Oxidative stress and lipid peroxidation by-products at the crossroad between adipose organ dysregulation and obesity-linked insulin resistance

Biochimie 95 (2013) 585e594

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Review

Oxidative stress and lipid peroxidation by-products at the crossroad between adipose organ dysregulation and obesity-linked insulin resistance Giuseppe Murdolo a, *, Marta Piroddi b, Francesca Luchetti c, Cristina Tortoioli d, Barbara Canonico c, Chiara Zerbinati e, Francesco Galli b, Luigi Iuliano e a

Department of Internal Medicine, Assisi Hospital, Via Valentin Muller 1, I-06081 Assisi, Perugia, Italy Department of Internal Medicine, Section of Applied Biochemistry and Nutritional Sciences, Perugia University, Perugia, Italy Department of Earth, Life and Environmental, University Carlo Bo, Urbino, Italy d Department of Internal Medicine, Section of Internal Medicine, Endocrine and Metabolic Sciences, Perugia University, Perugia, Italy e Department of Medic-Surgical Sciences and Biotechnologies, Unit of Vascular Medicine, Sapienza University of Rome, Latina, Italy b c

a r t i c l e i n f o

a b s t r a c t

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

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 force for the development of insulin resistance (IR) and type 2 diabetes, mounting evidence points to “adipose dysregulation”, rather than fat mass accrual per se, as a key pathophysiological trigger of the obesitylinked metabolic complications. The dysfunctional fat, besides hypertrophic adipose cells and inflammatory 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 the ectopic overflow/deposition of lipids in non-adipose targets (lipotoxicity) or by inducing a dysregulated secretion of different adipose-derived hormones (ie, adipokines and lipokines). This novel and provocative paradigm (“expandability hypothesis”) further extends current “adipocentric view” implicating a reduced adipogenic capacity as a missing link between “unhealthy” fat expansion and impairment of metabolic homeostasis. Hitherto, reactive oxygen species have been implicated in multiple forms of IR. However, the effects of stress on adipogenesis remain controversial. Compelling circumstantial data indicate that lipid peroxidation by-products (ie, oxysterols and 4-hydrononenal) may detrimentally affect adipose homeostasis partly by impairing (pre)adipocyte differentiation. In this scenario, it is tempting to speculate that a fine tuning of the adipose redox status may provide new mechanistic insights at the interface between fat dysregulation and development of metabolic dysfunctions. Yet, in humans, the molecular “signatures” of oxidative stress in the dysregulated fat as well as the pathophysiological effects of adipose (per)oxidation on glucose homeostasis remain poorly investigated. In this review we will summarize the potential mechanisms by which increased oxidative stress in fat may impair (pre)adipocyte differentiation and promote the adipose dysfunction. We will also attempt to highlight 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 potential target for novel therapeutic strategies to curb/prevent adiposity-linked insulin resistance. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Adipose tissue dysregulation Insulin-resistance Obesity Oxidative stress preadipocytes

1. Historical background: the regulation of energy balance and body weight

Abbreviations: AT, adipose tissue. * Corresponding author. Tel.: þ39 (0)75 578 3588; fax: þ39 75 573 0855. E-mail address: [email protected] (G. Murdolo). 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2012.12.014

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

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maintenance of a stable body weight [1,2]. Numerous control pathways, involving both neural networks and circulating signals, have evolved to maintain energy balance at an optimal level [1,2]. Adipose tissue (AT) serves a primary role in the setting of energy homeostasis because it represents the body’s main depot for energy storage and mobilization. Homeostatic circuits that regulate body fat mass include, among others, those based on insulin and leptin. These hormones, which circulating concentrations are proportional to fat mass, may act as “adiposity signals”. Insulin, on the one hand, displays adipogenic actions and stimulates the storage of triglycerides in adipose cells, which became enlarged. The size of adipocytes, on the other, may be “sensed” to the brain via the secretion of leptin [1,2], which, in turn, acts on the central nervous system to reduce feeding and increase energy expenditure to restrain fat mass expansion. Besides the “adiposity signals”, gut hormones, such as the gastric hormone ghrelin (see below), are also known to signal to the brain inducing a central integration according to the dietary intake and nutrient requirements. Evidence is mounting to suggest extensive cross-talks between adiposity signals, gut hormones and insulin [3,4]. Ghrelin, the orexigenic protein secreted by X/A-like cells of the gastric oxyntic glands, emerges as a suitable candidate linking fat mass expansion, control of food intake and glucose homeostasis. Independently of the orexigenic action, both the acylated and desacylated ghrelin forms may stimulate lipid accumulation in human visceral adipocytes and, as compared with obese normoglycemic subjects, obese patients with type 2 diabetes (T2D) exhibit higher acylated ghrelin concentrations [3]. Further insights into ghrelin effects on adipobiology have been provided by the discovery of aquaporins (AQP) [5]. Aquaporin-7 (AQP7), the adipose-specific watereglycerol transporter present in the plasma membrane of adipocytes, operates as a glycerol channel in vivo, whereby fat cell permeability and delivery of glycerol into plasma display a key role in the regulation of fat accumulation and insulin resistance [5]. Recent data indicate that, under physiological conditions insulin mediates a coordinated regulatory loop between fat-specific AQP7 and liver-specific AQP9 in order to maintain glucose homeostasis through modulation of glycerol output from adipocytes and glycerol uptake for hepatic gluconeogenesis according to the nutritional status [6]. On the other hand, the expression of AQP7 in human visceral adipocytes is repressed by acylated and desacyl ghrelin, supporting the view that ghrelin decreases lipolytic capacity and promotes fat cell enlargement [3]. Notably, insulin is also required for prandial ghrelin suppression in humans [7], and chronic hyperinsulinemia, as well as hyperleptinemia, have been indicated as the most important modulators of ghrelin secretion in obese individuals with or without T2D [4]. It is thus plausible that the disruption of the coordinated regulation between insulin, ghrelin and AQP7/AQP9 might impair glucose homeostasis by increasing glycerol release from adipocytes and glucose production from the liver, thereby perpetuating a vicious cycle. In this scenario, the occurrence of obesity may be seen as a result from the failure or breakdown of the homeostatic mechanisms regulating energy balance. Many attempts have thus been made to explain how genetic and environmental factors can overcome normal energy homeostatic control and cause obesity. This fertile area of research is continuously open to new areas of understanding and could potentially open novel therapeutic targets for tackling adiposity epidemic. 2. Obesity and type 2 diabetes: not only a matter of fat Overwhelming evidence has proven that obesity plays a central role 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 only instigator of the adiposity-associated unfavorable metabolic outcomes. Different epidemiological trials showed that some morbidly obese patients (and up to 25% of obese individuals) are “metabolically healthy”, while about 18% of non-obese subjects demonstrate biochemical characteristics of the insulin-resistance syndrome [12e14]. Accordingly, some authors have recently suggested a reappraisal of the cutoffs to diagnose overweight and obesity. Indeed, 29% and 80% of subjects classified as lean or overweight 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 cardiometabolic risk factor profile as obese patients [15]. Altogether, these observations support the assumption that fat mass expansion is neither sufficient nor necessary for development of the metabolic dysfunctions. AT is now emerging as a remarkably active “organ”, with functional pleiotropism and high remodeling capacity [16]. Although basically adipose tissues are generally regarded as connective tissues without a specific anatomy, accumulating data support the idea that fat tissues are organized to form a large “organ” with discrete anatomy, specific vascular and nerve supplies, complex cytology, and high physiological plasticity [17]. Obesity implies extensive changes in AT ultrastructure involving the enlargement of existing adipocytes, the formation of new fat cells from committed (pre)adipocytes, (adipogenesis), extracellular matrix proteolysis, and the coordinated development of the tissue vascular network (angiogenesis). Arguably, while fat is by far the most plastic organ in our body, the limit of fat mass expansion appears defined for any given individual [18]. Indeed, if insulin resistance would be a direct consequence of an increased fat mass, all the subjects would develop metabolic complications at the same degree of adiposity. In contrast, on an individual level, there is no clear cut point of “adiposity”, as conventionally ascertained by the BMI, clearly separating distinct insulin sensitive from insulin resistant sub-phenotypes [13]. Although different and partly conflicting hypotheses have been put forward to explain such an apparent paradox, one provocative and seemingly counterintuitive paradigm postulates that a reduced ability of the adipose organ to further expand, rather than fat mass accrual per se, may be the key determinant underlying the adiposity-induced metabolic dysfunctions (“expandability hypothesis”) [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 comprises adipocytes, which account for only 25% of the total cell population. Notably, diverse cell-types, which include the adipose precursor cells among others, are found in the so-called “stroma-vascular fraction”, and account for the remaining 75% of the whole cell population. Recent evidence points to substantial AT “remodeling” in obesity, which appears of particular interest in the setting of metabolic homeostasis [21]. Mature adipocytes are derived from a pool of undifferentiated precursors (ie, the adipose-derived mesenchymal stem cell, ASC), which become “committed” toward the adipogenic lineage (ie, preadipocytes). However, not all the progenitors become adipocytes simultaneously [22]. Our current understanding of AT development in humans is that the major pool of precursor cells is primarily recruited before puberty, while in adulthood there is a 10% annual adipocyte turn-over [23,24]. This implies that fat mass expansion in adult age is mainly a consequence of adipocyte

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enlargement (hypertrophy) rather than recruitment of new adipocytes (hyperplasia). The enlargement of existing adipocytes and the formation of new fat cells are however exquisitely regulated by the body’s demand of storing fat, and thus represent a prerequisite for maintaining energy and metabolic homeostasis [25]. Growth of a “healthy” or “unhealthy” fat organ appears to be determined by a complex, cell-type orchestrated, coordination, whereby adipocyte turnover is balanced by recruitment and differentiation of new fat cells, which, in turn, needs to be “titrated” to the requirement for energy 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”. The formation of new adipocyte from the precursors pool takes place once a maximum capacity is attained, which in humans averages 1000 pL [11]. In the face of a positive energy balance, the failure, or the lack thereof, of such a “feedback” mechanism, leads to hypertrophy of mature fat cells. Notably, the lipid-accumulating ability of human preadipocytes is inversely correlated with the size of mature fat cells of the donor, implying that adipocytes enlargement (hypertrophy) represents a consequence of a reduced ability to recruit/differentiate new (pre)adipocytes [25e27]. In keeping with this, the hypertrophic obesity is more strongly associated with insulin resistance than the hyperplastic form [14,28], and the enlargement of the adipocyte in the subcutaneous (sc) abdominal depot has previously been reported as an “obesity” independent predictor of insulin resistance and T2D development [29,30]. Furthermore, hypertrophic adipocytes, even in the absence of obesity per se, are associated with several features of “dysfunctional” fat and systemic insulin resistance [31]. Accordingly, in similarly obese individuals, only those diagnosed with T2D exhibit inappropriately enlarged sc adipocytes and decreased adipogenic capacity [32e34]. Even more, an impaired ability to recruit new fat cells and the inappropriate enlargement of pre-existing adipocytes also characterize a population of non-obese, but insulin resistant individuals with genetic predisposition for T2D and features of the metabolic syndrome, as compared with phenotypically “matched” subjects lacking diabetes heredity [35]. The adipose cell hypertrophia can be thus envisaged as a “marker” for increased susceptibility to the metabolic complications early and before “obesity” (as conventionally defined by BMI cut-point) develops [27]. Further insights into the complex interaction between (pre)adipocyte differentiation and metabolic outcomes have recently been provided by the observation that, in the setting of obesity, an enhanced adipogenic capacity of the sc fat depots may protect against the metabolic syndrome [36]. Altogether, these observations emphasize the concept that insulin 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 individuals possess a genetically and environmentally determined limit for AT expansion, beyond which lipids are deposited ectopically in non-adipose organs [37e39]. A key question is now why the impairment of (pre)adipocyte differentiation instigates adipose organ dysfunction and hampers glucose homeostasis. 4. Impaired adipogenesis induces adipose dysregulation and poses a “diabetogenic” milieu From a pathophysiological standpoint, dysfunctional adipocyte maturation may instigate a “diabetogenic” milieu through different and partly synergistic mechanisms.

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First, the inability to safely store “metabolically active” fatty acids derivatives in hypertrophic adipocytes induces “ectopic” accumulation of lipids within non-adipose “targets” (ie, skeletal muscle, liver, pancreatic islets) with toxic and detrimental consequences on tissue homeostasis (lipotoxicity) [40]. A general phenomenon for ectopic fat accumulation is that it occurs when the sc adipocytes become enlarged (around 0.8e1.0 mg lipid/cell), and thus “resistant” to further lipid accretion [41]. In harmony with the concept of lipotoxicity, systemic metabolic homeostasis has been consistently linked with a “lipid-mediated endocrine network” [42]. Experimental animal models, and, preliminary evidence in humans [43,44], suggest that a single serum lipid (ie, palmitoleic acid) may function as insulin sensitizing adipose-derived lipid hormone (“lipokine”). Therefore, in the face of positive energy balance, an impaired formation of new fat cells may lead to enlargement 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 studies demonstrate that AT content of specific fatty acids is positively correlated 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 insulin resistance/T2D [45,46]. Finally, in a population of similarly obese individuals with and without the metabolic syndrome, a comprehensive “lipidomic” characterization found that, in plasma, the shift between different saturated (ie, cerotic acid) and unsaturated (ie, nervonic acid) fatty acid derivatives was strongly associated with the “unhealthy” obese phenotype, while no significant differences in palmitoleic acid levels were seen (Iuliano L. unpublished data). It is thus conceivable that the pattern of specific fatty acids in the bloodstream may well reflect the degree of “adipose cell dysfunction” and drive the metabolic homeostasis. In this regard, an intriguing role in adipocyte biology has recently been provided by the characterization of caveolins [47e49]. These integral plasma membrane proteins serve as structural elements of caveolae having proven scaffolding, transport and signaling capabilities, and play a key role in shifting the focus of obesity and insulin resistance development to lipid dynamics [49]. Of note, isolated adipocytes from obese T2D patients display higher expression of caveolin-1 (CAV-1) when compared with those of obese normoglycemic subjects, and CAV-1 expression in sc abdominal depot appears inversely associated with fasting triglycerides and lipogenic genes [47,48]. These observations highlight the potential importance of CAV-1 in the regulation of intracellular cholesterol homeostasis and lipid storage ability of adipocytes, even though the clinical relevance of CAV-1 in the development of obesity-associated comorbidities needs to be further elucidated. Second, enlargement of pre-existing adipocytes instigates macrophage infiltration into the AT, which, in turn, promotes local inflammation and induces a dysregulated secretion of other prototypical adipose-derived hormones (ie, adipokines) [38]. The adipose inflammation appears orchestrated by complex auto/ paracrine cellular cross-talks. The increased production of proinflammatory molecules (ie, IL-6), along with the downregulation of adiponectin, an insulin-sensitizing and anti-diabetogenic hormone, leads to impairment of insulin signaling and promotes both the cardiovascular and metabolic dysfunctions [10,38,50]. Of interest, lipocalin-2 (LCN-2) has recently been reported as a novel proinflammatory adipokine with potential role in adiposity-linked insulin resistance [51]. The increased expression of LCL-2 in visceral fat depot of obese patients, as well as the association with several pro-inflammatory genes, implicate LCN-2 as an important autocrine/paracrine modulator of the inflamed fat. Accordingly, increased macrophage infiltration and reduced circulating adiponectin levels characterize the dysfunctional and hypertrophic fat of

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morbidly obese, but insulin resistant, individuals (“unhealthy obesity”), when compared with similarly obese, insulin-sensitive (“healthy obesity”), counterparts [14]. Intriguingly, the discovery of microRNA (miRNA) has recently opened new insights for better understanding the complicated regulatory network governing macrophage-mediated AT inflammation/dysfunction and metabolic outcomes. MiRNA are a family of small noncoding RNAs that have been demonstrated to be crucial regulators in multiple pathophysiological processes and importantly associated with chronic inflammatory diseases [52]. Experimental data in mice identified miR-233 as a novel regulator of macrophage “polarization”, which may protect against diet-induced AT inflammation and systemic insulin resistance by promoting phenotypic switching of resident macrophages from proinflammatory (M1) toward antiinflammatory (M2) responses [52]. It has thus been proposed a new miRNA-based paradigm for the regulation of insulinsensitivity, which provides the basis for using miRNA as analogs to treat insulin resistance related disorders. Third, the inflammatory microenvironment in inappropriately expanded fat organ negatively affects preadipocyte differentiation [53]. Moreover, locally released inflammatory molecules (ie, TNF-a) can either sustain a macrophage-like phenotype in undifferentiated precursor cells [25] or also impair the ability of mature adipose cells to safely store triglycerides [54], thereby perpetuating a “vicious cycle”. Together, these findings provide a robust mechanistic platform for linking impaired preadipocyte differentiation with the cues of adipose dysregulation (increased fat cell size, inflammation, dysfunctional trafficking/overflow of fatty acids, secretion of prodiabetogenic lipokines and adipokines), which, ultimately, initiates the insulin resistance and/or favors the progression toward frank diabetes. The key question is then why there is an impairment of preadipocyte differentiation. 5. The Wnt signaling network: a molecular driver of (pre) adipocyte differentiation Recently, the characterization of the adipogenic precursor cells in the context of “healthy” or “unhealthy” obesity, as well as the signal networks underlying the lineage “commitment”, recruitment and differentiation are attracting a great deal of interest. Adipocyte arises from undifferentiated mesenchymal precursor cells (ASCs). Although the molecular aspects of adipogenesis are beyond the purpose of this review, fat cell formation is a complex and well-orchestrated multistep process that mainly comprises a “commitment” step of the undifferentiated precursors into preadipocytes and a “terminal differentiation” to mature fat cells [55,56]. The molecular regulation of terminal differentiation has been well characterized, and relies on sequential activation of transcription factors where induction of PPARg and CCAT/ enhancer-binding protein alpha (C/EBPa) acts synergistically to produce and maintain the phenotype of the mature fat cell [55,56]. In contrast, there is yet a dearth of knowledge on the molecular events regulating the commitment of ASCs to the adipocyte lineage. A growing body of data indicates the wingless-type MMTV integration site family (Wnt) signaling as a critical regulator of adipogenic precursor cell fate [20,27,50,53,57e64]. Basically, Wnts are an evolutionary conserved family of secreted cysteine-rich glycosylated proteins that, through autocrine or paracrine 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, the binding 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 on the transcriptional regulator b-catenin (Fig. 1). In the absence of Wnts, cytoplasmic b-catenin is recruited to a “degradation complex”, whereby glycogen synthase kinase 3-b (GSK-3b), axin and adenomatosus polyposis coli (APC) cooperate in inducing sequential phosphorylation and subsequent proteasomal degradation of b-catenin. Binding of specific Wnt ligands to Fzd-LRP5/6 receptor complex results in activation of Dishevelled (Dvl) and recruitment of axin to LRP5/6 co-receptor. The sequestration of axin to the receptor complex also inactivates the “degradation complex and prevents proteasomal degradation of b-catenin, which becomes stabilized (hypophosphorylated) and accumulates in the cytosol. This ultimately coincides with the nuclear translocation and binding of b-catenin to the lymphoid enhancer-binding factor/ T-cell-specific transcription factors (LEF/TCF), family of transcription factors that activate specific Wnt target genes [20]. To date, more than 80 target genes are known to be regulated by the Wnt/b-catenin pathway, and among others they include those that promote cell-cycle progression, cellular differentiation, and metabolism [60]. Notably, AT contains several Wnt-producing cells, and the “canonical” Wnt/b-catenin pathway appears to be highly active in undifferentiated ASCs where it may direct the cell fate toward osteogenic/myogenic and aside the adipogenic differentiation [57e60]. The inability to adequately suppress Wnt activation in ASC has thus been regarded as a possible mechanism underlying the perturbed adipogenesis of the hypertrophic/dysregulated fat. Numerous studies demonstrated that the inhibition of Wnt signaling represents an absolute prerequisite either for ASC commitment toward the adipogenic lineage or for appropriate (pre)adipocyte differentiation and induction of PPARg and C/EBPa [27,50,53,61e63,67]. Furthermore, temporal expression profiling studies also indicate that while Wnt activation appears required early to increase the number of preadipocytes recruitment, this signal must be terminated before the newly recruited (pre)adipocytes can undergo terminal differentiation [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 energy storage demand. Notably, in the sc AT of non-obese, but insulinresistant individuals, the gene expression of canonical Wnt signaling components (ie, b-catenin and GSK-3b) is upregulated and, in line with the reduced abundance of essential adipogenic transcription factors (ie, C/EBPa and PPAR-g), appears inversely correlated with the size of adipocytes [68]. These findings reinforce the concept that inappropriate canonical Wnt signaling activation in AT might be an early and “adiposity-independent” instigator of impaired adipogenesis and systemic insulin resistance. Nonetheless, although Wnts are basically molecules secreted by cells that have not undergone full terminal differentiation (ie, the preadipocytes), also mature fat cells were reportedly shown to release factors that modulate Wnt signaling, implying a paracrine regulation and commitment of early precursor cells as the mature adipocytes expand [27,61]. Together, the prevailing view is that a coordinated regulation of Wnt/b-catenin signaling is required to prevent “unhealthy” AT expansion and consequent development of metabolic complications. What might thus be the pathophysiological factors responsible for uncoupling Wnt/b-catenin signaling and AT plasticity, and why Wnt is over-active in the dysregulated fat? Thus far, this remains an open question. While genetic factors are 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-adipogenic effects of TNF-a involve activation of the Wnt signal implicates

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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 to Frizzled 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 cell cycle and metabolism From Ref. [20].

the adipose inflammation as potential contributor to deregulation of Wnt/b-catenin signaling [25,50,53]. As a corollary to these findings, the Wnt signaling cascade can be envisaged as a redundant pathway upon which different modulators of (pre)adipocytes recruitment and differentiation can converge. In this scenario, reactive oxygen species (ROS) and free radicals have started to attract a great deal of attention since changes of intracellular redox state appear importantly involved in regulating the differentiation of the adipose precursor cells. Oxidative stress may thus represent a novel molecular cue at the interface between adipose dysregulation and obesity-linked metabolic complications. 6. Oxidative stress: a novel instigator of the adipose organ dysregulation? Oxidative stress (OS) can be defined as the steady state condition where the free radical/reactive oxygen species (ROS) flux is balanced 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 play a critical role in the pathogenesis of obesity, atherosclerosis, T2D and IR [74e76]. AT itself has been suggested as the major source of free radicals and ROS [75e77], leading to postulate that increased OS in accumulated fat might be an early “instigator” of the obesityassociated metabolic complications [75,77,78]. However, the inherent mechanisms linking oxidative damage to the adipose dysfunction remain largely unknown. ROS are highly reactive molecules generated either during physiological cellular processes or under various stress conditions [79]. Remarkably, ROS and free radicals are now emerging as “signaling molecules” that may engage different regulatory pathways controlling tissue homeostasis [80]. The redox-regulation of signal transduction may thus be of importance in adipogenesis

[81e83]. In line with the paradigm of the “expandability hypothesis”, the adipose oxidative stress may well represent a suitable candidate linking the failure of fat cell formation with adipose dysregulation and its associated complications. Yet, the literature remains controversial regarding the effects of stress on (pre)adipocyte recruitment and differentiation. Depending on model and experimental settings, increased oxidative stress has been associated with both pro- or anti-adipogenic attitudes [77,81,82,84,85]. One hypothesis that may reconcile such conflicting observations differentiates the effects of “physiological”, tightly regulated, and targeted intracellular redox changes from the “noxious effects” of increased, uncontrolled production of free radicals and ROS. The adipose stem cells are very sensitive to redox changes. Under physiological conditions, when OS is balanced, free radical signaling works in accordance with cellular function to maintain long-term regenerative potential and survival of stem cells [82,86]. ROS are generally assumed to promote cell proliferation. It may thus be anticipated that an initial “burst” in ROS production may be a prerequisite for the initiation of adipogenic differentiation, by inducing signaling events that switch committed (pre)adipocytes from proliferation to differentiation. On the other hand, an excessive and inappropriate redox balance may detrimentally affect precursor cells recruitment/differentiation, thereby promoting the development of “unhealthy” fat expansion [75,81,85,87]. In harmony with this, ROS production was found to be markedly increased during differentiation of 3T3-L1 cells into adipocytes [75]. However, an elegant work by Mouche et al. demonstrated that (pre)adipocytes exhibit limited, if any, “spontaneous” ROS production, which, in turn, becomes upregulated in mature fat cells [87]. Accordingly, in a mouse model of obesity-linked IR, the activation of the NADPH oxidase (Nox) complex has been indicated as the major source of ROS in adipocytes [75]. However, although decreased Nox4 mRNA

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content was reported as a hallmark of (pre)adipocyte differentiation [87], the expression of Nox4 has been elsewhere proposed as a “molecular switch” promoting insulin-induced differentiation of the (pre)adipocyte [85]. It is thus plausible that Nox4-mediated ROS production may either trigger adipogenesis, when released intracellularly, or provide a cellular cross-talk between different celltypes within fat tissue, when secreted in the extracellular space [87]. In this regard, in 3T3-L1 (pre)adipocytes ROS downregulate adiponectin and PPAR-g, while increase mRNA expression of IL-6 and monocyte chemoattractant protein-1 (MCP-1), a well-known regulator of monocyte recruitment to sites of inflammation [75]. In addition, macrophages are also leading source of ROS, and byproducts of lipid peroxidation are themselves potent chemoattractant [88]. Thus, the cross-talk between (pre)adipocyte and macrophages, which importantly contributes to adipose inflammation, may concur to regulate OS in accumulated fat, as previously postulated [38]. Finally, in AT of obese rodents, decreased expression of selenoprotein P, a selenium transporter with antioxidant protection from ROS, was linked with AT inflammation, impaired preadipocyte differentiation, and, as expected, the development of insulin resistance [89]. It is thus plausible that free radicals and ROS in accumulated fat may impair (pre)adipocyte differentiation and induce macrophage infiltration, providing a mutually inductive “feedback loop” by which inflammation and oxidative stress conspire to induce adipose dysregulation and metabolic dysfunctions. Taken together, from these data an appealing scenario arises where the fine tuning of the redox balance in AT emerges as a prerequisite for maintaining local and systemic homeostasis by complex auto/paracrine and endocrine ROS-mediated cross-talks between different cell-types of the fat organ. By which mechanisms however the “adipose stress” may hamper recruitment and differentiation of the precursor cells. Novel and intriguing insights into this topic were recently gained by the demonstration that the Wnt signaling pathway may undergo a redox-dependent regulation [83]. Briefly, ROS-stimulated Wnt cascade appears orchestrated in a temporal manner by a thioredoxin-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 different experimental settings, Funato et al. clearly demonstrated that oxidative stress induces the dissociation of NRX from Dvl, thus transactivating downstream Wnt signaling pathway [83]. It is thus tantalizing to speculate that aberrant ROS production in the adipose micromilieu may engage Wnt or other stress sensing pathways (ie, the mitogen-activated protein kinases pathway; MAPKs), which transactivation hampers (pre)adipocyte recruitment and differentiation [90]. In humans, to the best of our knowledge, direct evidence to support such a hypothesis is still lacking. Interestingly, data of the literature implicate that, in mesenchymal osteoblast progenitor cells, acute increase in ROS antagonizes the osteogenic and favors adipogenic differentiation by diverting the pool of b-catenin away from prototypical Wnt-mediated (TCF/LEF) transcription factors [86]. Such a “Wnt antagonism” fits well with the decrease of bone formation and the increase of marrow adiposity observed with increasing age, but it is unlikely to represent a more generalized phenomenon of the adult pluripotent stem cells. In human marrow stromal cells, specific products of cholesterol oxidation, namely the oxysterols, have importantly been implicated as “endogenous signals” modulating lineage-specific differentiation in favor of osteogenesis and against adipogenic differentiation [91,92]. Even more, the oxysterol-associated anti-adipogenic actions were consistently linked with selective activation of Wnt target genes [93]. Finally, a major oxidation product of membrane lipids containing polyunsaturated n-6 acyl groups, namely 4-hydroxynonenal (4-HNE), has also emerged as a potent activator of the canonical Wnt pathway in retinal cell lines and in a rat model of diabetic retinopathy [94]. Collectively, these data underscore the biological importance of the “adipose oxidative stress” in regulating precursor cell commitment and differentiation partly by modulating the Wnt signaling pathway. In this context, the lipid peroxidation byproducts may serve as reliable “molecular signatures” of the adipose 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 peroxidation products 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 transduction pathways involved in adipogenesis and metabolic homeostasis. 7. Lipid peroxidation as “markers” of the adipose oxidation and “makers” of systemic insulin-resistance Lipid peroxidation refers to the oxidative degradation of lipids, a process initiated by free radicals/ROS escaping the antioxidant system. By-products of lipid peroxidation, while initially identified as “toxic” end products, at nontoxic and “physiological” levels may serve as “signaling molecules” regulating various cell functions [95e98]. These “signaling” effects primarily result from the adductforming capacity with different macromolecules such as phospholipids, proteins and nucleotides, which ultimately disrupt their biological activity [97e99]. Among lipid peroxidation products oxysterols and 4-hydroxynonenal (4-HNE) received most attention since cross-sectional studies and experimental data indicate their mechanistic implication in the pathophysiology of obesity-linked metabolic diseases [96,97,100e107]. 7.1. Oxysterols Oxysterols are oxidized derivatives of cholesterol resulting from non enzymatic (ie, “autoxidation”) or enzymatic cholesterol oxidation [73]. In line with their role as part of the cellular machinery that governs cell function and integrity, oxysterols are known to exert a multitude of (patho)physiologic effects by acting as signaling molecules at transcriptional, translational- and posttranslational level. A number of key proteins implicated in the control of metabolic homeostasis are recognized targets for oxysterols [108e112]. AT plays a key role in regulating the trafficking of lipids and peroxidation by-products. Indeed, AT stores over half of total body cholesterol [113], the progenitor of oxysterols, and this proportion increases when fat cells become enlarged [114]. Moreover, adipocytes remove serum oxidized low-density lipoproteins (oxLDLs), action that appears beneficial for glucose metabolism and homeostasis [113e115]. Thus, as far as AT acts as a “sink” to safely store harmful cholesterol metabolites, it can be anticipated that the loss of these protective qualities may instigate obesity-linked insulin resistance [105]. In line with such a hypothesis, serum concentrations of 7-ketocholesterol (7k-C) and 7b-hydroxycholesterol (7b-HC), the most abundant oxysterols produced by oxidative stress and carried on oxLDL [116], have been consistently associated with the occurrence 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 adipogenic and in favor of the osteogenic lineage through transactivation of Wnt-mediated signaling pathway [91,120]. It is thus possible to postulate that, while under normal conditions adipocytes provide a protective mechanism for trapping the harmful oxysterols, the increased oxysterols content in hypertrophic fat cells may induce adipocyte dysfunction and contribute to impair precursor cells recruitment/differentiation, leading ultimately to the unfavorable cardio-metabolic outcomes. 7.2. 4-Hydroxynonenal Besides cholesterol, the polyunsaturated fatty acids (PUFA) may also undergo free radical-mediated oxidation generating a series of lipid aldehydes [98]. Interestingly, the pivotal role of such reactive

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molecules on different outcomes of insulin resistance has recently been outlined [96]. One of the most intensively investigated lipid peroxidation product is 4-hydroxynonenal (4-HNE), a major oxidation product of membrane lipids containing n-6 polyunsaturated acyl groups. Experimental data and human studies underscore the theoretical likelihood that 4-HNE actually serves as a key “diabetogenic” molecule at different levels. Indeed, 4-HNE: 1) impairs insulin signaling/action in skeletal muscle by inactivating critical components of the insulin signaling pathway; 2) blunts glucose-induced insulin secretion in pancreatic b-cells; and, 3) disrupts the insulin biological 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, at the adipose level. In line with such a “adipocentric” view, recent data failed to find differences in skeletal muscle proteineHNE content between similarly obese, insulin-sensitive and insulinresistant patients [122]. Furthermore, the inverse relationship found between muscle proteineHNE content and glucose uptake was independent from BMI [121], implying that muscle lipid peroxidation characterizes insulin resistance above and beyond fat mass expansion. This paradigm extends the previous concept that fat dysregulation initiates the IR [39,124], suggesting the adipose lipoxidation as putative instigator of muscle IR partly through 4-HNE-mediated mechanisms. Crescent scientific interest has thus been focused on the characterization of the adipose lipid peroxidation in the setting of obesity and IR. From a chemical standpoint, 4-HNE is highly reactive because of its aldehyde function that covalently modifies lysine residues of proteins. Since proteineHNE modifications are relatively “long-lived footprints” of lipid peroxidation, this aldehyde adduct appears a suitable and metastable biomarker of adipose oxidative stress in vivo [121,125]. Accordingly, in experimental mice models, adipose 4-HNE accumulation per se was shown either to promote the obese state or to induce the development of IR through carbonylation of key adipocytes proteins involved in lipid metabolism [106,126]. Moreover, exposure of adipose cell to increasing 4-HNE concentrations leads to a dysfunctional and “unhealthy” phenotype characterized by selective impairment of insulin signaling (downregulation of IRS-1; upregulation of p38MAPK), increased lactate and reduced adiponectin production, biological events underlying the development of IR [127e129]. Nonetheless, in differentiating (pre)adipocytes, oxidative stress can induce intracellular 4-HNE production, which, in turn, activates the MAPK pathway (p38MAPK) [130]. Along with the previously reported effects of Wnt signal, p38 activity is also known to inhibit adipocyte differentiation, fostering the noxious effect of 4-HNE on adipose homeostasis [90]. Finally, 4-HNE may also stimulate ROS production in mature adipose cells, thus establishing a “self-propelled” oxidative loop between (pre)adipocyte and differentiated fat cells or different other cell-types (ie, macrophages) within the adipose organ [127]. Despite this rationale, there is yet a dearth of knowledge about the occurrence of lipid peroxidation in human obesity as well as the occurrence of adipose oxidation in the dysregulated fat. In order to address this question, we recently undertook a study to characterize 4-HNE expression in the sc abdominal depot of similarly obese insulin-sensitive and IR/T2D patients. Notably, SDS-PAGE analysis revealed that the expression of HNE-adducts of many proteins (size range 25e100 kDa) in T2D patients was higher than that of similarly obese, nondiabetic controls (Murdolo G et al., unpublished). Furthermore, in keeping with the increased proteine 4-HNE adducts content, the adipose precursor cells of the “diabetic” fat exhibited an inherently reduced ability to undergo full adipogenic differentiation. A similar effect was also recapitulated when

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precursor cells isolated from lean healthy controls, with normal adipogenic potential, were challenged with nontoxic 4-HNE concentrations (1e10 mM). Taken together, current results point to 4-HNE as a potential marker of fat oxidation and dysregulation (“diabetic” adiposopathy), as well as a putative driver of impaired adipogenesis and systemic insulin resistance. 8. Potential role of antioxidants in the treatment of insulin resistance The mechanisms by which oxidative stress induces insulin resistance are complex and still debated. Notably, attempts to crudely use antioxidant supplementation as a means of alleviating oxidative stress and thereby improving metabolic dysfunctions in obesity and T2D have proven to be partly disappointing. A number of human intervention studies found that dietary supplementation with antioxidant a-lipoic acid, glutathione, vitamin E, vitamin C, and flavonols may ameliorate insulinsensitivity in patients with insulin resistance, T2D and/or atherosclerotic cardiovascular disease [131]. However, many of these trials have been relatively small and of short duration, and this has hindered their interpretation. A recent meta-analysis of the effects of dietary supplementation with vitamin C or E revealed that, in T2D patients, despite the lack of improvement of glycemic control and degree of insulin resistance, antioxidant supplementation may reduce the early glycation end-product (ie, HbA1c), suggesting a potential benefit of antioxidants in protecting against diabetes complications [134]. Thus, appropriate double-blind, randomized, placebo-controlled studies, with a comprehensive evaluation of different markers of oxidative stress and assessment of individual insulin sensitivity/resistance status are warranted. Nonetheless, better definitions of the specific oxidative species, as well as their cellular target(s) and type of cellular modification(s) such targets undergo, are also required to design more efficient means of alleviating oxidative damage in obesity-linked insulin resistance. Here, we outlined a scientific rationale for targeting the “adipose oxidation” as a potential target for novel therapeutic strategies to prevent/tackle adiposity-linked metabolic dysfunctions. Circumstantial data from experimental animal models, as well as anecdotic observations in humans, partly support the assumption that selective control of cellular redox balance in AT may serve as a critical link between adipose organ dysfunction and derangements of glucose homeostasis [75,101,127,132,133]. Clearly, unlocking the adiposespecific mechanisms underlying the impairment of redox balance and its consequence on hampered precursor cells recruitment and differentiation is attractive for targeted therapeutics. Wish as we might, the possibility that such a strategy can hopefully play a role in preventing, reverting or halting the progression of adiposity-linked cardiometabolic complications is still awaiting. Disclosures The authors have nothing to disclose. Acknowledgments This work was partly funded by grant from “Fondazione Cassa di Risparmio di Perugia; Ricerca Scientifica e Tecnologica”. References [1] G. Frühbeck, J. Gómez-Ambrosi, Control of body weight: a physiologic and transgenic perspective, Diabetologia 46 (2003) 143e172.

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