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Review

Oxidative stress and lipid peroxidation by-products at the crossroadbetween adipose organ dysregulation and obesity-linked insulinresistance

Giuseppe Murdolo a,*, Marta Piroddi b, Francesca Luchetti c, Cristina Tortoioli d,Barbara Canonico c, Chiara Zerbinati e, Francesco Galli b, Luigi Iuliano e

aDepartment of Internal Medicine, Assisi Hospital, Via Valentin Muller 1, I-06081 Assisi, Perugia, ItalybDepartment of Internal Medicine, Section of Applied Biochemistry and Nutritional Sciences, Perugia University, Perugia, ItalycDepartment of Earth, Life and Environmental, University Carlo Bo, Urbino, ItalydDepartment of Internal Medicine, Section of Internal Medicine, Endocrine and Metabolic Sciences, Perugia University, Perugia, ItalyeDepartment of Medic-Surgical Sciences and Biotechnologies, Unit of Vascular Medicine, Sapienza University of Rome, Latina, Italy

a r t i c l e i n f o

Article history:Received 6 July 2012Accepted 13 December 2012Available online 26 December 2012

Keywords:Adipose tissue dysregulationInsulin-resistanceObesityOxidative stress preadipocytes

a b s t r a c t

Obesity has been proposed as an energy balance disorder in which the expansion of adipose tissue (AT)leads to unfavorable health outcomes. Even though adiposity represents the most powerful driving forcefor the development of insulin resistance (IR) and type 2 diabetes, mounting evidence points to “adiposedysregulation”, rather than fat mass accrual per se, as a key pathophysiological trigger of the obesity-linked metabolic complications. The dysfunctional fat, besides hypertrophic adipose cells and inflam-matory cues, displays a reduced ability to form new adipocytes from the undifferentiated precursor cells(ie, the preadipocytes). The failure of adipogenesis poses a “diabetogenic” milieu either by promoting theectopic overflow/deposition of lipids in non-adipose targets (lipotoxicity) or by inducing a dysregulatedsecretion of different adipose-derived hormones (ie, adipokines and lipokines). This novel and provoc-ative paradigm (“expandability hypothesis”) further extends current “adipocentric view” implicatinga reduced adipogenic capacity as a missing link between “unhealthy” fat expansion and impairment ofmetabolic homeostasis.

Hitherto, reactive oxygen species have been implicated in multiple forms of IR. However, the effects ofstress on adipogenesis remain controversial. Compelling circumstantial data indicate that lipid perox-idation by-products (ie, oxysterols and 4-hydrononenal) may detrimentally affect adipose homeostasispartly by impairing (pre)adipocyte differentiation. In this scenario, it is tempting to speculate that a finetuning of the adipose redox status may provide new mechanistic insights at the interface between fatdysregulation and development of metabolic dysfunctions. Yet, in humans, the molecular “signatures” ofoxidative stress in the dysregulated fat as well as the pathophysiological effects of adipose (per)oxidationon glucose homeostasis remain poorly investigated.

In this review we will summarize the potential mechanisms by which increased oxidative stress in fatmay impair (pre)adipocyte differentiation and promote the adipose dysfunction. We will also attempt tohighlight the conundrum with the adipose redox changes and the regulation of glucose homeostasis.Finally, we will briefly discuss the scientific rationale for proposing the adipose redox state as a potentialtarget for novel therapeutic strategies to curb/prevent adiposity-linked insulin resistance.

� 2013 Elsevier Masson SAS. All rights reserved.

1. Historical background: the regulation of energy balanceand body weight

The maintenance of adequate nutrition and energy stores isessential for survival and reproductive capacity of individuals.Humans, like other mammals, are characterized by a tight controlof energy homeostasis by coordinated changes in food intakeand energy expenditure through mechanisms that allow the

Abbreviations: AT, adipose tissue.* Corresponding author. Tel.: þ39 (0)75 578 3588; fax: þ39 75 573 0855.

E-mail address: gmurdolo@tiscali.it (G. Murdolo).

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maintenance of a stable body weight [1,2]. Numerous control path-ways, involving both neural networks and circulating signals, haveevolved to maintain energy balance at an optimal level [1,2].

Adipose tissue (AT) serves a primary role in the setting of energyhomeostasis because it represents the body’smain depot for energystorage and mobilization. Homeostatic circuits that regulate bodyfat mass include, among others, those based on insulin and leptin.These hormones, which circulating concentrations are proportionalto fat mass, may act as “adiposity signals”. Insulin, on the one hand,displays adipogenic actions and stimulates the storage of triglyc-erides in adipose cells, which became enlarged. The size of adipo-cytes, on the other, may be “sensed” to the brain via the secretion ofleptin [1,2], which, in turn, acts on the central nervous system toreduce feeding and increase energy expenditure to restrain fatmass expansion. Besides the “adiposity signals”, gut hormones,such as the gastric hormone ghrelin (see below), are also known tosignal to the brain inducing a central integration according to thedietary intake and nutrient requirements. Evidence is mounting tosuggest extensive cross-talks between adiposity signals, guthormones and insulin [3,4].

Ghrelin, the orexigenic protein secreted by X/A-like cells of thegastric oxyntic glands, emerges as a suitable candidate linking fatmass expansion, control of food intake and glucose homeostasis.Independently of the orexigenic action, both the acylated anddesacylated ghrelin forms may stimulate lipid accumulation inhuman visceral adipocytes and, as compared with obese normo-glycemic subjects, obese patients with type 2 diabetes (T2D) exhibithigher acylated ghrelin concentrations [3]. Further insights intoghrelin effects on adipobiology have been provided by the discoveryof aquaporins (AQP) [5]. Aquaporin-7 (AQP7), the adipose-specificwatereglycerol transporter present in the plasma membrane ofadipocytes, operates as a glycerol channel in vivo, whereby fat cellpermeability and delivery of glycerol into plasma display a key rolein the regulation of fat accumulation and insulin resistance [5].Recent data indicate that, under physiological conditions insulinmediates a coordinated regulatory loop between fat-specific AQP7and liver-specific AQP9 in order to maintain glucose homeostasisthroughmodulation of glycerol output from adipocytes and glyceroluptake for hepatic gluconeogenesis according to the nutritionalstatus [6]. On the other hand, the expression of AQP7 in humanvisceral adipocytes is repressed by acylated and desacyl ghrelin,supporting the view that ghrelin decreases lipolytic capacity andpromotes fat cell enlargement [3]. Notably, insulin is also requiredfor prandial ghrelin suppression in humans [7], and chronic hyper-insulinemia, as well as hyperleptinemia, have been indicated as themost importantmodulators of ghrelin secretion in obese individualswith orwithout T2D [4]. It is thus plausible that the disruption of thecoordinated regulation between insulin, ghrelin and AQP7/AQP9might impair glucose homeostasis by increasing glycerol releasefrom adipocytes and glucose production from the liver, therebyperpetuating a vicious cycle.

In this scenario, the occurrence of obesity may be seen asa result from the failure or breakdown of the homeostatic mecha-nisms regulating energy balance. Many attempts have thus beenmade to explain how genetic and environmental factors can over-come normal energy homeostatic control and cause obesity. Thisfertile area of research is continuously open to new areas ofunderstanding and could potentially open novel therapeutic targetsfor tackling adiposity epidemic.

2. Obesity and type 2 diabetes: not only a matter of fat

Overwhelming evidence has proven that obesity plays a centralrole in the development of insulin resistance and T2D [8e10].While in obesity AT expands to accommodate increased lipid

partitioning [11], fat mass accrual by itself is unlikely to be the onlyinstigator of the adiposity-associated unfavorable metabolicoutcomes. Different epidemiological trials showed that somemorbidly obese patients (and up to 25% of obese individuals) are“metabolically healthy”, while about 18% of non-obese subjectsdemonstrate biochemical characteristics of the insulin-resistancesyndrome [12e14]. Accordingly, some authors have recently sug-gested a reappraisal of the cutoffs to diagnose overweight andobesity. Indeed, 29% and 80% of subjects classified as lean or over-weight according to their body mass index (BMI), respectively,are actually “obese” as estimated by their body fat percentage.More importantly, these subjects already exhibit similar car-diometabolic risk factor profile as obese patients [15]. Altogether,these observations support the assumption that fat mass expansionis neither sufficient nor necessary for development of themetabolicdysfunctions.

AT is now emerging as a remarkably active “organ”, with func-tional pleiotropism and high remodeling capacity [16]. Althoughbasically adipose tissues are generally regarded as connectivetissues without a specific anatomy, accumulating data support theidea that fat tissues are organized to form a large “organ” withdiscrete anatomy, specific vascular and nerve supplies, complexcytology, and high physiological plasticity [17]. Obesity impliesextensive changes in AT ultrastructure involving the enlargementof existing adipocytes, the formation of new fat cells fromcommitted (pre)adipocytes, (adipogenesis), extracellular matrixproteolysis, and the coordinated development of the tissue vascularnetwork (angiogenesis). Arguably, while fat is by far the mostplastic organ in our body, the limit of fat mass expansion appearsdefined for any given individual [18]. Indeed, if insulin resistancewould be a direct consequence of an increased fat mass, all thesubjects would develop metabolic complications at the samedegree of adiposity. In contrast, on an individual level, there is noclear cut point of “adiposity”, as conventionally ascertained by theBMI, clearly separating distinct insulin sensitive from insulinresistant sub-phenotypes [13].

Although different and partly conflicting hypotheses havebeen put forward to explain such an apparent paradox, oneprovocative and seemingly counterintuitive paradigm postulatesthat a reduced ability of the adipose organ to further expand, ratherthan fat mass accrual per se, may be the key determinant under-lying the adiposity-inducedmetabolic dysfunctions (“expandabilityhypothesis”) [19e21].

3. Impaired adipogenesis characterizes the “hypertrophic”,metabolically unhealthy obesity

The cellular composition of AT is heterogeneous [11]. In adults,the majority (w35e70%) of adipose mass volume comprisesadipocytes, which account for only 25% of the total cell population.Notably, diverse cell-types, which include the adipose precursorcells among others, are found in the so-called “stroma-vascularfraction”, and account for the remaining 75% of the whole cellpopulation.

Recent evidence points to substantial AT “remodeling” in obesity,which appears of particular interest in the setting of metabolichomeostasis [21]. Mature adipocytes are derived from a pool ofundifferentiated precursors (ie, the adipose-derived mesenchymalstem cell, ASC), which become “committed” toward the adipogeniclineage (ie, preadipocytes). However, not all the progenitors becomeadipocytes simultaneously [22]. Our current understanding of ATdevelopment in humans is that the major pool of precursor cells isprimarily recruited before puberty, while in adulthood there is a 10%annual adipocyte turn-over [23,24]. This implies that fat massexpansion in adult age is mainly a consequence of adipocyte

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enlargement (hypertrophy) rather than recruitment of new adipo-cytes (hyperplasia). The enlargement of existing adipocytes and theformation of new fat cells are however exquisitely regulated by thebody’s demand of storing fat, and thus represent a prerequisite formaintaining energy and metabolic homeostasis [25]. Growth ofa “healthy” or “unhealthy” fat organ appears to be determined bya complex, cell-type orchestrated, coordination, whereby adipocyteturnover is balanced by recruitment and differentiation of new fatcells, which, in turn, needs to be “titrated” to the requirement forenergy storage [21].

The adipocyte is the only cell whose size may vary dramatically;about 20-fold in diameter, and, thus 2000-fold in volume [11].However, the enlargement of fat cell is not “indefinite”. Theformation of new adipocyte from the precursors pool takes placeonce a maximum capacity is attained, which in humans averages1000 pL [11]. In the face of a positive energy balance, the failure, orthe lack thereof, of such a “feedback” mechanism, leads to hyper-trophy of mature fat cells. Notably, the lipid-accumulating ability ofhuman preadipocytes is inversely correlated with the size ofmature fat cells of the donor, implying that adipocytes enlargement(hypertrophy) represents a consequence of a reduced ability torecruit/differentiate new (pre)adipocytes [25e27]. In keeping withthis, the hypertrophic obesity is more strongly associated withinsulin resistance than the hyperplastic form [14,28], and theenlargement of the adipocyte in the subcutaneous (sc) abdominaldepot has previously been reported as an “obesity” independentpredictor of insulin resistance and T2D development [29,30].Furthermore, hypertrophic adipocytes, even in the absence ofobesity per se, are associated with several features of “dysfunc-tional” fat and systemic insulin resistance [31]. Accordingly, insimilarly obese individuals, only those diagnosed with T2D exhibitinappropriately enlarged sc adipocytes and decreased adipogeniccapacity [32e34]. Even more, an impaired ability to recruit new fatcells and the inappropriate enlargement of pre-existing adipocytesalso characterize a population of non-obese, but insulin resistantindividuals with genetic predisposition for T2D and features of themetabolic syndrome, as compared with phenotypically “matched”subjects lacking diabetes heredity [35]. The adipose cell hyper-trophia can be thus envisaged as a “marker” for increased suscep-tibility to the metabolic complications early and before “obesity”(as conventionally defined by BMI cut-point) develops [27]. Furtherinsights into the complex interaction between (pre)adipocytedifferentiation and metabolic outcomes have recently beenprovided by the observation that, in the setting of obesity, anenhanced adipogenic capacity of the sc fat depots may protectagainst the metabolic syndrome [36].

Altogether, these observations emphasize the concept thatinsulin resistance syndrome is a pathophysiological cue initiated in,and sustained by, a dysregulated fat with impaired adipogenesis(“adipocentric view”). The failure of (pre)adipocytes differentiation,in turn, supports the novel and provocative paradigm of the“adipose expandability” hypothesis, which proposes that individ-uals possess a genetically and environmentally determined limit forAT expansion, beyond which lipids are deposited ectopically innon-adipose organs [37e39].

A key question is now why the impairment of (pre)adipocytedifferentiation instigates adipose organ dysfunction and hampersglucose homeostasis.

4. Impaired adipogenesis induces adipose dysregulation andposes a “diabetogenic” milieu

From a pathophysiological standpoint, dysfunctional adipocytematuration may instigate a “diabetogenic”milieu through differentand partly synergistic mechanisms.

First, the inability to safely store “metabolically active” fattyacids derivatives in hypertrophic adipocytes induces “ectopic”accumulation of lipids within non-adipose “targets” (ie, skeletalmuscle, liver, pancreatic islets) with toxic and detrimental conse-quences on tissue homeostasis (lipotoxicity) [40]. A generalphenomenon for ectopic fat accumulation is that it occurs when thesc adipocytes become enlarged (around 0.8e1.0 mg lipid/cell), andthus “resistant” to further lipid accretion [41]. In harmony with theconcept of lipotoxicity, systemic metabolic homeostasis has beenconsistently linked with a “lipid-mediated endocrine network”[42]. Experimental animal models, and, preliminary evidence inhumans [43,44], suggest that a single serum lipid (ie, palmitoleicacid) may function as insulin sensitizing adipose-derived lipidhormone (“lipokine”). Therefore, in the face of positive energybalance, an impaired formation of new fat cells may lead toenlargement of pre-existing adipocytes, which, in turn, become“dysfunctional”, “insulin-resistant” [38] and secrete a pattern of“diabetogenic” lipokines. In line with this, recent humans studiesdemonstrate that AT content of specific fatty acids is positivelycorrelated with insulin sensitivity (ie, myristic and stearic acid),while the enrichment of “harmful” lipid species (ie, palmitic acids)is associated with both increased adipose cell size and insulinresistance/T2D [45,46]. Finally, in a population of similarly obeseindividuals with and without the metabolic syndrome, a compre-hensive “lipidomic” characterization found that, in plasma, the shiftbetween different saturated (ie, cerotic acid) and unsaturated (ie,nervonic acid) fatty acid derivatives was strongly associated withthe “unhealthy” obese phenotype, while no significant differencesin palmitoleic acid levels were seen (Iuliano L. unpublished data). Itis thus conceivable that the pattern of specific fatty acids in thebloodstream may well reflect the degree of “adipose cell dysfunc-tion” and drive the metabolic homeostasis. In this regard, anintriguing role in adipocyte biology has recently been provided bythe characterization of caveolins [47e49]. These integral plasmamembrane proteins serve as structural elements of caveolae havingproven scaffolding, transport and signaling capabilities, and playa key role in shifting the focus of obesity and insulin resistancedevelopment to lipid dynamics [49]. Of note, isolated adipocytesfrom obese T2D patients display higher expression of caveolin-1(CAV-1) when compared with those of obese normoglycemicsubjects, and CAV-1 expression in sc abdominal depot appearsinversely associated with fasting triglycerides and lipogenic genes[47,48]. These observations highlight the potential importance ofCAV-1 in the regulation of intracellular cholesterol homeostasis andlipid storage ability of adipocytes, even though the clinical rele-vance of CAV-1 in the development of obesity-associated co-morbidities needs to be further elucidated.

Second, enlargement of pre-existing adipocytes instigatesmacrophage infiltration into the AT, which, in turn, promotes localinflammation and induces a dysregulated secretion of otherprototypical adipose-derived hormones (ie, adipokines) [38]. Theadipose inflammation appears orchestrated by complex auto/paracrine cellular cross-talks. The increased production of pro-inflammatory molecules (ie, IL-6), along with the downregulationof adiponectin, an insulin-sensitizing and anti-diabetogenichormone, leads to impairment of insulin signaling and promotesboth the cardiovascular and metabolic dysfunctions [10,38,50]. Ofinterest, lipocalin-2 (LCN-2) has recently been reported as a novelproinflammatory adipokine with potential role in adiposity-linkedinsulin resistance [51]. The increased expression of LCL-2 in visceralfat depot of obese patients, as well as the association with severalpro-inflammatory genes, implicate LCN-2 as an important auto-crine/paracrine modulator of the inflamed fat. Accordingly,increased macrophage infiltration and reduced circulating adipo-nectin levels characterize the dysfunctional and hypertrophic fat of

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morbidly obese, but insulin resistant, individuals (“unhealthyobesity”), when compared with similarly obese, insulin-sensitive(“healthy obesity”), counterparts [14]. Intriguingly, the discoveryof microRNA (miRNA) has recently opened new insights for betterunderstanding the complicated regulatory network governingmacrophage-mediated AT inflammation/dysfunction and meta-bolic outcomes. MiRNA are a family of small noncoding RNAs thathave been demonstrated to be crucial regulators in multiplepathophysiological processes and importantly associated withchronic inflammatory diseases [52]. Experimental data in miceidentified miR-233 as a novel regulator of macrophage “polariza-tion”, whichmay protect against diet-induced AT inflammation andsystemic insulin resistance by promoting phenotypic switching ofresident macrophages from proinflammatory (M1) toward anti-inflammatory (M2) responses [52]. It has thus been proposeda new miRNA-based paradigm for the regulation of insulin-sensitivity, which provides the basis for using miRNA as analogsto treat insulin resistance related disorders.

Third, the inflammatory microenvironment in inappropriatelyexpanded fat organ negatively affects preadipocyte differentiation[53]. Moreover, locally released inflammatory molecules (ie, TNF-a)can either sustain amacrophage-like phenotype in undifferentiatedprecursor cells [25] or also impair the ability of mature adipose cellsto safely store triglycerides [54], thereby perpetuating a “viciouscycle”.

Together, these findings provide a robust mechanistic platformfor linking impaired preadipocyte differentiation with the cues ofadipose dysregulation (increased fat cell size, inflammation,dysfunctional trafficking/overflow of fatty acids, secretion of pro-diabetogenic lipokines and adipokines), which, ultimately, initi-ates the insulin resistance and/or favors the progression towardfrank diabetes.

The key question is then why there is an impairment of pre-adipocyte differentiation.

5. The Wnt signaling network: a molecular driver of (pre)adipocyte differentiation

Recently, the characterization of the adipogenic precursor cellsin the context of “healthy” or “unhealthy” obesity, as well as thesignal networks underlying the lineage “commitment”, recruit-ment and differentiation are attracting a great deal of interest.

Adipocyte arises from undifferentiated mesenchymal precursorcells (ASCs). Although the molecular aspects of adipogenesis arebeyond the purpose of this review, fat cell formation is a complexand well-orchestrated multistep process that mainly comprisesa “commitment” step of the undifferentiated precursors into pre-adipocytes and a “terminal differentiation” to mature fat cells[55,56]. The molecular regulation of terminal differentiation hasbeen well characterized, and relies on sequential activation oftranscription factors where induction of PPARg and CCAT/enhancer-binding protein alpha (C/EBPa) acts synergistically toproduce and maintain the phenotype of the mature fat cell [55,56].In contrast, there is yet a dearth of knowledge on the molecularevents regulating the commitment of ASCs to the adipocyte lineage.A growing body of data indicates the wingless-type MMTV inte-gration site family (Wnt) signaling as a critical regulator of adipo-genic precursor cell fate [20,27,50,53,57e64].

Basically,Wnts are an evolutionary conserved family of secretedcysteine-rich glycosylated proteins that, through autocrine orparacrine network influence numerous developmental processes,control cell fate determination, cell proliferation/differentiation,regulating adult tissues remodeling [20,64e66]. In the “classical”(formerly referred to as “canonical”) Wnt-signaling pathway, thebinding and cross-link of Wnts to a receptor complex, which

comprises the Frizzled (Fzd) and LDL receptor-related protein(LRP5/6) co-receptors, initiate signaling cascades that converge onthe transcriptional regulator b-catenin (Fig. 1). In the absence ofWnts, cytoplasmic b-catenin is recruited to a “degradationcomplex”, whereby glycogen synthase kinase 3-b (GSK-3b), axinand adenomatosus polyposis coli (APC) cooperate in inducingsequential phosphorylation and subsequent proteasomal degra-dation of b-catenin. Binding of specific Wnt ligands to Fzd-LRP5/6receptor complex results in activation of Dishevelled (Dvl) andrecruitment of axin to LRP5/6 co-receptor. The sequestration of axinto the receptor complex also inactivates the “degradation complexand prevents proteasomal degradation of b-catenin, whichbecomes stabilized (hypophosphorylated) and accumulates in thecytosol. This ultimately coincides with the nuclear translocationand binding of b-catenin to the lymphoid enhancer-binding factor/T-cell-specific transcription factors (LEF/TCF), family of transcrip-tion factors that activate specific Wnt target genes [20]. Todate, more than 80 target genes are known to be regulated by theWnt/b-catenin pathway, and among others they include thosethat promote cell-cycle progression, cellular differentiation, andmetabolism [60].

Notably, AT contains several Wnt-producing cells, and the“canonical” Wnt/b-catenin pathway appears to be highly active inundifferentiated ASCs where it may direct the cell fate toward oste-ogenic/myogenic and aside the adipogenic differentiation [57e60].

The inability to adequately suppress Wnt activation in ASC hasthus been regarded as a possible mechanism underlying the per-turbedadipogenesis of thehypertrophic/dysregulated fat. Numerousstudies demonstrated that the inhibition ofWnt signaling representsan absolute prerequisite either for ASC commitment toward theadipogenic lineage or for appropriate (pre)adipocyte differentiationand induction of PPARg and C/EBPa [27,50,53,61e63,67]. Further-more, temporal expression profiling studies also indicate that whileWnt activation appears required early to increase the number ofpreadipocytes recruitment, this signalmust be terminatedbefore thenewly recruited (pre)adipocytes can undergo terminal differentia-tion [58]. Thus, the fine tuning of the Wnt signaling within specific“time-windows” allows that the differentiation of nascent (pre)adipocytes and growth of pre-existing fat cells meet the energystorage demand. Notably, in the sc AT of non-obese, but insulin-resistant individuals, the gene expression of canonical Wntsignaling components (ie, b-catenin andGSK-3b) is upregulated and,in line with the reduced abundance of essential adipogenic tran-scription factors (ie, C/EBPa and PPAR-g), appears inversely corre-lated with the size of adipocytes [68]. These findings reinforce theconcept that inappropriate canonical Wnt signaling activation in ATmight be an early and “adiposity-independent” instigator ofimpaired adipogenesis and systemic insulin resistance. Nonetheless,althoughWnts are basicallymolecules secreted by cells that have notundergone full terminal differentiation (ie, the preadipocytes), alsomature fat cells were reportedly shown to release factors thatmodulate Wnt signaling, implying a paracrine regulation andcommitment of early precursor cells as the mature adipocytesexpand [27,61]. Together, the prevailing view is that a coordinatedregulation of Wnt/b-catenin signaling is required to prevent“unhealthy” AT expansion and consequent development of meta-bolic complications.

What might thus be the pathophysiological factors responsiblefor uncoupling Wnt/b-catenin signaling and AT plasticity, and whyWnt is over-active in the dysregulated fat?

Thus far, this remains an open question. While genetic factorsare likely to play a role [69,70], a dysregulated secretion of“endogenous” Wnt antagonists has recently been postulated[27,61,71,72]. Interestingly, the finding that the anti-adipogeniceffects of TNF-a involve activation of the Wnt signal implicates

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the adipose inflammation as potential contributor to deregulationof Wnt/b-catenin signaling [25,50,53].

As a corollary to these findings, theWnt signaling cascade can beenvisaged as a redundant pathway uponwhich different modulatorsof (pre)adipocytes recruitment and differentiation can converge. Inthis scenario, reactive oxygen species (ROS) and free radicals havestarted to attract a great deal of attention since changes of intra-cellular redox state appear importantly involved in regulating thedifferentiation of the adipose precursor cells. Oxidative stress maythus represent a novel molecular cue at the interface betweenadipose dysregulation and obesity-linked metabolic complications.

6. Oxidative stress: a novel instigator of the adipose organdysregulation?

Oxidative stress (OS) can be defined as the steady state conditionwhere the free radical/reactive oxygen species (ROS) flux isbalanced by antioxidant defenses [73]. The upregulation of OS,which mainly occurs in conditions of increased flux of free radicals/ROS and/or reduced antioxidant levels, is now accepted to playa critical role in the pathogenesis of obesity, atherosclerosis, T2Dand IR [74e76]. AT itself has been suggested as the major source offree radicals and ROS [75e77], leading to postulate that increasedOS in accumulated fat might be an early “instigator” of the obesity-associated metabolic complications [75,77,78]. However, theinherent mechanisms linking oxidative damage to the adiposedysfunction remain largely unknown.

ROS are highly reactive molecules generated either duringphysiological cellular processes or under various stress conditions[79]. Remarkably, ROS and free radicals are now emerging as“signaling molecules” that may engage different regulatory path-ways controlling tissue homeostasis [80]. The redox-regulation ofsignal transduction may thus be of importance in adipogenesis

[81e83]. In line with the paradigm of the “expandability hypoth-esis”, the adipose oxidative stress may well represent a suitablecandidate linking the failure of fat cell formation with adiposedysregulation and its associated complications.

Yet, the literature remains controversial regarding the effects ofstress on (pre)adipocyte recruitment and differentiation. Depend-ing on model and experimental settings, increased oxidative stresshas been associated with both pro- or anti-adipogenic attitudes[77,81,82,84,85]. One hypothesis that may reconcile such conflict-ing observations differentiates the effects of “physiological”, tightlyregulated, and targeted intracellular redox changes from the“noxious effects” of increased, uncontrolled production of freeradicals and ROS.

The adipose stem cells are very sensitive to redox changes. Underphysiological conditions, when OS is balanced, free radical signalingworks in accordance with cellular function to maintain long-termregenerative potential and survival of stem cells [82,86]. ROS aregenerally assumed to promote cell proliferation. It may thus beanticipated that an initial “burst” in ROS production may bea prerequisite for the initiation of adipogenic differentiation, byinducing signaling events that switch committed (pre)adipocytesfromproliferation to differentiation. On the other hand, an excessiveand inappropriate redox balancemay detrimentally affect precursorcells recruitment/differentiation, thereby promoting the develop-ment of “unhealthy” fat expansion [75,81,85,87]. In harmony withthis, ROS production was found to be markedly increased duringdifferentiation of 3T3-L1 cells into adipocytes [75]. However, anelegant work by Mouche et al. demonstrated that (pre)adipocytesexhibit limited, if any, “spontaneous”ROSproduction,which, in turn,becomes upregulated in mature fat cells [87]. Accordingly, ina mouse model of obesity-linked IR, the activation of the NADPHoxidase (Nox) complex has been indicated as the major source ofROS in adipocytes [75]. However, although decreased Nox4 mRNA

Fig. 1. The canonical Wnt/b-catenin signaling. Panel A: Wnt in the unstimulated state. Panel B: Wnt-stimulated induction of b-catenin/TCF target gene expression. Wnt binding toFrizzled and LRP5/6 co-receptors results in hypophosphorylation of b-catenin that translocates to the nucleus, where it activates a number of Wnt target-genes involved in the cellcycle and metabolism From Ref. [20].

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content was reported as a hallmark of (pre)adipocyte differentiation[87], the expression of Nox4 has been elsewhere proposed asa “molecular switch” promoting insulin-induced differentiation ofthe (pre)adipocyte [85]. It is thus plausible that Nox4-mediated ROSproduction may either trigger adipogenesis, when released intra-cellularly, or provide a cellular cross-talk between different cell-types within fat tissue, when secreted in the extracellular space[87]. In this regard, in 3T3-L1 (pre)adipocytes ROS downregulateadiponectin and PPAR-g, while increase mRNA expression of IL-6and monocyte chemoattractant protein-1 (MCP-1), a well-knownregulator of monocyte recruitment to sites of inflammation [75]. Inaddition, macrophages are also leading source of ROS, and byprod-ucts of lipid peroxidation are themselves potent chemoattractant[88]. Thus, the cross-talk between (pre)adipocyte andmacrophages,which importantlycontributes to adipose inflammation,may concurto regulate OS in accumulated fat, as previously postulated [38].Finally, in AT of obese rodents, decreased expression of selenopro-tein P, a selenium transporter with antioxidant protection from ROS,was linked with AT inflammation, impaired preadipocyte differen-tiation, and, as expected, the development of insulin resistance [89].It is thus plausible that free radicals and ROS in accumulated fatmayimpair (pre)adipocyte differentiation and induce macrophage infil-tration, providing a mutually inductive “feedback loop” by whichinflammation and oxidative stress conspire to induce adipose dys-regulation and metabolic dysfunctions.

Taken together, from these data an appealing scenario ariseswhere the fine tuning of the redox balance in AT emerges asa prerequisite for maintaining local and systemic homeostasis bycomplex auto/paracrine and endocrine ROS-mediated cross-talksbetween different cell-types of the fat organ.

By which mechanisms however the “adipose stress” mayhamper recruitment and differentiation of the precursor cells.

Novel and intriguing insights into this topicwere recently gainedby the demonstration that theWnt signaling pathway may undergoa redox-dependent regulation [83]. Briefly, ROS-stimulated Wntcascade appears orchestrated in a temporal manner by a thio-redoxin-related protein, nucleoredoxin (NRX) (Fig. 2). NRX usually

blocks Wnt pathway activation by interacting with Dishevelled(Dvl), an essential adapter protein for Wnt signaling. In differentexperimental settings, Funato et al. clearly demonstrated thatoxidative stress induces the dissociation of NRX from Dvl, thustransactivating downstream Wnt signaling pathway [83]. It is thustantalizing to speculate that aberrant ROS production in the adiposemicromilieu may engage Wnt or other stress sensing pathways (ie,the mitogen-activated protein kinases pathway; MAPKs), whichtransactivation hampers (pre)adipocyte recruitment and differenti-ation [90].

In humans, to the best of our knowledge, direct evidence tosupport such a hypothesis is still lacking. Interestingly, data of theliterature implicate that, in mesenchymal osteoblast progenitorcells, acute increase in ROS antagonizes the osteogenic and favorsadipogenic differentiation by diverting the pool of b-catenin awayfrom prototypical Wnt-mediated (TCF/LEF) transcription factors[86]. Such a “Wnt antagonism” fits well with the decrease of boneformation and the increase of marrow adiposity observed withincreasing age, but it is unlikely to represent a more generalizedphenomenon of the adult pluripotent stem cells. In humanmarrowstromal cells, specific products of cholesterol oxidation, namely theoxysterols, have importantly been implicated as “endogenoussignals” modulating lineage-specific differentiation in favor ofosteogenesis and against adipogenic differentiation [91,92]. Evenmore, the oxysterol-associated anti-adipogenic actions wereconsistently linked with selective activation of Wnt target genes[93]. Finally, a major oxidation product of membrane lipids con-taining polyunsaturated n-6 acyl groups, namely 4-hydroxynonenal(4-HNE), has also emerged as a potent activator of the canonicalWntpathway in retinal cell lines and in a rat model of diabetic retinop-athy [94].

Collectively, these data underscore the biological importanceof the “adipose oxidative stress” in regulating precursor cellcommitment and differentiation partly by modulating the Wntsignaling pathway. In this context, the lipid peroxidation by-products may serve as reliable “molecular signatures” of theadipose oxidative stress and, besides the potential “toxicity”, they

Fig. 2. Model of redox-dependent regulation of Wnt/b-catenin signal transduction. Panel A: ROS-stimulated Wnt cascade may be regulated by a thioredoxin-related protein,nucleoredoxin (NRX), which usually blocks Wnt pathway activation by interacting with Dishevelled (Dvl), an essential adapter protein for Wnt signaling. Panel B: Lipid peroxidationproducts may transactivate upstream or downstream the Wnt signaling cascade at different steps From Ref. [83].

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may well act as signaling molecules engaging critical transductionpathways involved in adipogenesis and metabolic homeostasis.

7. Lipid peroxidation as “markers” of the adipose oxidationand “makers” of systemic insulin-resistance

Lipid peroxidation refers to the oxidative degradation of lipids,a process initiated by free radicals/ROS escaping the antioxidantsystem. By-products of lipid peroxidation, while initially identifiedas “toxic” end products, at nontoxic and “physiological” levels mayserve as “signaling molecules” regulating various cell functions[95e98]. These “signaling” effects primarily result from the adduct-forming capacity with different macromolecules such as phos-pholipids, proteins and nucleotides, which ultimately disrupt theirbiological activity [97e99].

Among lipid peroxidation products oxysterols and 4-hydroxyno-nenal (4-HNE) received most attention since cross-sectional studiesand experimental data indicate their mechanistic implicationin the pathophysiology of obesity-linked metabolic diseases[96,97,100e107].

7.1. Oxysterols

Oxysterols are oxidized derivatives of cholesterol resultingfrom non enzymatic (ie, “autoxidation”) or enzymatic cholesteroloxidation [73]. In line with their role as part of the cellularmachinery that governs cell function and integrity, oxysterols areknown to exert a multitude of (patho)physiologic effects by actingas signaling molecules at transcriptional, translational- and post-translational level. A number of key proteins implicated in thecontrol of metabolic homeostasis are recognized targets for oxy-sterols [108e112].

AT plays a key role in regulating the trafficking of lipids andperoxidation by-products. Indeed, AT stores over half of total bodycholesterol [113], the progenitor of oxysterols, and this proportionincreases when fat cells become enlarged [114]. Moreover, adipo-cytes remove serum oxidized low-density lipoproteins (oxLDLs),action that appears beneficial for glucose metabolism andhomeostasis [113e115]. Thus, as far as AT acts as a “sink” to safelystore harmful cholesterol metabolites, it can be anticipated that theloss of these protective qualities may instigate obesity-linkedinsulin resistance [105].

In line with such a hypothesis, serum concentrations of7-ketocholesterol (7k-C) and 7b-hydroxycholesterol (7b-HC), themost abundant oxysterols produced by oxidative stress and carriedon oxLDL [116], have been consistently associated with the occur-rence of T2D and correlated with coronary multiple risks [104,117].Moreover, oxLDL and 7k-C were also reported to impair (pre)adipocyte differentiation and induce IR in adipocytes [105,118,119].Accordingly, oxysterols can also behave as “anti-adipogenic signals”by diverting pluripotent mesenchymal cells fate away from adi-pogenic and in favor of the osteogenic lineage through trans-activation of Wnt-mediated signaling pathway [91,120].

It is thus possible to postulate that, while under normal condi-tions adipocytes provide a protective mechanism for trapping theharmful oxysterols, the increased oxysterols content in hypertro-phic fat cells may induce adipocyte dysfunction and contribute toimpair precursor cells recruitment/differentiation, leading ulti-mately to the unfavorable cardio-metabolic outcomes.

7.2. 4-Hydroxynonenal

Besides cholesterol, the polyunsaturated fatty acids (PUFA) mayalso undergo free radical-mediated oxidation generating a series oflipid aldehydes [98]. Interestingly, the pivotal role of such reactive

molecules on different outcomes of insulin resistance has recentlybeen outlined [96]. One of the most intensively investigatedlipid peroxidation product is 4-hydroxynonenal (4-HNE), a majoroxidation product of membrane lipids containing n-6 poly-unsaturated acyl groups.

Experimental data and human studies underscore the theoret-ical likelihood that 4-HNE actually serves as a key “diabetogenic”molecule at different levels. Indeed, 4-HNE: 1) impairs insulinsignaling/action in skeletal muscle by inactivating critical compo-nents of the insulin signaling pathway; 2) blunts glucose-inducedinsulin secretion in pancreatic b-cells; and, 3) disrupts the insulinbiological activity through its direct adduction [102,103,121e123].However, since AT is the major organ equipped to store lipids,peroxidation would be expected to occur first, and more likely, atthe adipose level. In line with such a “adipocentric” view, recentdata failed to find differences in skeletal muscle proteineHNEcontent between similarly obese, insulin-sensitive and insulin-resistant patients [122]. Furthermore, the inverse relationshipfound between muscle proteineHNE content and glucose uptakewas independent from BMI [121], implying that muscle lipid per-oxidation characterizes insulin resistance above and beyond fatmass expansion. This paradigm extends the previous concept thatfat dysregulation initiates the IR [39,124], suggesting the adiposelipoxidation as putative instigator of muscle IR partly through4-HNE-mediated mechanisms.

Crescent scientific interest has thus been focused on the char-acterization of the adipose lipid peroxidation in the setting ofobesity and IR. From a chemical standpoint, 4-HNE is highly reac-tive because of its aldehyde function that covalently modifies lysineresidues of proteins. Since proteineHNE modifications are rela-tively “long-lived footprints” of lipid peroxidation, this aldehydeadduct appears a suitable and metastable biomarker of adiposeoxidative stress in vivo [121,125]. Accordingly, in experimental micemodels, adipose 4-HNE accumulation per se was shown either topromote the obese state or to induce the development of IR throughcarbonylation of key adipocytes proteins involved in lipid metab-olism [106,126]. Moreover, exposure of adipose cell to increasing4-HNE concentrations leads to a dysfunctional and “unhealthy”phenotype characterized by selective impairment of insulinsignaling (downregulation of IRS-1; upregulation of p38MAPK),increased lactate and reduced adiponectin production, biologicalevents underlying the development of IR [127e129]. Nonetheless,in differentiating (pre)adipocytes, oxidative stress can induceintracellular 4-HNE production, which, in turn, activates the MAPKpathway (p38MAPK) [130]. Along with the previously reportedeffects ofWnt signal, p38 activity is also known to inhibit adipocytedifferentiation, fostering the noxious effect of 4-HNE on adiposehomeostasis [90]. Finally, 4-HNE may also stimulate ROS produc-tion in mature adipose cells, thus establishing a “self-propelled”oxidative loop between (pre)adipocyte and differentiated fat cellsor different other cell-types (ie, macrophages) within the adiposeorgan [127].

Despite this rationale, there is yet a dearth of knowledge aboutthe occurrence of lipid peroxidation in human obesity as well as theoccurrence of adipose oxidation in the dysregulated fat. In order toaddress this question, we recently undertook a study to charac-terize 4-HNE expression in the sc abdominal depot of similarlyobese insulin-sensitive and IR/T2D patients. Notably, SDS-PAGEanalysis revealed that the expression of HNE-adducts of manyproteins (size range 25e100 kDa) in T2D patients was higher thanthat of similarly obese, nondiabetic controls (Murdolo G et al.,unpublished). Furthermore, in keeping with the increased proteine4-HNE adducts content, the adipose precursor cells of the “diabetic”fat exhibited an inherently reduced ability to undergo full adipo-genic differentiation. A similar effect was also recapitulated when

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precursor cells isolated from lean healthy controls, with normaladipogenic potential, were challenged with nontoxic 4-HNEconcentrations (1e10 mM).

Taken together, current results point to 4-HNE as a potentialmarker of fat oxidation and dysregulation (“diabetic” adiposop-athy), as well as a putative driver of impaired adipogenesis andsystemic insulin resistance.

8. Potential role of antioxidants in the treatment of insulinresistance

The mechanisms by which oxidative stress induces insulinresistance are complex and still debated. Notably, attempts tocrudely use antioxidant supplementation as a means of alleviatingoxidative stress and thereby improving metabolic dysfunctions inobesity and T2D have proven to be partly disappointing.

A number of human intervention studies found that dietarysupplementation with antioxidant a-lipoic acid, glutathione,vitamin E, vitamin C, and flavonols may ameliorate insulin-sensitivity in patients with insulin resistance, T2D and/or athero-sclerotic cardiovascular disease [131]. However, many of these trialshave been relatively small and of short duration, and this hashindered their interpretation. A recent meta-analysis of the effectsof dietary supplementationwith vitamin C or E revealed that, in T2Dpatients, despite the lack of improvement of glycemic control anddegree of insulin resistance, antioxidant supplementation mayreduce the early glycation end-product (ie, HbA1c), suggestinga potential benefit of antioxidants in protecting against diabetescomplications [134]. Thus, appropriate double-blind, randomized,placebo-controlled studies, with a comprehensive evaluation ofdifferent markers of oxidative stress and assessment of individualinsulin sensitivity/resistance status are warranted. Nonetheless,better definitions of the specific oxidative species, as well as theircellular target(s) and type of cellular modification(s) such targetsundergo, are also required to design more efficient means of alle-viating oxidative damage in obesity-linked insulin resistance.

Here, we outlined a scientific rationale for targeting the “adiposeoxidation” as a potential target for novel therapeutic strategies toprevent/tackle adiposity-linked metabolic dysfunctions. Circum-stantial data from experimental animalmodels, as well as anecdoticobservations in humans, partly support the assumption that selec-tive control of cellular redox balance in ATmayserve as a critical linkbetween adipose organ dysfunction and derangements of glucosehomeostasis [75,101,127,132,133]. Clearly, unlocking the adipose-specific mechanisms underlying the impairment of redox balanceand its consequence on hampered precursor cells recruitment anddifferentiation is attractive for targeted therapeutics.

Wish as we might, the possibility that such a strategy can hope-fully play a role in preventing, reverting or halting the progression ofadiposity-linked cardiometabolic complications is still awaiting.

Disclosures

The authors have nothing to disclose.

Acknowledgments

This work was partly funded by grant from “Fondazione Cassa diRisparmio di Perugia; Ricerca Scientifica e Tecnologica”.

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