKT activation by growth factors such as insulin might activate mTORC1 in a TSC2-independent manner [59] (Fig. 2). On the other hand, mTORC2 induces basal AKT phosphorylation on Ser473 that precedes and facilitates Thr 308 phosphorylation by PDK1 [60].

image

Figure 2. Schematic diagram of mTOR activation and mTORC1 functions on adipogenesis. mTOR exists in association with two distinct multiprotein complexes: mTORC1 consists of mTOR and four regulatory associated proteins (Raptor, mLST8, PRAS40, and Deptor), whereas mTORC2 complex consists of mTOR and five associated proteins (Rictor, mSin1, Protor-1, mLST8, and Deptor). The heterodimer composed of TSC1 and TSC2 (TSC) is one of the most important sensors involved in the regulation of mTORC1 activity. Growth factors inactivate the TSC1/TSC2 complex by an AKT-dependent mechanism. Inactivation of TSC2 results in activation of mTORC1 via Rheb. Additionally, AKT activation by growth factors such as insulin (and also by amino acids) can activate mTORC1 in a TSC1/2-independent manner (through negative phosphorylation of mTORC1 suppressor, PRAS40). mTORC2 is activated by growth factors in a TSC1/TSC2-independent manner and induces basal AKT phosphorylation on Ser473. The activation of mTORC1 by growth factors such as insulin has key roles controlling white and brown adipogenesis. mTORC1 positively regulates commitment of MSC to white adipogenic program as well as adipogenesis/lipogenesis. In contrast, temporal control of mTOR is required for a proper brown adipocyte differentiation. Early activation of the mTOR signaling pathway followed by a subsequent downregulation of this pathway by an AMPK-dependent mechanism is an absolute requirement to reach a fully differentiated brown phenotype. mTORC2 implication in the control of lipogenesis/adipogenesis seems to be limited in mammals. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Expansion of fat mass in obesity is associated with marked activation of mTOR in adipose tissue, whereas fat mass retraction owing to caloric restriction and fasting is associated with adipose tissue mTOR inhibition. Accordingly, chronic pharmacological inhibition of the mTORC1 signaling pathway is associated with a reduction in adipose tissue mass owing to both reduced adipocyte size and number [61]. Downstream of mTORC1 signaling pathway, deletion of p70S6K protects against age- and diet-induced obesity, and genetic models of obesity exhibit markedly increased p70S6K activity [62]. However, although there is a direct association between mTORC1 activity and adiposity, the mechanism by which mTOR modulates fat mass is far from clear. In this context, the role of mTORC1 in regulating lipid synthesis, which is required for cell growth but also adipocyte differentiation, is starting to be appreciated. In addition, the important role of mTORC1 in mitochondrial metabolism and biogenesis uncovers new signaling pathways in brown fat function and development (see below).

Role of mTor in Adipogenesis

Primarily through the use of the mTORC1-specific inhibitor rapamycin, several studies have concluded that mTORC1 activity is required for proper differentiation of preadipocyte cell lines and primary cultures. Thus, a constitutive activation of mTOR might be required for both the clonal expansion at the early stage of the process and for the execution of the adipogenic program [63, 64]. Rapamycin significantly reduced the expression of most adipocyte marker genes including PPARγ, adipsin, FABP4, and FAS, as well as decreased intracellular lipid accumulation in 3T3-L1 and 3T3-F442A cells, suggesting that rapamycin would affect both lipogenesis and adipogenesis [65] (Fig. 2). Studies performed with stem cells as cellular models also indicate that the mTOR/p70S6K pathway may act downstream of the PI3K/Akt cascade in mediating the adipogenic conversion of MSCs [66]. In fact, although p70S6K might be dispensable for terminal adipocyte differentiation, it is clearly involved in the commitment of embryonic stem cells to early adipocyte progenitors [67]. Mice lacking the specific and essential mTORC1 component Raptor in adipose tissue have confirmed the important role of this complex in adipocyte metabolism and full-body energy homeostasis. Thus, Raptorad−/− mice were protected against diet-induced obesity owing to elevated energy expenditure [68]. Despite the well-established positive role of mTORC1 in regulating adipogenesis and adipose cell maintenance, DEPTOR, which is an endogenous inhibitor of mTORC1 signalling, has also been revealed as a new regulator of adipogenesis as it promotes adipose tissue expansion and adipocyte differentiation by regulating the balance between mTORC1 and AKT activity [69]. It should be noted, however, that DEPTOR impairs mTORC1 action to a much lower degree than rapamycin or Raptor loss. Consequently, the impact of mTORC1 inhibition on the regulation of adipogenesis and WAT accumulation may be different. On the other hand, despite p70S6K activity being markedly increased in models of obesity, the complete blockage of this kinase by rapamycin partially reduced differentiation. It has been hypothesized that adipogenic mTORC1 signaling occurs via the 4E-BP1/eIF4E pathway, rather than through p70S6K [70]. Specifically, the deletion of 4E-BP1 results in a reduction in adipose tissue partially link to the conversion of WAT into BAT, as detected by the expression of UCP1 and PGC-1α in WAT [71]. A molecular link between nutrient status and adipogenesis has been defined as PPARγ activity has been revealed also to be dependent on amino acid sufficiency. Rapamycin has been described to specifically disrupt the positive transcriptional feedback loop between C/EBPα and PPARγ. Thus, a model has been proposed in which the mTOR pathway and the PI3K/Akt pathway would act in parallel to regulate PPARγ activation during adipogenesis by mediating nutrient availability and insulin signals, respectively. Interestingly, mouse knockouts affecting specific mTOR targets suggest that both p70S6K activation and 4E-BP1/2 inhibition might contribute to a proadipogenic role for mTORC1 activation [62, 72]. To summarize, mTORC1 promotes adipogenesis and adipocyte maintenance by several mechanisms including the control of lipogenesis through SREBP-1, PPARγ, and lipin. By contrast and despite two recent reports suggest that mTORC2 function may also affect fat accumulation in yeast, the implication of mTORC2 in the control of lipogenesis/adipogenesis appears to be limited in mammals (reviewed in ref. [52]).

Little is known about specific role of mTOR in brown adipogenesis. Mostly, data from our lab demonstrated that early activation of the mTOR–p70S6K signaling pathway is an absolute requirement to reach a fully differentiated brown phenotype [50]. However, subsequent downregulation of this pathway also seems crucial for the process (Fig. 2). mTORC1 inhibition by rapamycin and siRNA decreased cell proliferation and precluded brown adipocyte differentiation [50]. These negative regulatory effects on brown adipogenesis may be exerted through a mechanism that depends on the control of cell growth, in a similar way to that described in human preadipocytes [65]. Nevertheless, a potential link between mTOR and mitochondrial activities should also be taken into account. The complete inhibition of mTORC1 by loss of Raptor in adipose tissue increases mitochondrial uncoupling [68], whereas studies in an immortalized line of T lymphocytes suggest that mTORC1 activity may play an important role in determining the relative balance between mitochondrial and nonmitochondrial sources of ATP generation [73]. In contrast, in skeletal muscle, mTORC1 is necessary for the maintenance of mitochondrial oxidative function through a mechanism that seems to be dependent on the transcription factor yin-yang 1 (YY1) which is a common target of mTORC1 and PGC-1α [74]. Additionally, TSC1 deletion in hematopoietic stem cells increased mitochondrial biogenesis and elevated levels of reactive oxygen species [75]. Therefore, the influence of mTORC1 in mitochondrial activity seems to be tissue specific. Whether this pathway plays a role in mitochondrial activity in BAT should be clarified and deserves further studies.

AMPK–mTOR Crosstalk in Adipogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

As described above, while AMPK is activated under low-energy conditions, mTOR activity depends on diverse growth-positive signals such as high-energy levels, amino acids, and growth factors. In this regard, energy status of the cell is signaled to mTOR through AMPK. Several studies have revealed the convergence of AMPK and mTOR signaling pathways, pointing to mTOR as a central signal integrator that receives signals arising from growth factors, nutrients, and cellular energy metabolism (Fig. 3). In response to energy depletion (low ATP:ADP ratio), activated AMPK is thought to inhibit mTORC1 activity primarily in the opposite way that growth factors stimulate it, mainly by phosphorylation and activation of the negative regulator TSC2. Additionally, AMPK can reduce mTORC1 activity in response to energy depletion by directly phosphorylating Raptor, which might be crucial for the role of AMPK as a metabolic checkpoint coordinating cell growth with energy status [76]. In vitro, cellular levels of ATP increase mTOR signaling, and mTORC1 itself is thought to serve as an ATP sensor. Thus, AMPK regulates the activities of both p70S6K and 4E-BP1, indicating a convergence of AMPK and mTOR signaling pathways (reviewed in [56]). The interplay of mTOR and AMPK provides a more exact mechanism for mammals to coordinate with the environmental conditions.

image

Figure 3. mTOR as a central signal integrator of the energy status of the cell. mTOR activity is highly controlled by energy levels, amino acids, or growth factors through several mechanisms. (1) In response to low-energy conditions mTORC1 is inhibited directly by AMPK (through Raptor inhibition) or indirectly by TSC2 phosphorylation, resulting in energy preservation. (2) TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3. Inhibition of the serine/threonine kinase GSK-3 in response to Wnt or AKT activation leads to the inactivation of the tumor suppressor complex TSC1/2, followed by activation of the mTOR signaling pathway. GSK3 phosphorylation of TSC2 follows the AMPK-priming phosphorylation. GSK3 and AMPK depend on each other to regulate mTOR signaling pointing to TSC2 as a key node. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

As described above, Akt is a positive regulator of mTOR that mediates the activation of mTORC1 by growth factors but it is also a key regulator of energy metabolism that inhibits AMPK [56]. Historically, the activation of mTORC1 via Akt-mediated phosphorylation of TSC2 and via inhibition of AMPK was viewed as two separate pathways, leading to the activation of mTOR. However, currently it has been proved that AKT lies upstream of both pathways and induces full inhibition of TSC2 and activation of mTORC1 both through direct phosphorylation and by inhibition of AMPK-mediated phosphorylation of TSC2 [77]. TSC2 has been described as a physiological substrate of GSK3 and similar to AKT, it is known that Wnt stimulates the mTORC1 signaling pathway via inhibiting GSK3 phosphorylation of TSC2. More importantly, GSK3 phosphorylates TSC2 after the AMPK-priming phosphorylation and inhibition of GSK3 compromises AMPK-induced TSC2 phosphorylation. Thus, GSK3 and AMPK depend on each other to regulate mTORC1 signaling, pointing to TSC2 as a key node, which integrates Wnt and energy signals via phosphorylation [78] (Fig. 3).

Although it is clear that AMPK and mTOR are key factors in the adipocyte differentiation process and that fat loss induced by fasting or caloric restriction correlates with mTOR inhibition as well as AMPK activation, no such data presently exist for the AMPK–mTOR interplay in this process. Only the study in brown adipocytes reported by our group has investigated the contribution of AMPK–mTOR crosstalk in brown fat adipogenesis [50]. In fact, we have recently shown that the brown adipocyte differentiation program is sequentially controlled by LKB1–AMPK and TSC2–mTOR–p70S6K intracellular signaling pathways. Via pharmacological inhibition as well as genetic knockdown approaches, our study reveals that at early stages the mTOR–p70S6K signaling pathway is essential for brown adipocyte differentiation. The activation of AMPK at later stages, also necessary for brown adipogenesis, would be involved in mTOR–p70S6K shutdown. Our data confirm that inactivation of mTOR throughout the differentiation process is dependent on AMPK activation. Thus, at the beginning of the process, a transitory phosphorylation of TSC2 at the Thr1462 residue, probably induced by an AKT-dependent mechanism, is found, resulting in TSC2 inhibition and consequently in mTOR-dependent p70S6K activation. However, once the LKB1–AMPK cascade is activated through brown adipogenesis, a decrease in the inactive form of TSC2 is detected [50]. Surprisingly, although it has been reported that the Wnt pathway positively regulates mTOR activity [79], our study suggests negative feedback regulation between these cascades. In this regard, the Wnt signaling pathway, known to be involved in brown adipocyte differentiation, is rapidly and transiently activated through the brown differentiation process. On the other hand, it was observed that rapamycin activated the Wnt pathway by delaying β-catenin degradation in which it may represent a novel mTOR–Wnt feedback loop [50].

Conclusions and Future Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

In view of the worldwide epidemic of obesity and associated metabolic disorders, it is important to identify pathways that can be manipulated either genetically or pharmacologically to regulate weight gain. Inhibition of white adipocyte differentiation as well as the promotion of BAT development should be considered as promising strategies in the field of antiobesity therapies. Emerging data reveal that temporal control of several signaling pathways such as AMPK and mTOR is required for a proper differentiation process of fat cells. Specifically, AMPK–mTOR crosstalk has been revealed as one of the mechanisms that differentially regulates brown and white differentiation programs. How these events are integrated in the cells to regulate both adipogenic and a thermogenic program is an exciting topic and requires future studies. On the other hand, AMPK, but also mTOR, seems to be a vital link between immune function and metabolism. In fact, emerging data might reveal a critical role of these kinases in promoting the differentiation, activation, and function of some immune cells [80]. Several studies have established adipose tissue as a new important player of the immune response and recent studies point to accumulation of inflammatory T cells in adipose tissue an early event in obesity even prior to macrophage infiltration [81]. In fact, cells of both innate and adaptive immune system such as macrophages, B and T cells, and dendritic-like cells massively infiltrate adipose tissue in an obese environment. Nevertheless, adipose tissue is well-spread through the body and it should not be ruled out that in physiological conditions this organ could provide immune cells which participate to immune response as has been recently proved for ADSC [82]. In this context, adipose tissue may also be considered as an extramedullary reservoir for immune cells, opening new and fascinating perspectives on a potential role of this tissue not only in obesity-associated metabolic disorders but also in host defense and inflammatory disease.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES

Our lab is supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2008-04043 and SAF2012-36186 to S.F.-V.; PI11/0085 and CB07/08/0012 to J.V.). S.F.-V. acknowledges support from the “Miguel Servet” tenure track program (CP10/00438) from the Fondo de Investigación Sanitaria (FIS) and co-financed by the European Regional Development Fund (ERDF). A.V.-C. and V.C.-M. are supported by FPI and “Juan de la Cierva” fellowships respectively, from the Spanish Ministry.

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  2. Abstract
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  4. The Adipose Organ
  5. Adipogenesis
  6. The Energy-Sensing Kinase AMPK and Adipose Tissue
  7. The Nutrient-Sensing Kinase mTOR and Adipose Tissue
  8. AMPK–mTOR Crosstalk in Adipogenesis
  9. Conclusions and Future Perspectives
  10. Acknowledgments
  11. REFERENCES
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