(AMP)-Activated Protein Kinase: a New Target for Nutraceutical Compounds

International Journal of Molecular Sciences

Review Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds

Fabiola Marín-Aguilar 1, Luis E. Pavillard 1, Francesca Giampieri 2, Pedro Bullón 1 and Mario D. Cordero 1,*

1 Research Laboratory, Oral Medicine Department, University of Sevilla, Sevilla 41009, Spain; [email protected] (F.M.-A.); [email protected] (L.E.P.); [email protected] (P.B.) 2 Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche—Sez. Biochimica, Università Politecnica delle Marche, Ancona 60100, Italy; [email protected] * Correspondence: [email protected]; Tel.: +34-954-481-120; Fax: +34-954-486-784

Academic Editor: Rosa M. Lamuela-Raventós Received: 17 November 2016; Accepted: 23 January 2017; Published: 29 January 2017

Abstract: Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inﬂammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identiﬁed as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases.

Keywords: adenosine monophosphate-activated protein kinase (AMPK); nutraceutical compounds; cancer; type II diabetes; neurodegenerative diseases; cardiovascular diseases

1. Introduction Adenosine monophosphate-activated protein kinase (AMPK), is a heterotrimeric protein kinase consisting of an alpha (α) catalytic subunit in combination with scaffolding beta (β) and regulatory gamma (γ) subunits (Figure1). These subunits, encoded by seven genes: Protein Kinase AMP-Activated Catalytic Subunit α1 (PRKAA1), Protein Kinase AMP-Activated Catalytic Subunit α2 (PRKAA2), Protein Kinase AMP-Activated Non-Catalytic Subunit β1 (PRKAB1), Protein Kinase AMP-Activated Non-Catalytic Subunit β2 (PRKAB2), Protein Kinase AMP-Activated Non-Catalytic Subunit γ1 (PRKAG1), Protein Kinase AMP-Activated Non-Catalytic Subunit γ2 (PRKAG2), Protein Kinase AMP-Activated Non-Catalytic Subunit γ3 (PRKAG3) can theoretically combine to form twelve different possible isoforms that may differ in tissue-speciﬁc expression and activation. AMPK is known as the fuel of the cell, working to ensure that adenosine triphosphate (ATP) levels are maintained under energetic stress situations such as exercise, starvation, hypoxia or rapid cell growth [1].

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The co-expression of the three different subunits (α,, β, and γ) of AMPK is absolutely necessary to generate a a functionally functionally active active protein. protein. The The ββ-subunit-subunit is isthe the smallest smallest am amongong the the three three and and serves serves as asa scaffold a scaffold for for anchoring anchoring α αandand γγ-subunits-subunits by by its its C-terminal C-terminal module, module, conforming conforming the the “AMPK regulatory core”. The carbohydrate-bindingcarbohydrate-binding module (CBM) of the β-subunits sits above the protein kinase module of the α-subunit. The The interface interface between between both both creates creates a a novel allosteric binding site, recently described as the “ADaM" (allosteric drug and metabolite) site, binding some of the known synthetic AMPK activators [[1].1]. The The catalytic catalytic domain domain on on the α-subunit (Thr172) is followed by an auto-inhibitory domaindomain (AID) (AID) and and regulatory regulatory interacting interacting motifs motifs (α-RIMS), (α-RIMS), which are which ﬂexible are regulatory flexible segmentsregulatory triggering segments conformational triggering conformational changes in response changes to in adenosine response monophosphate to adenosine monophosphate (AMP) binding to(AMP) the AMPK bindingγ-subunit. to the AMPKγ-subunits γ-subunit. contain γ-subunits four tandem contain cystathionine four tandemβ synthase cystathionine domains β synthase (CBS´s), whichdomains are (CBS´s), known which as bateman are known domains. as batemanγ1, γ2 and domains.γ3 contain γ1, γ four2 and potential γ3 contain nucleotide four potential binding nucleotide binding sites, mainly CBS1 and CBS3, known to bind AMP, adenosine diphosphate sites, mainly CBS1 and CBS3, known to bind AMP, adenosine diphosphate (ADP) or ATP, inducing a (ADP) or ATP, inducing a conformational switch that allosterically activates AMPK as well as conformational switch that allosterically activates AMPK as well as protecting pTr172 in α- subunits protecting pTr172 in α- subunits from dephophosrylation by phosphatases [2]. from dephophosrylation by phosphatases [2].

Thr-172 P

N Catalytic domain AID β-binding C AMPKα subunits: α-1, α-2 domain α-RIM Ser-108 P

N Glycogen interacting α-γ binding domain C AMPKβ subunits: β-1, β-2 domain

ATP/ADP/AMP

AMPKγ subunits: γ1, γ2, γ3 Site 1 Site 2 Site 3 Site 4 β-binding N CBS1 CBS2 CBS3 CBS4 C γ-1 domain

N β-binding CBS1 CBS2 CBS3 CBS4 C γ-2 domain

N β-binding CBS1 CBS2 CBS3 CBS4 C γ-3 domain

Figure 1. Diagram of the adenosine monophosphate-activated protein kinase (AMPK) domain structure. Figure 1. Diagram of the adenosine monophosphate-activated protein kinase (AMPK) domain Two α subunits, two β and three γ subunits have been described to date. The α-subunit is conformed structure. Two α subunits, two β and three γ subunits have been described to date. The α-subunit is by a catalytic domain containing Thr172 kinase for the activation by upstream kinases, Liver Kinase conformed by a catalytic domain containing Thr172 kinase for the activation by upstream kinases, B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKb), an auto-inhibitory Liver Kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKb), an domain (AID), two regulatory interacting motifs (α-RIMs), and a C-terminal domain that firmly binds to auto-inhibitory domain (AID), two regulatory interacting motifs (α-RIMs), and a C-terminal domain β and γ subunits. The β-subunit contains a N-terminal domain rich in glycine, a carbohydrate binding that firmly binds to β and γ subunits. The β-subunit contains a N-terminal domain rich in glycine, a module (CBM) containing Ser108, important for some direct activators of AMPK, and a C-terminal carbohydrate binding module (CBM) containing Ser108, important for some direct activators of domain that attaches to α and γ subunits. The γ-subunit consist of three γ isoforms and variable length AMPK, and a C-terminal domain that attaches to α and γ subunits. The γ-subunit consist of three γ N-terminal domains and four cystathionine β-synthase domains (CBS) forming bateman domains isoforms and variable length N-terminal domains and four cystathionine β-synthase domains (CBS) that create adenosine monophosphate (AMP)/ adenosine diphosphate (ADP)/adenosine triphosphate forming bateman domains that create adenosine monophosphate (AMP)/ adenosine diphosphate (ATP) binding sites. All amino acid numbers refer to human AMPK sequences. (ADP)/adenosine triphosphate (ATP) binding sites. All amino acid numbers refer to human AMPK sequences. 1.1. Mechanism of AMPK Activation 1.1. MechanismAs discussed of AMPK above, Activation AMPK is regulated both allosterically and by post-translational modifications. ThereAs are discussed different well-definedabove, AMPK mechanisms is regulated of AMPKboth activationallosterically by whichand by several post-translational small drugs havemodifications. been developed There are to inducedifferent AMPK well-defined phosphorylation. mechanisms The of AMPK direct phosphorylationactivation by which at a several kinase α site,small fordrugs example, have been Thr172 developed of the to -subunitinduce AMPK by upstream phosphorylation. kinases LiverThe direct Kinase phosphorylation B1 (LKB1) and at 2+ β β Caa kinase/calmodulin-dependent site, for example, Thr172 protein of kinase-the α-subunit(CaMKKb) by upstream or, Ser108 kinases of the Liver-subunit, Kinase is theB1 (LKB1) mechanism and Ca2+/calmodulin-dependent protein kinase-β (CaMKKb) or, Ser108 of the β-subunit, is the

Int. J. Mol. Sci. 2017, 18, 288 3 of 24 used by salicylate and A769662. Another small-molecule allosteric activator of AMPK is compound 2, C2 (5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid). It is thought that C2 activates both AMPK α1 and α2 isoforms by binding to the γ-subunit. This is a new AMP-mimetic compound similar to 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a nucleoside that is taken up into cells by adenosine transporters, both are prodrugs converted into AMP analogs inside cells. Finally, AMP and/or ADP binding to nucleotide binding sites on C-terminal modules of γ-subunits induced conformational changes to protect AMPK from dephosphorylation or inactivation by phosphatases, which suggests that AMPK is a sensor of AMP/ATP or ADP/ATP ratios [1,2]. In order to preserve body energy homeostasis, AMPK, once activated, phosphorylates to activate the pathways corraleted to ATP production, such as glucose transport in muscle via the tre-2/Bub2/Cdc16 domain family member (TBC1D1), fatty acid oxidation via acetyl-CoA carboxylase (ACC2), or autophagy via Unc-51 Like Autophagy Activating Kinase 1 (ULK1). Simultaneously, AMPK inhibits the pathways involved in ATP consumption, for example cholesterol via 3-hydroxy-3methyl-glutaryl-coenzyme A (HMG-CoA) reductase, fatty acid synthesis (ACC1) and protein synthesis (mammalian target of rapamycin complex 1, mTORC1) [1]. Apart from the regulation of metabolic enzymes, AMPK is involved in long-term adaptive changes through the regulation of co-activators (peroxisome proliferator-activated receptor γ coactivator 1-α, PGC1α) and transcription factors (histone deacetylase, HDAC). Importantly, AMPK regulates the activity of another serine/threonine protein kinase, mammalian target of rapamycin (mTOR) [3], enabling cells to react to metabolic stress as well as to regulate autophagy and protein translation through its effects on tuberous sclerosis complex 2 (TSC2) and raptor. AMPK also plays a crucial role in the regulation of mitochondrial homeostasis, controlling major steps in mitochondrial biogenesis and degradation. It has been demonstrated that AMPK is directly connected with the process of mitophagy, by which it recycles essential nutrients from dysfunctional mitocondria. When this occurs, oxidative damage induced by mitochondria poisons [4] indirectly activates AMPK leading to the phosphorylation of mitochondrial ﬁssion factor (MFF) protein, which triggers mitochondrial fragmentation, and in turn, mitophagy. Many naturally-occurring compounds, such as resveratrol or quercetin, are assumed to activate AMPK through this indirect process. They induce mitochondrial dysfunction through inhibiting mitochondrial ATP production and increasing cellular AMP or ADP, indirectly activating AMPK. However, it should be noted that AMPK is usually activated following one of the mechanisms below mentioned [5]:

1. Activation through the AMPK γ-subunit: Prodrugs that are converted into AMP analogs following cellular uptake by intracellular enzymes, such as 5-aminoimidazole-4-carboxamide riboside (AICAR). Upon entry the cell, AICAR is phosphorylated to generate Phosphorylated AICAriboside (ZMP), which works as an AMP mimetic. Another prodrug is compound 13 (C13), that generates isoxazole or C2, a potent activator and protector of α isoforms from dephosforylation. 2. Direct activators binding between the β-CBM domain and kinase domain Ser108. Examples of these AMPK activating compounds are thienopyridone (A-769662), salicylate and 911 compound. 3. A number of agents (metformin or hydrogen peroxide) and natural occurring products (berberin or resveratrol) act as mitochondria poisons (above mentioned) increasing the AMP:ATP ratio or energy charge and therefore activating AMPK indirectly.

AMPK can reach near-activation by any modulator or compound causing AMP accumulation. Whereas indirect activators induce a signiﬁcant change in cellular ATP, ADP or AMP levels, direct ones induce conformational changes in the AMPK structural complex because a direct interaction between the modulator and AMPK subunits is needed [6]. Due to the major importance of AMPK in body energy homeostasis, this enzyme has drawn the attention of many researchers as a suitable candidate for the treatment of several metabolic diseases [7]. Int. J. Mol. Sci. 2017, 18, 288 4 of 24 Int. J. Mol. Sci. 2017, 18, 288 4 of 24

1.2. Metabolic Functions and Phys Physiologicaliological Regulation of AMPK When AMPK is activated, a number of signaling pathways are initiated, having effects effects on lipid, lipid, glucose andand proteinprotein homeostasis. homeostasis. These These effects effects are are crucial crucial for for the the regulation regulation of metabolic of metabolic events events in liver, in heart,liver, heart, skeletal skeletal muscle, muscle, brain and brain adipose and adipose tissue (Figure tissue2 ).(Figure AMP-activated 2). AMP-activated protein kinase protein is considered kinase is asconsidered a fuel sensor as a forfuel glucose sensor andfor glucose lipid metabolism. and lipid metabolism.

(A)

(B)

Figure 2. (A) Diagram of metabolic functions of AMPK in various tissues; Some key metabolic effects are shown.shown. TheThe adipocyte-derived adipocyte-derived hormones hormones leptin leptin and and adiponectin, adiponectin, as well as well as exercise, as exercise, activate activate (grey arrow)(grey arrow) AMPK AMPK in skeletal in skeletal muscle, muscle, stimulating stimulating fatty fatty acid acid oxidation. oxidation. Moreover, Moreover, leptin’sleptin’s activation of AMPK in in skeletal skeletal muscle muscle involves involves the the hypothalamic-sympathetic hypothalamic-sympathetic nervous nervous system system (SNS) (SNS) axis. axis. In Inhypothalamus, hypothalamus, AMPK AMPK activity activity plays plays a role a role in in regulati regulationon of of food food intake. intake. AMPK AMPK inhibits inhibits (black (black T-bar) T-bar) insulin secretion from pancreatic β cells, and insulin inhibits AMPK activation in ischemic heart and hypothalamus, whilst it has no effect on AMPK in skeletal muscle or adipocytes (*). ( B) Physiological regulation of AMPK in terms of related diseases andand different situations. Overnutrition, inactivity or genetic factors (dashed arrows) can result in a state ofof dysregulationdysregulation characterized by inﬂammationinflammation and insulin resistance, that in turn can predispose toto oneone oror moremore ofof thethe disordersdisorders shown.shown.

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The therapeutic potential of AMPK activators to treat type 2 diabetes mellitus (T2DM) [8] was suggested when it was discovered that physical exercise activated AMPK in skeletal muscle, which led to increase glucose uptake. Activation of AMPK stimulates glucose transporter type 4 (GLUT4) translocation to the plasma membrane to actively promote increased glucose uptake in skeletal muscle, enabling ATP production through glycolysis. [9]. It is thought that translocation of GLUT4 from skeletal muscle to the plasma membrane is mediated through phosphorylation of TBC1D1 [10] which increases the activity of Rab family G proteins and induces fusion of GLUT4 vesicles with the plasma membrane. The liver also plays a key role in the maintenance of glucose homeostasis by modulating hepatic glucose during periods of fasting (ATP levels decrease and AMPK is activated as a consequence of energy demand) and feeding. In postprandial situations, hepatic glycogen storage are restored and excess of carbohydrates become triglycerides, originating long-term energy storage. During that process, AMPK is inactivated and anabolic pathways are restored, increasing fatty acid synthesis through ACC activation. AMPK is then a master metabolic regulator, being responsible for the regulation of anabolic and catabolic processes and whole-body energy homeostasis. Under physiological conditions, the activation of AMPK is related to changes in energy balance. This energy sensor, activated when cellular energy levels are low, results in activation of catabolic processes, and an inactivation of anabolic processes, having, for example, a beneficial effect on glycemia and therefore on diabetes. AMPK also has functions as a regulator of proliferative signals such as mammalian target of rapamycin (mTOR), tuberous sclerosis complex (TSC), ribosomal protein S6 kinase (p70S6) and elongation factor-2, indicating that cancer cell proliferation can be modified via modulating the signaling network through AMPK [11]. Since this kinase is involved in multiple signaling pathways, deregulation of AMPK is associated with a large number of human pathologies, so that the current focus lies in finding AMPK activators. According to this, growing attention is nowadays given to the possible preventive capacity of nutraceutical compounds to prevent illness onset through the modulation of AMPK pathway. Some foods include several dietary compounds with many health advantages which present a high-grade chance for well-being optimization. Since ancient times, the benefits of foods have been investigated. Epidemiological studies have shown a close relation between the consumption of foods from plants (such as some fruits, vegetables, and grains), and direct health benefits. Glucosinolates, for example, are a group of dietary phytochemicals that have been associated with health benefits, as well as other compounds such as sulfur-containing compounds belonging to the Alliaceae family, terpenoids (carotenoids, monoterpenes, and phytosterols), and, in particular, polyphenols (anthocyanins, flavones, isoflavones, stilbenoids). This wide range of products cannot be truly classified as “food” and a new hybrid term between nutrients and pharmaceuticals, known as “nutraceuticals,” has been used to designate them [12]. A nutraceutical compound is by definition “a food that provides benefits on health in addition to its nutritional content”. Taking that into account, nutraceutical compounds define a precise category, in the frontier between drugs and food. Polyphenols are phytochemical compounds that constitute an important part of human diet. Many different nutraceuticals belonging to this group have been identified in a wide variety of fruits, vegetables and other plant-based foods, such as grains or roots [13]. Chemically, polyphenols contain one or more benzene rings joined to hydroxyl groups that confer the molecule the so-called antioxidant capacity. Based on differences in generic structure of the aromatic rings and the chemical groups attached to them, polyphenols are classified into five different subclasses (Figure3): flavonoids, phenolic acids (derived from hydroxybenzoic acid or hydroxycinnamic acid), phenolic alcohols, stilbenes and lignans [14]. Flavonoids are the widest group of polyphenols, which in turn, are classified into another five different subgroups: isoflavones, flavonols, anthocyanins, flavones and flavonones [13], quercetin being the best example to explain flavonoids actions concerning to its molecular structure (Figure4). Int. J. Mol. Sci. 2017, 18, 288 6 of 24

Int. J. Mol. Sci. 2017, 18, 288 6 of 24 Taking into account that ﬂavonoids are the widest group of polyphenols, the structural characteristicsTaking into in aaccount ﬂavonoid that molecule flavonoids that determineare the widest its antioxidant group of power polyph are:enols, the structural characteristics in a flavonoid molecule that determine its antioxidant power are: 1. The presence of 30-40-O-dihydroxy structure on the B aromatic ring (cathecol), which confers 1. Themore presence stability of and 3′-4 participates′-O-dihydroxy in electron structure delocalization. on the B aromatic Studies ring have (cathecol), reported the which relationship confers morebetween stability cathecols and participates and its involvement in electron in delocalization. the inhibition of Studies lipid peroxidation have reported [15 the]. relationship between cathecols and its involvement in the inhibition of lipid peroxidation [15]. 2. Hydroxyl groups in 5 and 7 positions on the A ring. 2. Hydroxyl groups in 5 and 7 positions on the A ring. 3. Double bond localized in 2,3 positions conjugated with a 4-oxo group and the presence of 3-OH 3. Double bond localized in 2,3 positions conjugated with a 4-oxo group and the presence of 3-OH in C ring, responsible for the delocaization from B ring. in C ring, responsible for the delocaization from B ring.

Figure 3. General structure of main groups of polyphenols.polyphenols. Substituents corre correspondingsponding to concrete structures of some compounds are underlined. Nucl Nuclearear carbonic atoms corresponding to flavonoids ﬂavonoids structure are listed.

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Figure 4. Quercetin structure. Flavonoid that own maximum antioxidant potential. Cathecol structure Figure 4. Quercetin structure. Flavonoid that own maximum antioxidant potential. Cathecol in the B ring, with hydroxyl groups in 5 and 7 positions, double bond in 2,3 position, conjugated with structure in the B ring, with hydroxyl groups in 5 and 7 positions, double bond in 2,3 position, an 4-oxo group, and 3-OH group in the C ring are represented. conjugated with an 4-oxo group, and 3-OH group in the C ring are represented.

For maximum antioxidant effectiveness, the presence of all these functions functions are are required. required. In In fact, fact, the absence of some of them induceinduce changes in its activity [[16].16]. 0 0 Quercetin (3,3(3,3,4′,4,5,7-pentahydroxyflavone)′,5,7-pentahydroxyflavone) satisfies satisfies the structuralthe structural requirements requirements that a flavonoid that a flavonoidmust own must to express own to the express maximum the maximum antioxidant antioxidant potential. potential. It is known worldwide that a healthy lifestyle, including a suitable diet combined with regular exercise is vital to promote well-being and preventprevent onset of pathology.pathology. Metabolic syndrome is a resulting example example due due to to bad bad habits habits in in lifestyle. lifestyle. This This worldwide worldwide epidemic epidemic threat threat is characterized is characterized by cardio-metabolicby cardio-metabolic risk risk factors, factors, such such as asobesity, obesity, insulin insulin resistance, resistance, hypertension, hypertension, and and dyslipemia. dyslipemia. Prevention is therefore, a powerful strategy for an effective medical treatment.treatment. Nutraceuticals are a great tool to manage pre-clinical he healthalth conditions since they can be included in daily diet to use as illness-onset prophylactics [17]. [17]. This This review review highlights some some recent findings findings related to nutraceutical compounds presentpresent in in daily daily diet diet targeting targeting AMPK AMPK pathway, pathway, that that could could have have a beneficial a beneficial effect oneffect health on healthand particularly, and particularly, on illness on onsetillness prevention. onset prevention.

2. Nutraceutical Compounds and Cancer Despite the numerous advances in biomedical an andd clinical research, cancer remains a leading cause of of death death worldwide. worldwide. The The progression progression of of cancer cancer is isa continual a continual unregulated unregulated process process resulting resulting in manyin many accumulated accumulated abnormalities abnormalities with with gradual gradual progression, progression, eventually eventually leading leading to to cancerous cancerous cells growing and dividing in an uncontrolled manner manner and and spreading spreading through through tissues tissues and and organs organs [12]. [12]. AMPK is proposed asas aa mastermaster controllercontroller of of cancer, cancer, since since it it plays plays an an important important role role in in the the prevention prevention of ofits its development. development. This This protein protein kinase kinase is responsible is responsible for cancer for cancer cell proliferation cell proliferation and apoptosis. and apoptosis. Natural Naturalcomponents, components, such as polyphenols such as polyphenols and in particular and in ﬂavonoids, particular targetflavonoids, AMPK target to induce AMPK apoptosis to induce and apoptosisto avoid cell and proliferation to avoid cell [18 proliferation]. [18]. Targeting the mTOR pathway has currently emerge emergedd as an interesting tool to control cancer with nutraceuticals, since mTOR promotes tumorigenesis.tumorigenesis. Modulating the AMPK/mTORAMPK/mTOR (Figure (Figure 55)) pathway with phytochemical compou compoundsnds could be one useful strate strategygy for cancer prevention and control. Some Some studies studies performed performed in in human human colore colorectalctal carcinoma carcinoma cell cell line line HCT-116 HCT-116 and and inin vivo, vivo, on BALB/C AnN-Foxn1 AnN-Foxn1 nude nude mice mice treated treated with with HCT-11 HCT-1166 cells, cells, have demonstrated the capacity of phenolic acids (caffeic acid; Table 1 1)) toto induce induce apoptosis apoptosis throughthrough thethe AMPK/mTORAMPK/mTOR pathwaypathway [[19].19]. On the other hand, it has been reported in variousvarious studies that grape ﬂavonols,flavonols, such as quercetin (Table1 1),), have have beenbeen involvedinvolved inin cancercancer protectionprotection duedue toto theirtheir strongstrong antioxidantantioxidant andand pro-apoptoticpro-apoptotic effects [20]. [20]. Anti-carcinoge Anti-carcinogenicnic activity of quercetin, quercetin, present present in grapes grapes and and other other fruits fruits and and vegetables, vegetables, is the most studied activity of flavonoids in cancer. In humans, grape consumption has been

Int. J. Mol. Sci. 2017, 18, 288 8 of 24 isInt. the J. Mol. most Sci. studied 2017, 18, activity288 of ﬂavonoids in cancer. In humans, grape consumption has been associated8 of 24 with reduced breast cancer risk [21] and it has been demonstrated that MCF-7 breast cancer cells associated with reduced breast cancer risk [21] and it has been demonstrated that MCF-7 breast treatment with this compound exerted anti-proliferative effects and induced apoptosis through AMPK cancer cells treatment with this compound exerted anti-proliferative effects and induced apoptosis activation, that in turn modulated apoptotic pathways such as apoptosis signal-regulating kinase through AMPK activation, that in turn modulated apoptotic pathways such as apoptosis 1/p38 MAP Kinase (MAPK) and cyclooxygenase 2 (COX-2), and inhibited the adenosin/threonine signal-regulating kinase 1/p38 MAP Kinase (MAPK) and cyclooxygenase 2 (COX-2), and inhibited (Akt) pathway [18,22,23]. the adenosin/threonine (Akt) pathway [18,22,23].

Figure 5.5. Targeting thethe AMPKAMPK activationactivation throughthrough nutraceuticals.nutraceuticals. Resveratrol activates AMPKAMPK leadingleading toto apoptosisapoptosis ofof coloncolon cancercancer cells,cells, enhanceenhance ofof cancercancer cellcell responseresponse toto ionizingionizing radiation,radiation, andand mTOR-dependent and independentindependent autophagy. ResveratrolResveratrol also activates SIRT1, whichwhich improvesimproves AMPKAMPK activation,activation, leading leading in turnin turn to downregulation to downregulation of mTOR. of mTOR:mTOR. mammalianmTOR: mammalian target of rapamycin, target of SIRT1:rapamycin, Sirtuin-1. SIRT1: Grey Sirtuin-1. and blue arrowsGrey indicateand blue activation, arrows T-bars indicate indicate activation, downregulation. T-bars indicate downregulation. The expression of Bcl-2 and Bax, which are crucial proteins in cell cycle arrest and apoptosis, were The expression of Bcl-2 and Bax, which are crucial proteins in cell cycle arrest and apoptosis, modified via AMPK in HT-29 colon cancer cells. In vivo studies were performed to support in vitro were modified via AMPK in HT-29 colon cancer cells. In vivo studies were performed to support in evidence by oral administration of quercetin to mice with HT-29 tumor xenografts showing a significant vitro evidence by oral administration of quercetin to mice with HT-29 tumor xenografts showing a induction of apoptosis and a reduction in tumor volume [24]. Moreover, although flavones are the significant induction of apoptosis and a reduction in tumor volume [24]. Moreover, although less common flavonoids found in diet, some, such as luteolin or hispidulin (Table1), have also shown flavones are the less common flavonoids found in diet, some, such as luteolin or hispidulin (Table 1), beneficial effects on cancer cells through AMPK activation. On one side, luteolin causes Reactive have also shown beneficial effects on cancer cells through AMPK activation. On one side, luteolin Oxygen Species (ROS) release, inducing cell death and inhibiting nuclear factor-κ β (NF-κB) [25]. causes Reactive Oxygen Species (ROS) release, inducing cell death and inhibiting nuclear factor-κ β On the other side, the treatment of human glioblastoma multiform cells and human ovarian cells with (NF-κB) [25]. On the other side, the treatment of human glioblastoma multiform cells and human hispidulin was able to arrest the cell cycle at the G1 phase, inducing apoptosis [26]. The anticancer ovarian cells with hispidulin was able to arrest the cell cycle at the G1 phase, inducing apoptosis [26]. properties promoted by flavonols like epillocatechin gallate (EGCG; Table1) rely on a strong activation The anticancer properties promoted by flavonols like epillocatechin gallate (EGCG; Table 1) rely on a of the AMPK pathway by promoting apoptosis in rat hepatoma cells. It has been reported that liver strong activation of the AMPK pathway by promoting apoptosis in rat hepatoma cells. It has been hepatocellular cells (HepG2) treatment with EGCG exerted cell cytotoxicity through stimulation of reported that liver hepatocellular cells (HepG2) treatment with EGCG exerted cell cytotoxicity AMPK while in Hep3B cells the activation of AMPK resulted in a decrease in COX-2 expression [27,28]. through stimulation of AMPK while in Hep3B cells the activation of AMPK resulted in a decrease in According to the evidence, EGCG could be used as an adjuvant in combination with chemotherapy, COX-2 expression [27,28]. According to the evidence, EGCG could be used as an adjuvant in increasing therapy efficacy in patients. combination with chemotherapy, increasing therapy efficacy in patients. Isoflavones like genistein (Table1) have great anticarcinogenic effects through the modulation Isoflavones like genistein (Table 1) have great anticarcinogenic effects through the modulation of AMPK pathway and COX-2 expression. In MCF-7 breast cancer cell lines, genistein has been of AMPK pathway and COX-2 expression. In MCF-7 breast cancer cell lines, genistein has been demonstrated to be directly involved in COX-2 downregulation [29]. In HT29 colon cancer cells demonstrated to be directly involved in COX-2 downregulation [29]. In HT29 colon cancer cells genistein was an effective phytochemical to combine with 5-fluouracil, commonly used in colon genistein was an effective phytochemical to combine with 5-fluouracil, commonly used in colon cancer treatment. This combination led to an increase of ROS and consequently to AMPK activation, cancer treatment. This combination led to an increase of ROS and consequently to AMPK activation, decreasing COX-2 expression [30]. decreasing COX-2 expression [30]. Another group of polyphenols of interest are lignans and particularly, magnolol (Table1), Another group of polyphenols of interest are lignans and particularly, magnolol (Table 1), a a compound isolated from roots and barks of Magnolia officinalis that has recently shown interesting compound isolated from roots and barks of Magnolia officinalis that has recently shown interesting anticancer activity through AMPK modulation [31–33]. The treatment of HCT-116 human colon cancer anticancer activity through AMPK modulation [31–33]. The treatment of HCT-116 human colon cancer cells with this compound induced apoptosis and exerted anti-proliferative effects in a dose and time dependent manner.

Int. J. Mol. Sci. 2017, 18, 288 9 of 24 cells with this compound induced apoptosis and exerted anti-proliferative effects in a dose and time dependent manner. To date, a number of studies have explained the role of AMPK in tumorigenesis [34]. Initially, the connection between AMPK and cancer biology was through the discovery of tumor suppressor LKB1 as a major AMPK upstream kinase [35]. Gene mutations of the LKB1 gene are responsible for inherited Peutz–Jeghers syndrome, which is characterized by the development of harmartomatous polyps in the intestine [36]. Since then, numerous in vitro and in vivo studies have proposed that AMPK deeply mediates the tumor suppressor effects of LKB1. This is backed by ﬁndings that some particular drugs are capable of activating AMPK, such as metformin, phenformin or A-769662, as they are able to delay the onset of tumorigenesis in in vivo models [37] or reduce the rate of cancer risk. [38,39]. However, it is important to note that AMPK functions as either an anti- or pro-tumorigenic regulator depending on the molecular pathway involved. Tumor suppressor p53 is activated upon oxidative stress and in turn inhibits cell proliferation through particular target genes. Cell proliferation is positively regulated by mTOR, whose action is controlled by the TSC1/TSC2 complex. However, the mechanism by which p53 and oxidative stress negatively control cell growth via TSC1/TSC2/mTOR core is not solidly established. It has been demonstrated that the products of two p53 target genes [40], known as sestrin1 and sestrin2, activate AMPK and target it to phosphorylate TSC2 and, thus inhibit mTOR and cell proliferation. On the other hand, some authors have reported that mTORC1 [41] and RNA polymerase I transcription initiation factor (TIF-1A) [42], both of which are involved in rapidly growth cells, are controlled by AMPK. For instance, under metabolic stress conditions, tumor cells require AMPK overcome hypoxia and nutrient limitation driven by their uncontrolled proliferation. Normal cells rely less on AMPK than tumor cells, suggesting that sometimes it would be a more effective strategy to inhibit AMPK to combat tumors instead of promoting its activation. Conclusively, the application of AMPK activators in oncology depend on patients circumstances and kind of tumor. Int. J. Mol. Sci. 2017, 18, 288 10 of 24

Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEW 10 of 24 Int.Int. J.J. Mol.Mol. Sci.Sci. 20172017,,, 1818,,, xxx FORFORFOR PEERPEERPEER REVIEWREVIEWREVIEW 1010 ofof 2424 Table 1. List of nutraceutical compounds targeting AMPK pathways in cancer. Table 1. List of nutraceutical compounds targeting AMPK pathways in cancer. TableTable 1.1. ListList ofof nutraceuticalnutraceutical compoundscompounds tatargetingrgeting AMPKAMPK pathwayspathways inin cancer.cancer. NC Classiﬁcation Pathway Experimental Model Comments Nutrient References NC Classification Pathway Experimental Model Comments Nutrient References NCNC ClassificationClassification Pathway Pathway Experimental Experimental ModelModelModel Comments Comments Comments Nutrient Nutrient Nutrient References References References

- HepG2 Hepatocarcinoma cells -- HepG2-HepG2 HepG2 HepatocarcinomaHepatocarcinoma Hepatocarcinoma cells cellscells 1. InhibitoryInhibitory effect effect on NF- onκ NF-B κB Flavone - AMPK/NF-κB - HepG2 Hepatocarcinoma cells 1.1. InhibitoryInhibitory effecteffect onon NF-NF-κκBB Celery, parsley [14] FlavoneFlavone -- AMPK/NF-AMPK/NF-κκBB -- Five-week-oldFive-week-old- Five-week-old malemale nude mice 1.2.1. ReducesInhibitoryInhibitory tumor effecteffect size onon NF-NF-κκBB Celery,Celery, parsleyparsley [14] [14] FlavoneFlavone -- AMPK/NF-AMPK/NF-κκBB -- Five-week-oldFive-week-old malemale 2. Reduces tumor size Celery,Celery, parsleyparsley [14] [14] nude mice 2.2. ReducesReduces tumortumor sizesize nudenude micemice Luteolin LuteolinLuteolin

- H4IIE rat cells 1. Stimulation of apoptosis -- H4IIE-H4IIE H4IIE ratrat rat cellscells cells 1. StimulationStimulation of apoptosisofof apoptosisapoptosis -- -AMPK/p53 AMPK/p53AMPK/p53 expressionexpression - H4IIEH4IIE ratrat cellscells 1.1. StimulationStimulation ofof apoptosisapoptosis FlavonolFlavonol -- AMPK/p53AMPK/p53 expressionexpression -- p53-positive- p53-positive HepG2 cells cells 2. ExertsExerts cell cell cytotoxicity cytotoxicity Fruits, vegetables, vegetables, tea tea [16,17] [16 ,17] FlavonolFlavonol - - AMPK/COX-2AMPK/COX-2 -- p53-positivep53-positive HepG2HepG2 cellscells 2.2. ExertsExerts cellcell cytotoxicitycytotoxicity Fruits,Fruits, vegetables,vegetables, teatea [16,17] [16,17] -- AMPK/COX-2AMPK/COX-2 - p53-negative- p53-negative Hep3BHep3B cells cells 3. DecreasesDecreases COX-2 COX-2 expression expression - AMPK/COX-2 -- p53-negativep53-negative Hep3BHep3B cellscells 3.3. DecreasesDecreases COX-2COX-2 expressionexpression - p53-negative Hep3B cells 3. Decreases COX-2 expression

EGCG EGCGEGCG 1. Inhibits cell growth 1.1. InhibitsInhibits cellcell growthgrowth 1.1. InhibitsInhibitsInhibits cell cellcell growth growthgrowth -- AMPKα1/COX-2 2. Cells cycle arrest Apple, grape, berries, -- AMPKAMPKαα1/COX-21/COX-2 - MCF7 breast cancer cells 2.2. Cells Cells cycle cyclecycle arrest arrestarrest Apple,Apple, grape,grape, berries,berries, Flavonol - - AMPKAMPKα1/COX-21/ASK1/p38 -- MCF7-MCF7 MCF7 breastbreast breast cancercancer cells cellscells 3. Induction of ROS onion,Apple, grape,red wine, berries, beans, onion, red [12,13,19,22] FlavonolFlavonolFlavonol -- AMPKAMPKαα1/ASK1/p381/ASK1/p38 -- MCF7 breast cancer cells 3.3. InductionInduction of of ROSof ROSROS onion,onion, redred wine,wine, beans,beans, [12,13,19,22][12,13,19,22][12,13,19 , 22] FlavonolFlavonol - - AMPKAMPKAMPKαα1/ASK1/p381/ASK1/p38 pathway -- HT29HT29- HT29 coloncolon colon cancer cancer cells cellscells 3.3. InductionInduction ofof ROSROS onion,onion,wine, beans, redred wine,wine, broccoli, beans,beans, parsley [12,13,19,22][12,13,19,22] pathway -- HT29HT29 coloncolon cancercancer cellscells 4. InductionInduction of apoptosisof apoptosis broccoli, parsley pathwaypathway 4.4. InductionInduction ofof apoptosisapoptosis broccoli,broccoli, parsleyparsley pathway 4.5. ReductionInduction of of tumor apoptosis volume broccoli, parsley Quercetin 5. Reduction of tumor volume QuercetinQuercetin 5.5. ReductionReduction ofof tumortumor volumevolume

-- MCF-7 breast cancer cells 1. Reduction COX-2 expression Isoflavone - AMPKα1/COX-2 -- MCF-7-MCF-7 MCF-7 breastbreast breast cancercancer cells cellscells 1.1. Reduction Reduction COX-2 COX-2COX-2 expression expressionexpression Legumes [33] IsoflavoneIsoflavoneIsoﬂavone -- AMPKAMPKαα1/COX-21/COX-2 - MCF-7 breast cancer cells 1. Reduction COX-2 expression LegumesLegumes [33][33] [33] IsoflavoneIsoflavone - - AMPKAMPKAMPKαα1/COX-21/COX-2 -- HT29HT29 coloncolon cancercancer cellscells 2.2. ApoptosisApoptosis inductioninduction LegumesLegumes [33][33] -- HT29-HT29 HT29 coloncolon colon cancer cells cellscells 2.2. Apoptosis Apoptosis induction inductioninduction Genistein GenisteinGenistein

- Human CRC cells: 1. Cell cycle arrest Coffee, argan oil, thyme, -- Human Human CRCCRC cells:cells: 1.1. CellCell cyclecycle arrestarrest Coffee,Coffee, arganargan oil,oil, thyme,thyme, - - Human- Human CRC CRC cells: cells: 1. CellCell cycle cycle arrest arrest Coffee,Coffee, arganargan oil, oil, thyme, thyme, sage, PhenolicPhenolic acid acid -- PI3K/Akt/AMPK/pmTOR HTC-116 and BALB/C 2. Augmentation of sage,spearmint, spearmint, ceylon cinnamon,ceylon [8] [8] PhenolicPhenolic acidacid -- -PI3K/Akt/AMPK/pmTOR PI3K/Akt/AMPK/pmTORPI3K/Akt/AMPK/pmTOR HTC-116HTC-116HTC-116 andand and BALB/C BALB/C 2.2. Augmentation AugmentationAugmentation of ofof sage,sage, spearmint,spearmint, ceylonceylon [8][8] AnN-Foxn1nudeAnN-Foxn1nude micemice apoptoticapoptotic pathways pathways cinnamon,star anise star anise AnN-Foxn1nudeAnN-Foxn1nude micemice apoptoticapoptotic pathwayspathways cinnamon,cinnamon, starstar aniseanise

Caffeic acid CaffeicCaffeic acid acidacid

Roots and barks of - RootsRoots andand barksbarks ofof -- -AMPK/pro-apoptotic AMPK/pro-apoptoticAMPK/pro-apoptotic proteins -- HCTHCT 116116 ColonColon RootsRoots and andand barks barksbarks of ofof species of LignanLignan -- AMPK/pro-apoptoticAMPK/pro-apoptotic -- HCT-HCT HCT 116116 116 ColonColon Colon cancer cells 1. ApoptosisApoptosis induction induction species of [20] [20] LignanLignan proteins(p53/Bax) (p53/Bax) cancer cells 1.1. ApoptosisApoptosis inductioninduction speciesspeciesMagnolia ofof ofﬁcinalis [20][20] proteinsproteins (p53/Bax)(p53/Bax) cancercancer cellscells Magnolia officinalis MagnoliaMagnolia officinalisofficinalis Magnolol MagnololMagnolol MagnololNC: Nutraceutical compound; EGCG: Epigallocatechin gallate; AMPK: Adenosin monophosphate protein kinase; NF-κB: Nuclear factor-κ β; HepG2: Liver hepatocellular cells; NC: Nutraceutical compound; EGCG: Epigallocatechin gallate; AMPK: Adenosin monophosphate protein kinase; NF-κB: Nuclear factor-κ β; HepG2: Liver COX-2:NC:NC: CyclooxygenaseNutraceuticalNutraceutical compound;compound; 2; mTOR: Mammalian EGCG:EGCG: EpigEpig targetallocatechinallocatechin of rapamycin; gallate;gallate; ROS: AMPK:AMPK: Reactive AdenosinAdenosin oxygen species; monophosphatemonophosphate 4EBP1: 4E Binding proteinprotein protein kinase;kinase; 1; PI3K: NF-NF-κκ PhosphoinositolB:B: NuclearNuclear factor-factor- 3-kinase;κκ ββ;; HepG2:HepG2: Akt: Adenosin/threonine; LiverLiver hepatocellular cells; COX-2: Cyclooxygenase 2; mTOR: Mammalian target of rapamycin; ROS: Reactive oxygen species; 4EBP1: 4E Binding protein 1; PI3K: ASK1:hepatocellularhepatocellular Apoptosis signal-regulating cells;cells; COX-2:COX-2: CyclooxygenaseCyclooxygenase kinase-1; CRC:Colorectal 2;2; mTOR:mTOR: cancer MammalianMammalian cells. targettarget ofof rapamycin;rapamycin; ROS:ROS: ReactiveReactive oxygenoxygen species;species; 4EBP1:4EBP1: 4E4E BinBindingding proteinprotein 1;1; PI3K:PI3K: Phosphoinositol 3-kinase; Akt: Adenosin/threonine; ASK1: Apoptosis signal-regulating kinase-1; CRC: Colorectal cancer cells. PhosphoinositolPhosphoinositol 3-kinase;3-kinase; Akt:Akt: Adenosin/thrAdenosin/threonine;eonine; ASK1:ASK1: ApoptosisApoptosis signal-regulatingsignal-regulating kikinase-1;nase-1; CRC:CRC: ColorectalColorectal cancercancer cells.cells.

Int. J. Mol. Sci. 2017, 18, 288 11 of 24

3. Nutraceutical Compounds and Cardiovascular Disease Cardiovascular disease (CVDs), including heart disease and stroke, represent the principal cause of death in western countries [9]. It has been reported that the increased incidence of CVDs by 2030 will lead to more than 23.6 million cardiovascular event-related deaths [43]. This fact has stimulated the research of substances that can improve cardiovascular health. CVD is a worldwide problem that can be prevented by simply developing healthy habits. Among nutraceutical compounds, resveratrol (Table2) is a very potent antioxidant and anti-inflammatory stilbene present in grapes and red wine that has the ability to up-regulate endothelial NO synthase (eNOS), protecting cardiovascular function through the AMPK pathway [44]. Moreover, AMPK activation is required to attenuate the expression of the intracellular adhesion molecule 1 (ICAM-1), which is involved in atherogenesis [45]. AMPK plays a crucial role in cardiac function, since its inactivation could lead to heart failure. Cardiac dysfunction can be prevented by resveratrol through AMPK modulation, since it has been shown that resveratrol treatment on cardiac function is closely related to its capacity to improve AMPK activity via Sirtuin-1 (SIRT1) activation [46], as shown in vitro on treated cardiomyocytes. A great beneficial effect after treatment with resveratrol was also found in an in vivo model of heart failure of myocardial infarction, enhancing AMPK expression [46]. AMPK activation is also necessary for vascular relaxation, which is mediated by resveratrol, improving endothelium dependent vasodilation [47,48]. AMPK activation has also shown an effect on hypertrophy, inhibiting hypertrophic growth [48]. Some studies have reported that resveratrol may reduce the hypertrophic growth preventing the inhibition of LKB1 and AMPK activity in isolated cardiomyocytes [49,50]. Another group exerting beneficial effects on cardiac function are anthocyanins [51]. In this context, Yang et al. focus on the direct effect of delphinidin-3-glucoside (dp-3-glu) (Table2) on CVD. Dp-3-glu is the principal pigment present in bilberry fruits and the treatment with this polyphenol shows in both in vitro and in vivo models an inhibition of platelet aggregation and prolonged time required for thrombus formation. It is thought that dp-3-glu surprisingly down-regulates AMPK pathway, attenuating in that way the activation of the cytoplasmic tail of integrin aIIbb3, which is a protein required for clot retraction and thrombus stability. This event inhibits platelet aggregation and thrombus growth, although further studies are necessary to associate anthocyanin consumption and CVD prevention. Recent reports show that also flavonols, and especially quercetin (Table2), seem to exert protective effects through AMPK activation [52–54]. Quercetin and its metabolites seem to protect endothelial and vascular function. A study performed in mouse endothelial dysfunction-induced with hypochlorous acid (HOCl) showed that quercetin enhanced acetylcholine-mediated endothelial relaxation through the AMPK pathway [55]. These findings were confirmed by inhibiting AMPK with compound C treatment of the aortic rings, which blocked the protective effects of quercetin [56,57]. These results suggest that flavonols targeting AMPK pathway are an interesting tool to manage or prevent cardiac dysfunction onset. Finally, hydroxytyroxol (HT; Table2) was also found to ameliorate endothelial functionality, since this phenolic alcohol is able to reduce intracellular ROS levels in porcine pulmonary artery endothelial cells. HT is able to increase the antioxidant activity of catalase through the AMPK/forkhead transcription factor 3a (FOXO3) pathway, proposing HT as an effective phenolic compound for reducing endothelial dysfunction and atherosclerosis [58]. As discussed above, AMPK activators could be deleterious in the treatment of some chronic human pathologies. Recent research have reported that mutations on the PRKAG2 gene could have cardiovascular adverse effects due to chronic activation of AMPK. For instance, naturally occurring mutations of human γ2 have been associated with enhanced glycogen storage in cardiomyocytes, cardiac hypertrophy, and electrophysiological abnormalities [59]. Then, one should be cautious when AMPK is considered as a therapeutic target. Int.Int.Int. J. J.J. Mol. Mol.Mol. Sci. Sci.Sci. 2017,, 1818,,, xx 288 FORFOR PEERPEER REVIEWREVIEW 12 of 24 12 of 24 Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEW 12 of 24

TableTable 2. List 2. List of nutraceutical of nutraceutical compounds compounds targeting targeting AMPK AMPK pathways pathways in cardiovascular in cardiovascular disease. disease. TableTable 2.2. ListList ofof nutraceuticalnutraceutical compoundscompounds targetingtargeting AMPKAMPK pathwayspathways inin cardiovascularcardiovascular disease.disease. NC Classification Pathway Experimental Model Comments Nutrient References NCNC ClassificationClassiﬁcation Pathway Pathway Experimental Experimental Model Model Comments Comments Nutrient Nutrient References References

1. Regulation of -- Porcin pulmonary artery 1. Regulation of Extra virgin olive oil, leaves Phenolic alcohol -- AMPK/FOXO3 - Porcin- Porcin pulmonary pulmonary artery artery antioxidant1. Regulation defense of antioxidant Extra virgin oliveExtra oil, virgin leaves olive oil,[38] leaves PhenolicPhenolic alcohol alcoholalcohol - - AMPK/FOXO3AMPK/FOXO3AMPK/FOXO3 endothelial cells (VECs) antioxidantantioxidant defensedefense from Olea Europea L. [38][38] [38] endothelialendothelialendothelial cellscells cells(VECs)(VECs) (VECs) systemdefense in VECs system in VECsfromfrom OleaOlea EuropeaEuropeafrom L.OleaL. Europea L. system in VECs Hydroxytyrosol system in VECs HydroxytyrosolHydroxytyrosol

1. InhibitionInhibition ofof bothboth -- C57BL/6J mice 1. Inhibition of both - C57BL/6J- C57BL/6J mice mice murine1. Inhibition and human of both murine and -- AMPK/integrin -- Human blood cells who had murine and human Bilberry fruits,Bilberry cacao, fruits, cacao, Anthocyanin - - AMPK/integrinAMPK/integrin aIIbb3 - Human- Human blood blood cells cellswho who had had notplatelethuman aggregation platelet aggregation Bilberry fruits, cacao, [31][31] [31] Anthocyanin aIIbb3 not takentaken any any platelet platelet medication platelet2. Reduction aggregation of thrombus pomegranate growth pomegranate [31] aIIbb3 not taken any platelet 2. Reduction of pomegranate medication 2.2. ReductionReduction ofof medicationmedication thrombus growth Dp-3-glu thrombus growth Dp-3-gluDp-3-glu thrombus growth

1. Prevention1. Prevention of cardiac of Skin of grapes,Skin of grapes, blueberries, - AMPK/SIRT1- In vitro 1. Preventioncardiac of dysfunction cardiac Skin of grapes, Stilbene -- AMPK/SIRT1 -- InIn vitrovitro dysfunction blueberries, raspberries,raspberries, mulberries [24][24] and [24] Stilbene - AMPK/SIRT1 - In vitro dysfunction2. Upregulates eNOS blueberries, redraspberries, wine [24] 2. Upregulates eNOS mulberries and red wine 2. Upregulates eNOS mulberries and red wine Resveratrol ResveratrolResveratrol

1. InductionInduction ofof eNOSeNOS - HAECs 1. Induction1. Induction of eNOS of eNOS activity -- HAECs activity Apple, grape,Apple, berries, grape, berries, onion, - AMPK/eNOS - HAECs- Isolated aortic rings from activity2. Increase of NO productionApple, grape, berries, Flavonol -- AMPK/eNOS -- IsolatedIsolated aorticaortic ringsrings fromfrom 2. IncreaseIncrease ofof NONO onion, red wine,red wine,beans, beans, [35][35] [35] Flavonol - AMPK/eNOS - IsolatedC57BL aortic mice rings from 2. Increase3. Increase of NO of relaxation onion, red wine,broccoli, beans, parsley [35] C57BL mice production broccoli, parsley C57BL mice production broccoli, parsley 3. Increase of relaxation Quercetin 3.3. IncreaseIncrease ofof relaxationrelaxation QuercetinQuercetin NC:NC: Nutraceutical Nutraceutical compound; compound; HT: HT: Hydroxytyrosol; Hydroxytyros AMPK:ol; AMPK: Adenosin Adenosin monophosphate monophosphate protein kinase;protein FOXO3: kinase; ForkheadFOXO3: Forkhead transcription transcription factor 3a; VECs: factor Vascular 3a; VECs: endothelial Vascular cells; Dp-3-glu: Delphinidin-3-glucoside;NC:NC: NutraceuticalNutraceutical compound;compound; NO: Nitric HT:HT: oxide; HydroxytyrosHydroxytyros eNOS: endothelialol;ol; AMPK:AMPK: nitric AdenosinAdenosin oxide synthase; monophosphatemonophosphate HAECs: Human proteinprotein aortic kikinase;nase; endothelial FOXO3:FOXO3: ForkheadForkhead cells. transcriptiontranscription factfactoror 3a;3a; VECs:VECs: VascularVascular endothelial cells; Dp-3-glu: Delphinidin-3-glucoside; NO: Nitric oxide; eNOS: endothelial nitric oxide synthase; HAECs: Human aortic endothelial cells. endothelialendothelial cells;cells; Dp-3-glu:Dp-3-glu: Delphinidin-3-glucoside;Delphinidin-3-glucoside; NO:NO: NitricNitric oxide;oxide; eNOS:eNOS: endothelialendothelial nitricnitric oxideoxide synthase;synthase; HAECs:HAECs: HumanHuman aaorticortic endothelialendothelial cells.cells.

Int. J. Mol. Sci. 2017, 18, 288 13 of 24

4. Nutraceutical Compounds and Type II Diabetes Mellitus Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by pancreatic β-cell dysfunction, hyperglycemia, and insulin resistance, resulting in glucose and lipid metabolism deregulation. Consequently, low-grade inflammation and oxidative stress arise, leading to micro- and macro-vascular severe complications, such as neuropathy, retinopathy and/or nephropathy, with quality of life considerably diminished [60–62]. Development of the disease can be prevented or delayed in people with impaired glucose tolerance by implementing changes in lifestyle, being diet the main modified factor. The interest of the scientific community in targeting the activation of AMPK pathway as a new treatment for metabolic disorders has arisen as a result of the value of this kinase in managing cellular metabolism and energy control. Phenolic compounds such as quercetin and resveratrol (Table3), have shown an increased glucose uptake in muscle cells and adipocytes by promoting translocation of GLUT4 via induction of AMPK in vitro [63,64]. Resveratrol has shown important beneficial effects in various in vitro and in vivo studies of human disease models of metabolic disorders [53,65–67]. These beneficial effects of stilbenes and more specifically of resveratrol, have been attributed to AMPK as a main target [53,68,69]. When resveratrol activates AMPK, translocation of GLUT4 is mediated, which is essential for the uptake of glucose from the blood to target different organs. Resveratrol is suggested to improve GLUT expression through AMPK phosphorylation in an assay performed in L6 myotubes cells enhancing glucose uptake and, overcoming in a certain way, insulin resistance [70]. Moreover, it have been demonstrated that resveratrol supplementation in db/db mice increased glycolytic activity and fatty acid oxidation [71] and decreased gluconeogenesis in liver, regulating in turn, glucose metabolism in hepatocytes and subsequently, improving hyperglycemia. Naringin and naringenin (Table3) are flavonoids present in citrus fruits, some berries, tomatoes and mint, and represent the most common studied compounds among flavonones [72]. In particular, naringin possesses powerful hypoglycemic effects [73]. Some studies have investigated the beneficial effect of naringin in primary hepatocytes exposed to high doses of glucose and in C57BL/6J mice fed with high-fat diet. They concluded that naringin protected against metabolic syndrome onset, up-regulating the AMPK pathway [74]. This activation by phosphorylating AMPK and insulin receptor substrate-1 (IRS-1) led to an improvement in insulin resistance, suppression of gluconeogenesis and an increase in mitochondrial oxidation. Naringenin, which is naringin aglycon, seems to be also involved in lipid and glucose metabolism, stimulating glucose uptake and increasing AMPK phosphorylation in L6 rat skeletal myotubes in a dose- and time-dependent way. Such events emphasize their potential as metabolic disorders preventives [75]. The antidiabetic properties of catechins lie on the protective effect of epigallocatechin gallate (EGCG; Table3), the major polyphenol present in green tea which seems to possess potentiating activity in glucose utilization [76–80]. EGCG activates AMPK, enhancing insulin signaling pathway by membrane translocation and phosphorylation of IRS-1, improving insulin sensitivity and secretion [81]. Int. J. Mol. Sci. 2017, 18, 288 14 of 24

Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEW 14 of 24 Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEW 14 of 24 Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEW Table 3. List of nutraceutical compounds targeting AMPK pathways in type 2 diabetes mellitus. 14 of 24 Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEW 14 of 24 Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEWTable 3. List of nutraceutical compounds targeting AMPK pathways in type 2 diabetes mellitus. 14 of 24 Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEWTable 3. List of nutraceutical compounds targeting AMPK pathways in type 2 diabetes mellitus. 14 of 24 NC ClassiﬁcationTable 3. List of nutraceutical Pathway compounds targeting AMPK Experimental pathways model in type 2 diabetes mellitus. Comments Nutrient References NC ClassificationTable 3. List ofPathway nutraceutical compounds Experimental targeting model AMPK pathways Comments in type 2 diabetes mellitus. Nutrient References NC ClassificationTable 3. List ofPathway nutraceutical compounds Experimental targeting model AMPK pathways Comments in type 2 diabetes mellitus. Nutrient References NC ClassificationTable 3. List ofPathway nutraceutical compounds Experimental targeting model AMPK pathways Comments in type 2 diabetes mellitus. Nutrient References NC ClassificationTable 3. List ofPathway nutraceutical compounds Experimental targeting model AMPK pathways Comments in type 2 diabetes mellitus. Nutrient References NC Classification Pathway Experimental model Comments Nutrient References NC Classification Pathway Experimental model Comments Nutrient References 1. Improvement of insulin NC Classification Pathway - ExperimentalHFD in C57BL/6 model- miceHFD in C57BL/61. Improvement mice Comments of insulin 1. ImprovementCitrus of fruits, insulin Nutrient some resistance berries, CitrusReferences fruits, some berries, FlavononaFlavonona - AMPK/IRS-1- AMPK/IRS-1 - HFD in C57BL/6 mice 1. Improvementresistance of insulin Citrus fruits, some berries, [4] [4] Flavonona - AMPK/IRS-1 -- HFDPrimary in C57BL/6 hepatocyte- micePrimary cells hepatocyte1.1. ImprovementresistanceImprovement cells ofof insulininsulin2. Stimulation Citrustomatoes, of glucose fruits, mint some uptake berries, tomatoes,[4] mint Flavonona - AMPK/IRS-1 -- HFDPrimary in C57BL/6hepatocyte mice cells 1. resistanceImprovement of insulin Citrustomatoes, fruits, mint some berries, [4] Flavonona - AMPK/IRS-1 -- PrimaryHFD in hepatocyteC57BL/6 mice cells 1.2. ImprovementresistanceStimulation of of glucose insulin uptake tomatoes,Citrus fruits, mint some berries, [4] Flavonona - AMPK/IRS-1 -- PrimaryHFD in C57BL/6hepatocyte mice cells 2. Stimulationresistance of glucose uptake tomatoes,Citrus fruits, mint some berries, [4] Flavonona - AMPK/IRS-1 - HFDPrimary in C57BL/6 hepatocyte mice cells 1.2. ImprovementStimulationresistance of glucoseof insulin uptake Citrustomatoes, fruits, mint some berries, [4] NaringinNaringin Flavonona - AMPK/IRS-1 -- Primary hepatocyte cells 2.2. resistanceStimulationStimulation ofof glucoseglucose uptakeuptake tomatoes, mint [4] Naringin - PrimaryHFD in C57BL/6hepatocyte mice cells 2. Stimulation of glucose uptake tomatoes,Citrus fruits, mint some berries, Naringin Flavonona - AMPK/IRS-1 2. Stimulationresistance of glucose uptake [4] NaringinNaringin - Primary hepatocyte cells tomatoes, mint Naringin 2. Stimulation of glucose uptake Naringin Citrus fruits, some berries, Naringin Flavonona - AMPK/IRS-1 - L6 rat skeletal myotubes 1. Stimulation of glucose uptake Citrus fruits, some berries, Citrus[4] fruits, some berries, FlavononaFlavonona - AMPK/IRS-1 - L6 rat skeletal myotubes 1. Stimulation of glucose uptake Citrus fruits, some berries, [4] [4] Flavonona - AMPK/IRS-1- AMPK/IRS-1 - L6- ratL6 skeletal rat skeletal myotubes myotubes 1. 1.Stimulation Stimulation of ofglucose glucose uptake uptake Citrustomatoes, fruits, mint some berries, tomatoes,[4] mint Flavonona - AMPK/IRS-1 - L6 rat skeletal myotubes 1. Stimulation of glucose uptake tomatoes,Citrus fruits, mint some berries, [4] Flavonona - AMPK/IRS-1 - L6 rat skeletal myotubes 1. Stimulation of glucose uptake tomatoes,Citrus fruits, mint some berries, [4] Naringenin Flavonona - AMPK/IRS-1 - L6 rat skeletal myotubes 1. Stimulation of glucose uptake Citrustomatoes, fruits, mint some berries, [4] Naringenin Flavonona - AMPK/IRS-1 - L6 rat skeletal myotubes 1. Stimulation of glucose uptake tomatoes, mint [4] Naringenin Citrustomatoes, fruits, mint some berries, Naringenin Flavonona - AMPK/IRS-1 - L6 rat skeletal myotubes 1. Stimulation of glucose uptake tomatoes, mint [4] Naringenin tomatoes, mint Naringenin 1. Increased glucose uptake in Skin of grapes, blueberries, Naringenin - L6 myotube cells 1. Increased glucose uptake in Skin of grapes, blueberries, Naringenin Stilbene - AMPK/GLUT4 - L6 myotube cells 1. Increasedmuscle cells glucose and adipocytes uptake1. Increased in Skinraspberries, glucose of grapes, uptake mulberries blueberries, in muscle and Skin[33] of grapes, blueberries, - - L6 myotube cells- L6 myotube1. cellsIncreased glucose uptake in Skin of grapes, blueberries, StilbeneStilbene - AMPK/GLUT4 -- L6db/db myotube mice cells 1. muscleIncreased cells glucose and adipocytes uptake in raspberries,Skin of grapes, mulberries blueberries, and raspberries,[33] mulberries and [33] Stilbene - AMPK/GLUT4- AMPK/GLUT4 -- db/dbL6 myotube mice cells 1.2. muscleIncreasedOvercome cells glucose insulin and adipocytes resistanceuptakecells in andraspberries, adipocytesSkinred wine of grapes, mulberries blueberries, and [33] StilbeneStilbene - AMPK/GLUT4AMPK/GLUT4 -- db/dbL6 myotube mice cells- db/db mice1.2. IncreasedmuscleOvercomemuscle cellscells glucose insulin andand adipocytesadipocytesresistance uptake in Skinraspberries,redraspberries, wine of grapes, mulberriesmulberries blueberries, andand [33][33] Resveratrol Stilbene - AMPK/GLUT4 -- L6db/db myotube mice cells 2. Overcomemuscle cells insulin and adipocytesresistance2. Overcome redraspberries, insulin wine resistance mulberries and red[33] wine Stilbene - AMPK/GLUT4 - db/db mice 2.1. muscleOvercomeIncreased cells glucoseinsulin and adipocytes resistance uptake in raspberries,redSkin wine of grapes, mulberries blueberries, and [33] Resveratrol -- L6db/db myotube mice cells 2. Overcome insulin resistance red wine Resveratrol Stilbene - AMPK/GLUT4 - db/db mice 2. muscleOvercome cells insulin and adipocytes resistance raspberries,red wine mulberries and [33] Resveratrol 2. Overcome insulin resistance red wine Resveratrol - db/db mice Resveratrol 2. Overcome insulin resistance redApple, wine grape, berries, onion, Apple, grape, berries, onion, Resveratrol 1. Increased glucose uptake in Apple, grape, berries, onion, Flavonol - AMPK/GLUT4 - In vitro 1. Increased glucose uptake in Apple,red wine, grape, beans, berries, broccoli, onion, [59–61] Flavonol - AMPK/GLUT4 - In vitro 1. Increasedmuscle cells glucose uptake in redApple, wine, grape, beans, berries, broccoli, onion, [59–61] Flavonol - AMPK/GLUT4 - In vitro 1.1. IncreasedmuscleIncreased cells glucoseglucose uptakeuptake1. Increased inin redApple,parsley glucose wine, grape, uptake beans, berries, inbroccoli, onion, Apple,[59–61] grape, berries, onion, red FlavonolFlavonol - AMPK/GLUT4- AMPK/GLUT4 -- InIn vitro vitro 1. muscleIncreased cells glucose uptake in Apple,redparsley wine, grape, beans, berries, broccoli, onion, [59–61] [59–61] Flavonol - AMPK/GLUT4 - In vitro 1. Increasedmuscle cells glucose uptakemuscle in cellsparsleyred wine, beans, broccoli, wine,[59–61] beans, broccoli, parsley Flavonol - AMPK/GLUT4 - In vitro muscle cells parsleyApple,red wine, grape, beans, berries, broccoli, onion, [59–61] Quercetin Flavonol - AMPK/GLUT4 - In vitro 1. Increasedmuscle cells glucose uptake in redparsley wine, beans, broccoli, [59–61] Quercetin - - muscle cells parsley Quercetin Flavonol AMPK/GLUT4 In vitro parsleyred wine, beans, broccoli, [59–61] Quercetin muscle cells Quercetin parsley Quercetin Quercetin 1. Potentiation on the Flavonol - AMPK/IRS-1 - In vitro 1. Potentiation on the Fruits, vegetables, tea [57] Flavonol - AMPK/IRS-1 - In vitro 1. Potentiationutilization of on glucose the Fruits, vegetables, tea [57] Flavonol - AMPK/IRS-1 - In vitro 1. Potentiationutilization of on glucose the Fruits, vegetables, tea [57] Flavonol - AMPK/IRS-1 - In vitro 1. utilization Potentiation of onglucose the 1. PotentiationFruits, on thevegetables, utilization tea [57] FlavonolFlavonol - AMPK/IRS-1- AMPK/IRS-1 - In- vitroIn vitro 1. utilization Potentiation of onglucose the Fruits, vegetables, tea Fruits, [57] vegetables, tea [57] Flavonol - AMPK/IRS-1 - In vitro 1. Potentiationutilization of on glucose the of glucoseFruits, vegetables, tea [57] EGCG Flavonol - AMPK/IRS-1 - In vitro utilization of glucose Fruits, vegetables, tea [57] EGCG 1. utilization Potentiation of onglucose the EGCG Flavonol - AMPK/IRS-1 - In vitro Fruits, vegetables, tea [57] EGCG utilization of glucose EGCG EGCG Phenolic 1. Insulin sensitivity Extra virgin olive oil, leaves EGCG Phenolic - AMPK/IRS-1 - 3T3-L1 adipocytes 1. Insulin sensitivity Extra virgin olive oil, leaves [63] EGCG Phenolicalcohol - AMPK/IRS-1 - 3T3-L1 adipocytes 1. Insulinimprovement sensitivity Extrafrom virginOlea Europea olive oil, L. leaves [63] PhenolicalcoholPhenolic - AMPK/IRS-1 - 3T3-L1 adipocytes 1.1. InsulinimprovementInsulin sensitivitysensitivity ExtrafromExtra Olea virginvirgin Europea oliveolive oil,oil,L. leavesleaves [63] HT alcoholPhenolic - AMPK/IRS-1 - 3T3-L1 adipocytes 1. improvementInsulin sensitivity fromExtra Olea virgin Europea olive oil,L. leaves Extra[63] virgin olive oil, leaves HT PhenolicalcoholPhenolic alcohol- AMPK/IRS-1 - 3T3-L1 adipocytes 1. Insulinimprovement sensitivity Extrafrom Oleavirgin Europea olive oil, L. leaves [63] [63] HT alcohol - AMPK/IRS-1- AMPK/IRS-1 - -3T3-L1 3T3-L1 adipocytes adipocytes 1.improvement Insulin sensitivity improvementfrom Olea Europea L. from[63]Olea Europea L. HT Phenolicalcohol - AMPK/IRS-1Indirect activation of 3T3-L1 adipocytes 1. Insulinimprovement sensitivity Extrafrom Oleavirgin Europea olive oil, L. leaves [63] HT alcohol - Indirect activation of - improvement from Olea Europea L. HT - AMPK/IRS-1Indirect activation of - 3T3-L1 Clinical adipocytes trial in newly [63] HT alcohol - IndirectAMPK byactivation inhibiting of - Clinical trial in newly 1. improvement Favorable effects on glucose, from Olea Europea L. HT Alkaloids - AMPKIndirect by activation inhibiting of - Clinicaldiagnosed trial type in newly 2 1. Favorable effects on glucose, Berberis spp. and other plants [82,83] Alkaloids - AMPKIndirectcomplex by activation I inhibitingof of -- Clinicaldiagnosed Clinical trialtrial type inin newlynewly2 1. Favorablelipids, HbA1c effects on glucose, Berberis spp. and other plants [82,83] Alkaloids - IndirectAMPKcomplex byactivation I ofinhibiting of - diagnosed Clinical trial type in newly2 1. Favorablelipids, HbA1c effects on glucose, Berberis spp. and other plants [82,83] Alkaloids complexAMPK by I of inhibiting - Clinicaldiagnoseddiabetes trial patients type in newly 2 1. lipids, Favorable HbA1c effects on glucose, Berberis spp. and other plants [82,83] Alkaloids - complexIndirectAMPKrespiratory- byIndirectactivation I of inhibiting chain activation of ofdiabetesdiagnosed AMPK bypatients type 2 1. lipids, Favorable HbA1c effects on glucose, Berberis spp. and other plants [82,83] Berberine Alkaloids AMPKrespiratorycomplex by I inhibitingof chain - Clinicaldiabetesdiagnosed trial patients type in newly- 2 Clinical trial1. in Favorablelipids, newly HbA1c diagnosed effects on glucose,1. Favorable Berberis effects onspp. glucose, and other plants [82,83] Berberine AlkaloidsAlkaloids respiratorycomplexinhibiting I of chain complex Idiagnoseddiabetes ofdiabetes patientspatients type 2 lipids, HbA1c Berberis spp. and other plants Berberis[82,83]spp. and other plants [82,83] Berberine - IndirectcomplexAMPKrespiratory activationby I of inhibiting chain of diabetes patients type 2 diabetes1. lipids, Favorable patients HbA1c effects on glucose,lipids, HbA1c BerberineBerberine Alkaloids - Indirectrespiratory activationrespiratory chain of chain- Clinicaldiabetesdiagnosed trial patients type in newly 2 Berberis spp. and other plants [82,83] Berberine - Indirectcomplexrespiratory activation I of chain of - lipids, HbA1c BerberineBerberine - IndirectAMPKrespiratory byactivation inhibiting chain of - Clinicaldiabetes trial patients in newly 1. Favorable effects on glucose, Alkaloids - AMPKIndirect by activation inhibiting of - Clinicaldiagnosed trial type in newly 2 diabetes 1. Favorable effects on glucose, Galega officinalis [82,83] Berberine Alkaloids - AMPKIndirectcomplexrespiratory by activation I inhibitingof chain of - ClinicaldiagnosedClinical trialtrial type inin newlynewly2 diabetes 1. Favorablelipids, HbA1c effects on glucose, Galega officinalis [82,83] Alkaloids - IndirectAMPKcomplex byactivation I ofinhibiting of - diagnosedClinical trial type in newly2 diabetes 1. Favorablelipids, HbA1c effects on glucose, Galega officinalis [82,83] Galegine Alkaloids complexAMPK by I of inhibiting - Clinicaldiagnosedpatients trial type in newly 2 diabetes 1. lipids, Favorable HbA1c effects on glucose, Galega officinalis [82,83] Galegine Alkaloids - complexIndirectAMPKrespiratory byactivation I of inhibiting chain of patientsdiagnosed type 2 diabetes 1. lipids, Favorable HbA1c effects on glucose, Galega officinalis [82,83] Galegine Alkaloids AMPKrespiratorycomplex -by IIndirect inhibitingof chain activation - ofClinicalpatientsdiagnosed AMPK trial by type in newly 2 diabetes 1. Favorablelipids, HbA1c effects on glucose, Galega officinalis [82,83] Galegine Alkaloids respiratorycomplex I of chain diagnosedpatientspatients type 2 -diabetes Clinical trial inlipids, newly HbA1c diagnosed 1. FavorableGalega effects officinalis on glucose, [82,83] NC:Galegine Nutraceutical compound;Alkaloids AMPK: AdenosincomplexrespiratoryAMPK monophosphateby I of inhibiting chain proteinpatients kinase; IRS-1: Insulin1. lipids,Favorable receptor HbA1c effects substrate on glucose, 1; GLUT4: Glucose transporter typeGalega 4; HFD: ofﬁcinalis High [82,83] NC:Galegine Nutraceutical compound;Alkaloids AMPK: Adenosinrespiratory monophosphateinhibiting chain complex proteinpatientsdiagnosed I of kinase; type 2 IRS-1:diabetes Insulin receptor substrate 1; GLUT4:Galega Glucose officinalis transporter type 4;[82,83] HFD: High NC:Galegine Nutraceutical compound; AMPK: Adenosinrespiratorycomplex monophosphate I of chain protein kinase; IRS-1:type 2Insulin diabetes lipids,receptor patients HbA1c substrate 2.1; lipids, GLUT4: HbA1c Glucose transporter type 4; HFD: High NC:fat diet;Galegine Nutraceutical T2DM: Type compound; 2 diabetes AMPK: mellitus; Adenosin EGCG: monophosphateEpillocatechinrespiratory chain gallate; proteinpatients HT: kinase; Hydroxytyrosol; IRS-1: Insulin HbA1c: receptor Glycated substrate haemoglobin. 1; GLUT4: Glucose transporter type 4; HFD: High fatNC: diet;Galegine Nutraceutical T2DM: Type compound; 2 diabetes AMPK: mellitus; Adenosin EGCG:respiratory Epillocatechinmonophosphate chain gallate; protein HT: kinase; Hydroxytyrosol; IRS-1: Insulin HbA1c: receptor Glycated substrate haemoglobin. 1; GLUT4: Glucose transporter type 4; HFD: High NC:fat diet; Nutraceutical T2DM: Type compound; 2 diabetes AMPK: mellitus; Adenosin EGCG: monophosphateEpillocatechin gallate; protein HT: kinase; Hydroxytyrosol; IRS-1: Insulin HbA1c: receptor Glycated substrate haemoglobin. 1; GLUT4: Glucose transporter type 4; HFD: High NC:fatNC: diet; Nutraceutical Nutraceutical T2DM: Type compound; compound; 2 diabetes AMPK: AMPK:mellitus; Adenosin Adenosin EGCG: monophosphateEpillocatechin monophosphate gallate; protein protein HT: kinase; kinase;Hydroxytyrosol; IRS-1: IRS-1: Insulin Insulin HbA1c: receptor receptor Glycated substrate substrate haemoglobin. 1; GLUT4: 1; GLUT4: Glucose Glucose transporter transporter typetype 4; HFD: 4; HFD: High High fat diet; T2DM: Type 2 fat diet; T2DM: Type 2 diabetes mellitus; EGCG: Epillocatechin gallate; HT: Hydroxytyrosol; HbA1c: Glycated haemoglobin. fatdiabetes diet; T2DM: mellitus; Type EGCG: 2 diabetes Epillocatechin mellitus; EGCG: gallate; Epillocatechin HT: Hydroxytyrosol; gallate; HT: HbA1c: Hydroxytyrosol; Glycated haemoglobin. HbA1c: Glycated haemoglobin.

Int. J. Mol. Sci. 2017, 18, 288 15 of 24

Several studies on food containing flavonoids have been reported to mediate blood glucose levels, being helpful for T2DM management. It has been identified that quercetin possesses α-glucosidase inhibitory activity in vitro [84,85]. Indeed, quercetin administration seems to reduce fasting and postprandial glycemia in diabetic mice and rats [86]. In addition to this, quercetin treatments resulted in interesting effects, improving insulin sensitivity in muscle cells through the AMPK pathway, which is involved in cellular glucose uptake by glucose transporter type 4. These findings suggest that quercetin constitutes a nutraceutical compound able to ameliorate insulin resistance in muscle cells through different events linked to AMPK phosphorylation and activation [87]. Phenolic alcohols, such as hydroxytyroxol (Table3) were also found to increase fatty acid oxidation and to improve insulin sensitivity through AMPK phosphorylation, as shown in 3T3-L1 adipocytes, suggesting its possible involvement in diabetes mellitus management [88]. Berberine and galegine are also natural occurring alkaloids that have been documented to exert several beneficial effects on human health and specially, on diebtes. Both berberine and galegine inhibit mitocondrial function through the inhibition of complex I of the respiratory chain [82] leading to an indirect activation of AMPK due to cessation of mitochondrial ATP production and, an increase in cellular AMP:ATP or ADP/ATP ratios in a similar manner to the biguanides [83]. Berberine has shown some favorable effects on plasma glucose, lipids and glycated haemoglobin (HbA1c) (Table3) in two clinical trials performed in patients newly diagnosed with type 2 diabetes [83].

5. Nutraceutical Compounds and Neurodegenerative Diseases Neurodegenerative diseases are characterized by progressive degeneration of nerve cells that eventually leads to dementia. Among these diseases, Alzheimer’s (AD), Parkinson’s (PD), Huntington’s (HD) and amyotrophic lateral sclerosis (ALS) can be found. Although they affect different neural populations, they share several characteristics in common. For example, they are characterized by the presence of proteins aggregates in degenerating neurons that certainly derive from defective purification mechanisms including proteosomal dysfunction and lysosomal clearance, and metabolic disturbances, excitotoxicity and oxidative stress are often described [89–91]. All of these aspects could participate in the deregulation of AMPK that has been reported to occur in these diseases [92]. In the brain, AMPK acts as a multifunctional metabolic sensor and, depending on the type of stress, cell type and duration of exposure, has a dual role in regulating cell death and survival: its activation incites cell death, while its inhibition produces a protective effect in different models exposed to different stressors [11]. The physiological functions of AMPK remain poorly studied, despite being highly expressed in neurons. Still, AMPK is vital for neuronal survival and genetic ablation of AMPK subunits γ (lochrig mutant, [93]) or β (alicorn mutant, [94]) in studies conducted in Drosophila, demonstrating that its genetic ablation induces progressive neurodegeneration. Given the demographic trend towards an aging population, the prevalence of these neurodegenerative diseases and, therefore, their socioeconomic burden will continue to increase dramatically in the coming decades. Current treatments are symptomatic only; there are no therapies available to cure these diseases [92]. In recent years, there have been considered several alternative approaches to delay the progression of these diseases and nutraceuticals are recently coming under the spotlight [95]. Excitotoxicity has been identified as one of the capital mechanisms involved in the onset and progression of a variety of neurodegenerative pathologies including AD, PD, and HD. Anthocyanins extracted from Korean black beans appear to strongly protect both mouse hippocampal HT22 cells and primary cultures of fetal rat hippocampal neurons against kainic acid (KA) induced excitotoxicity [96]. In fact, the treatment and consumption of isolated anthocyanins was able to inhibit KA-induced phosphorylation of AMPK, attenuate KA-induced deregulation of Ca2+ accumulation and ROS, and reduce the KA-induced increase in Bax content and decrease in Bcl-2 content, which slows the rate of apoptosis. These results suggest that anthocyanins may be involved in the neuroprotective Int. J. Mol. Sci. 2017, 18, 288 16 of 24 mechanism through modulation of the AMPK pathway, but more studies are needed to validate these hypotheses [96]. Recently, a novel mechanism has been proposed whereby, in old mice fed with a high cholesterol diet, quercetin may exert beneficial effects against cholesterol-induced neurotoxicity by activating AMPK and reducing protein phosphatase 2C (PP2C) expression [97]. In these animals, higher levels of brain ROS and carbonyl proteins were detected, along with a decrease in the activity of Cu-Zn superoxide dismutase (Cu-Zn SOD). Treatment with quercetin (Table4) was able to attenuate all these metabolic disorders, while these effects were attenuated by the administration of compound C. Oral administration of quercetin during a high-cholesterol diet blocked the cholesterol-induced activation of PP2C, promoted the activation of AMPK and subsequently the inactivation of ACC, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and FAS ligand. According to this study, quercetin supplementation decreased the density of CD11b-positive microglia cells (which are the first and main form of active immune defense in the central nervous system) in the hippocampus of these mice and reduced inflammatory markers such as COX-2, inducible nitric oxide synthase (iNOS), interleukin-1 beta (IL-1β), interlukin-6 (IL-6) and tumor necrosis factor α (TNF-α) through suppression of NF-κB in the brains of mice fed high cholesterol. In addition, it down-regulated the expression of the Aβ-converting enzyme 1, which decreased the levels of Aβ and Aβ deposits in the cerebral cortex and hippocampus. Finally, quercetin improved the cognitive deficit of mice fed high cholesterol [97]. Resveratrol (Table4) seems to exert also a neuroprotective effects through AMPK modulation. For example, its treatment inhibited extracellular signal-regulated kinases (ERK) and mTOR signaling in sensory neurons in a time- and concentration-dependent manner by AMPK activation, providing further evidence for AMPK expression as a novel treatment for acute and chronic pain states [98]. It also protected SH-SY5Y cells against rotenone-induced apoptosis and enhanced α-synuclein degradation in α-synuclein-expressing PC12 cell lines derived from pheocromocytoma of the rat adrenal medulla that promote autophagy via the AMPK/SIRT1 pathway in cellular models of PD [99]. In addition, in Neuro2a cells and primary neurons, resveratrol activated AMPK and stimulated mitochondrial biogenesis in an AMPK-dependent manner, independent of the activation of SIRT1, suggesting that resveratrol could affect homeostasis of neuronal energy [100]. In addition, the activation of the AMPK/SIRT1 axis could be potentially useful in reducing the risk of herpes simplex virus 1 (HSV-1) infection in neurons and the cellular damage associated with reactivation episodes [101]. Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEW 17 of 24 Int. J. Mol. Sci. 2017, 18, 288 17 of 24 Int.Int. J.J. Mol.Mol. Sci.Sci. 20172017,, 1818,, xx FORFOR PEERPEER REVIEWREVIEW 1717 ofof 2424

Table 4. List of nutraceutical compounds targeting AMPK pathways in neurodegenerative disease. Table 4. List of nutraceutical compounds targeting AMPK pathways in neurodegenerative disease. NC ClassificationTable Pathway 4. ListList ofof nutraceuticalnutraceutical compoundscompoundsExperimental targetingtargeting model AMAM PK pathwaysComments in neurodegenerative disease.Nutrient References NC NCClassification Classiﬁcation Pathway PathwayExperimental Experimental model modelComments CommentsNutrient NutrientReferences References NC Classification Pathway Experimental model Comments Apple,Nutrient berries, onion, References - Intraperitoneal 1. Reduction of neurotoxicity red wine, beans, Flavonol - PP2C/AMPKα/NF-κB injection in C57BL/6 Apple, berries, onion, [72] -- IntraperitonealIntraperitoneal Apple, berries, onion, red - Intraperitoneal injection in 1.2. ReductionNeuroprotective1. Reduction of neurotoxicity of effect neurotoxicity redbroccoli, wine, parsley, beans, Flavonol - PP2C/AMPKα/NF-κB aged mice 1. Reduction of neurotoxicity red wine,wine, beans, beans, broccoli, [72] Flavonol -- PP2C/AMPKα/NF-/NF-κB injectioninjectionC57BL/6 inin aged C57BL/6C57BL/6 mice 2. Neuroprotective effect [72][72] 2. Neuroprotective effect broccoli,green teaparsley, parsley, green tea Quercetin aged mice green tea Quercetin Quercetin Red raspberries, - Regulation of - HT22 cells 1. Attenuation of ROS accumulation Redsoybean, raspberries, peach, Anthocyanin - mitochondrial apoptotic - Primary hippocampal Red raspberries, [71] - Regulation of - HT22- HT22 cells cells 2. Decrease of apoptosis lychee,Red red raspberries, oranges soybean, (Bax/Bcl-2)- Regulation pathway of mitochondrial neuronal cells 1. Attenuation1. Attenuation of ROS of accumulation ROS accumulation soybean,soybean, peach,peach, AnthocyaninAnthocyanin mitochondrial apoptotic -- Primary- Primary hippocampal hippocampal peach, lychee, red oranges[71][71] [71] apoptotic (Bax/Bcl-2) pathway 2. Decrease2. Decrease of apoptosis of apoptosis lychee,andlychee, riceand redred rice orangesoranges Cyanidin (Bax/Bcl-2)(Bax/Bcl-2) pathwaypathway neuronalneuronal cells cells and rice Cyanidin 1. Autophagy-Mediated Neuroprotection - SH-SY5Y cells 1. Autophagy-Mediated 2. Increase1. Autophagy-Mediated viability of Neuroprotection - SH-SY5Y cells Neuroprotection -- SH-SY5YVero cells cells 2. Increase viability of Skin of grapes, SH-SY5Y- Vero cells cells HSV-1-infected neurons Skin of grapes, blueberries, - HT22 cells 2. IncreaseIncreaseHSV-1-infected viabilityviability ofof neurons blueberries, StilbeneStilbene - AMPK/SIRT1- AMPK/SIRT1 autophagy autophagy -- Vero- HT22 cells cells 3. Inhibit HSV-1 gene expression Skin ofraspberries, grapes, mulberries and[74,76][74 ,76] HSV-1-infected3. Inhibit HSV-1 neurons gene expression red wine -- HT22Rockefeller- Rockefeller cells mice mice embryos blueberries,raspberries, mulberries Stilbene - AMPK/SIRT1 autophagy 3.4. Inhibit4. HSV-1 HSV-1Inhibit HSV-1gene virion expression virion progeny productio [74,76] Resveratrol Stilbene - AMPK/SIRT1 autophagy embryosprimary primary cultured neurons 3. Inhibit HSV-1 gene expression and red wine [74,76] Resveratrol -- Rockefeller mice progeny5. Reduced productio neurodegenerative markersraspberries,raspberries, mulberriesmulberries cultured neurons 4. InhibitInhibit HSV-1HSV-1 virionvirion Resveratrol embryosembryos primaryprimary and red wine ResveratrolNC: Nutraceutical compound; PP2C: Protein phosphatase 2C; AMPK: Adenosin protein kinase; SIRT1:5. NAD-dependentprogenyReduced productioneurodegenerative deacetylase sirtuin-1; HSV-1: Herpes simplex virus-1; ROS: culturedcultured neuronsneurons Reactive oxygen species. 5. Reducedmarkers neurodegenerative NC: Nutraceutical compound; PP2C: Protein phosphatase 2C; AMPK: Adenosin protein kinasemarkers; SIRT1: NAD-dependent deacetylase sirtuin-1; HSV-1: Herpes NC:simplex Nutraceutical virus-1; ROS: compound; Reactive oxygenPP2C: Proteinspecies. ph osphatase 2C; AMPK: Adenosin protein kinase;; SIRT1:SIRT1: NAD-dependentNAD-dependent deacetylasedeacetylase sirtusirtuin-1;in-1; HSV-1:HSV-1: HerpesHerpes simplexsimplex virus-1;virus-1; ROS:ROS: ReactiveReactive oxygenoxygen species.species.

Int. J. Mol. Sci. 2017, 18, 288 18 of 24

6. Conclusions Given that AMPK is considered a master regulator of metabolism and has a potential effect in a lot of metabolic pathways, its modulation proposes to be a very important target for the treatment of different diseases. In this review, we have revised several of the most important nutraceutical compounds which have been shown to have an AMPK-mediated therapeutic effect. From these, several have shown an indirect effect on the AMPK activation, so new experiments about knockdown and knockout models will show a more direct perspective of the effect in AMPK activation. In this sense, berberine has been shown to lose effect by knockdown of AMPKα expression [102] with similar results in other compounds such as quercetin [103], proposing the need for this methodology in these studies. However, the effects shown for all these compounds could be evaluated for preventive potential. Could, for example, resveratrol have a preventive effect in the diet with respect to neurodegenerative disorders? Several of the diseases shown in this paper are related to aging, so the effect of these compounds included in the diet or as a supplement must be studied in animal models of age-related diseases in order to know the potential effect in the aging process. On the other hand, an important topic which will need study is the translational effect of doses from animal models to humans. For the in vitro and animal studies cited in the text, the experimental doses could translate into potential human doses depending on the pathology and on the patient requirements. Of course, more studies and clinical trials are needed to establish a therapeutical dose for the treatment of major diseases. To date, it has been documented that natural compounds can be used as prophylactics or to prevent onset of illness, and some experimental research suggests that they could be potential compounds for targeting AMPK. However, several of these compounds (such as resveratrol) require high doses in humans in order to show results, and new capsules with highly-puriﬁed doses are being commercialized. Of course, long-term treatment in humans will be needed with these compounds and their presentations.

Acknowledgments: This study was supported by a grant from the Andalusian regional government (Grupo de Investigacion Junta de Andalucia CTS113, Consejería de Salud de la Junta de Andalucia: PI-0036-2014). Fabiola Marín-Aguilar has the benefit of a “Formación de Personal Universitario” (FPU) Fellowship (FPU 13/03173) from the Spanish Ministry of Education. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ACC1 Acetyl-CoA carboxylase 1 ACC2 Acetyl-CoA carboxylase 2 ADP Adenosine diphosphate AMP Adenosine monophosphate AMPK Adenosine monophosphate-activated protein kinase ATP Adenosine triphosphate CaMKKb Ca2+/calmodulin-dependent protein kinase kinase-β EGCG Epillocatechin gallate ERK Extracellular signal-regulated kinases GLUT4 Glucose transporter type 4 HbA1c Glycated haemoglobin HDAC Histone deacetylase HMG-CoA reductase 3-hydroxy-3methyl-glutaryl-coenzyme A reductase HOCl Hypochlorous acid HSV-1 Herpex simplex virus 1 HT Hydroxytyrosol ICAM-1 Intracellular adhesion molecule 1 iNOS Inducible nitric oxide synthase LKB1 Liver kinase B1 mTOR Mammalian target of rapamycin mTORC1 Mammalian target of rapamycin complex 1 NF-κB Nuclear Factor-κ β PGC1α Peroxisome proliferator-activated receptor gamma coactivator 1-α PP2C Protein phosphatase 2C PRKAA1 Protein Kinase AMP-Activated Catalytic Subunit α 1 PRKAA2 Protein Kinase AMP-Activated Catalytic Subunit α 2 PRKAB1 Protein Kinase AMP-Activated Non-Catalytic Subunit β 1 PRKAB2 Protein Kinase AMP-Activated Non-Catalytic Subunit β 2 PRKAG1 Protein Kinase AMP-Activated Non-Catalytic Subunit γ 1 Int. J. Mol. Sci. 2017, 18, 288 19 of 24

PRKAG2 Protein Kinase AMP-Activated Non-Catalytic Subunit γ 2 PRKAG3 Protein Kinase AMP-Activated Non-Catalytic Subunit γ 3 Raptor Regulatory-associated protein of mTOR SIRT1 Sirtuin-1 TBC1D1 Tre-2/Bub2/Cdc16 domain family member TIF-1A Transcription initiation factor TNF-α Tumor necrosis factor α TSC2 Tuberous sclerosis complex 2 ULK1 Unc-51-like autophagy activating kinase 1 ZMP Phosphorylated AICAriboside

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