Lipokines and oxysterols: Novel adipose-derived lipid hormones linking adipose dysfunction and insulin resistance

Free Radical Biology and Medicine 65 (2013) 811–820

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Review Article

Lipokines and oxysterols: Novel adipose-derived lipid hormones linking adipose dysfunction and insulin resistance Giuseppe Murdolo a,b,n, Desirée Bartolini c, Cristina Tortoioli b, Marta Piroddi c, Luigi Iuliano d, Francesco Galli c a

Department of Internal Medicine, Assisi Hospital, I-06081 Assisi, Perugia, Italy Section of Internal Medicine, Endocrine, and Metabolic Sciences, Italy c Section of Applied Biochemistry and Nutritional Sciences, Department of Internal Medicine, Perugia University, Perugia, Italy d Unit of Vascular Medicine, Department of Medical–Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy b

art ic l e i nf o

a b s t r a c t

Article history: Received 11 June 2013 Received in revised form 7 August 2013 Accepted 8 August 2013 Available online 16 August 2013

The expansion of adipose tissue (AT) is, by definition, a hallmark of obesity. However, not all increases in fat mass are associated with pathophysiological cues. Indeed, whereas a “healthy” fat mass accrual, mainly in the subcutaneous depots, preserves metabolic homeostasis, explaining the occurrence of the metabolically healthy obese phenotype, “unhealthy” AT expansion is importantly associated with insulin resistance/type 2 diabetes and the metabolic syndrome. The development of a dysfunctional adipose organ may find mechanistic explanation in a reduced ability to recruit new and functional (pre)adipocytes from undifferentiated precursor cells. Such a failure of the adipogenic process underlies the “AT expandability” paradigm. The inability of AT to expand further to store excess nutrients, rather than obesity per se, induces a diabetogenic milieu by promoting the overflow and the ectopic deposition of fatty acids in insulin-dependent organs (i.e., lipotoxicity), the secretion of various metabolically detrimental adipose-derived hormones (i.e., adipokines and lipokines), and the occurrence of local and systemic inflammation and oxidative stress. Hitherto, fatty acids (i.e., lipokines) and the oxidation by-products of cholesterol and polyunsaturated fatty acids, such as nonenzymatic oxysterols and reactive aldehyde species, respectively, emerge as key modulators of (pre)adipocyte signaling through Wnt/βcatenin and MAPK pathways and potential regulators of glucose homeostasis. These and other mechanistic insights linking adipose dysfunction, oxidative stress, and impairment of glucose homeostasis are discussed in this review article, which focuses on adipose peroxidation as a potential instigator of, and a putative therapeutic target for, obesity-associated metabolic dysfunctions. & 2013 Elsevier Inc. All rights reserved.

Keywords: Adipose tissue Insulin resistance Obesity Oxidative stress Preadipocytes Oxysterols 4-Hydroxynonenal Free radicals

Contents Adipose (dys)function and type 2 diabetes: two facets of the same coin? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Impaired adipogenesis and metabolic dysfunctions: role of fatty acids and lipokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adipose precursor cell differentiation: the Wnt signaling and MAPK pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The wingless-type MMTV integration site (Wnt) family signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAPK involvement in normal and pathological adipogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress as a modulator of adipose biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress in dysfunctional fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Redox regulation of Wnt and MAPKs at the interface between oxidative stress and insulin resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid peroxidation at the crossroads of adipose dysfunction and insulin resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxysterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-Hydroxynonenal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author at: Department of Internal Medicine, Assisi Hospital, I-06081 Assisi, Perugia, Italy. Fax: þ 39 75 573 0855. E-mail address: [email protected] (G. Murdolo).

0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.08.007

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Adipose (dys)function and type 2 diabetes: two facets of the same coin? The global epidemic of obesity is one of the most important public health problems of our times. The World Health Organization classifies overweight and obesity as degrees of excess weight associated with “abnormal or excessive fat accumulation that may impair health” [1]. This definition highlights two important and still debated concepts, namely body fat increase and its associated health threats. Body mass index (BMI), also known as the Quételet index, is a simple index of body “fatness” that is commonly used to categorize overweight and obesity in adults. It is defined as a person′s weight in kilograms divided by the square of his or her height in meters (kg/m2). So far, overweight and obesity are defined by a BMI greater than or equal to 25 and 30 kg/m2, respectively. Moreover, the National Institutes of Health defined severe or “morbid” obesity as either a BMI of Z40 kg/m2 or a BMI of Z35 kg/m2 with two or more obesity-associated comorbidities [2]. The BMI thresholds for adiposity are derived from various epidemiological studies showing a direct relationship between increased BMI and unfavorable health outcomes or overall mortality. Accordingly, raised BMI is a major risk factor for various diseases such as cardiovascular and metabolic diseases (i.e., coronary heart disease, stroke, type 2 diabetes), musculoskeletal disorders (i.e., osteoarthritis), and some cancers (i.e., endometrial, breast, and colon). However, whereas BMI provides the most useful population-level estimate of adiposity, at the individual level the Quételet index should be considered a rough guide of adiposity because it may not reflect the same degree of fatness according to sex, age, and ethnic origin of the person. Recently, the American Medical Association recognized obesity as a “disease state” with multiple pathophysiological aspects requiring a range of interventions to advance its treatment and prevention [3]. This taxonomic upgrade challenged the concept of obesity and raised the level of interest surrounding this issue. However, whether obesity is a disease, a condition, or a risk factor remains still under debate. Regardless of these uncertainties and controversies, compelling evidence indicates that adiposity is associated with insulin resistance (IR) and type 2 diabetes (T2D) [4–6], which are regarded as the core defects of the metabolic syndrome, a cluster of cardiovascular risk factors centered on abdominal obesity and IR. Insulin resistance can be broadly defined as a subnormal biological response to normal insulin concentrations. Insulin has many diverse actions in the body, ranging from lipid and protein metabolism to arterial tone, sympathetic nervous system activation, and appetite control. By this definition, IR may thus pertain to many biological actions of insulin in many tissues. Typically, however, in clinical practice, IR refers to a state in which a given concentration of insulin is associated with a subnormal glucose response [7]. It is increasingly apparent that, although adiposity strongly predisposes to IR, not all obese patients are insulin resistant [8]. Various epidemiological trials showed how some morbidly obese patients (and up to 25% of obese individuals) are “metabolically healthy,” whereas about 18% of nonobese subjects demonstrate biochemical and metabolic features of the metabolic syndrome [8–10]. It can thus be argued that fat mass accrual per se is unlikely to be the sole determinant of the adiposity-associated unfavorable metabolic outcomes, and a preserved adipose tissue function, rather than a generalized expansion of fat mass, seems to be a prerequisite to maintaining insulin sensitivity. Adipose tissue (AT) is now emerging as a remarkably active organ, with functional pleiotropism and high remodeling capacity [11,12]. Obesity implies extensive changes in the ultrastructure of the fat organ involving the enlargement of existing adipocytes (i.e., hypertrophy), the formation of new fat cells from committed (pre) adipocytes (i.e., adipogenesis), extracellular matrix proteolysis, and

the coordinated development of the tissue vascular network (i.e., angiogenesis). One of the most remarkable features of AT is its capacity to expand in a nonneoplastic manner. Arguably, despite such a plasticity, the limit of fat mass expansion seems to be defined for any given individual [13]. Thus, in the face of positive energy balance, impaired adipose expandability may lead to metabolic dysfunctions. Indeed, if IR were a direct consequence of an increased fat mass, all 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 BMI, clearly separating distinct insulin-sensitive from insulin-resistant subphenotypes [10]. In harmony with such an assumption, 29 and 80% of subjects classified as “lean” or “overweight” according to their BMI, respectively, were found to be “obese” as estimated by body fat percentage, and, more importantly, these individuals already exhibit a cardiometabolic risk factor profile similar to that of obese patients [14]. By far, individuals possess a genetically and/or environmentally determined limit for AT expansion. It has hence been proposed that the impairment of new adipose cell formation in the subcutaneous fat depots (i.e., abdominal and tight fat) may play a pivotal role in the development of IR. This provocative paradigm (expandability hypothesis) partly explains the apparent paradox of an increased risk of IR in states of both expansion (adiposity) and reduction (lipodystrophy) of body fat [15–17]. Based on these observations, it is thus tantalizing to speculate that adiposity and T2D represent two facets of the same coin, the insulin-resistance syndrome, which, in turn, seems to be linked with the ability of appropriate or “healthy” subcutaneous fat mass expansion.

Impaired adipogenesis and metabolic dysfunctions: role of fatty acids and lipokines Growing observations support the concept that IR is a pathophysiological cue initiated in, and sustained by, dysregulated fat (adipocentric view) [8,18–30]. The main biological signatures that characterize the “unhealthy” fat organ are the following: (1) enlargement of existing fat cells; (2) selective impairment of insulin signaling in adipocytes (i.e., downregulation of IRS-1 and upregulation of mitogen-activated protein kinase (MAPK)-dependent signal); (3) adipose inflammation, characterized by extensive macrophage and lymphocyte infiltration; (4) limited angiogenesis; and (5) hampered adipogenic differentiation of the precursor cell. Although the dynamics of these steps remains still intricate and not fully defined, the failure of (pre)adipocyte differentiation may well be the initial trigger in the development of unhealthy fat mass expansion [8,18–30], which ultimately favors a diabetogenic milieu through various and partly synergistic mechanisms. First, the inability to safely store metabolically active fatty acids derivatives in hypertrophic adipocytes induces ectopic accumulation of lipids within nonadipose targets (i.e., skeletal muscle, liver, pancreatic islets) with detrimental consequences on tissue homeostasis (lipotoxicity) [31,32]. In harmony with such a concept, a lipid-mediated endocrine network underlying the regulation of systemic metabolic homeostasis has recently been postulated [33]. Evidence obtained in experimental animal models and humans [34,35] suggests that a single serum lipid (i.e., palmitoleic acid) may function as an insulin-sensitizing adipose-derived lipid hormone (“lipokine”). In the presence of a positive energy balance, the impaired formation of new adipocytes leads to enlargement of preexisting fat cells, which, in turn, become dysfunctional, insulin resistant [29], thus secreting a pattern of diabetogenic lipokines. In keeping with this, recent human data demonstrate that AT content of specific fatty acids (i.e., myristic and stearic acid) is

G. Murdolo et al. / Free Radical Biology and Medicine 65 (2013) 811–820

positively correlated with insulin sensitivity, whereas the enrichment of harmful lipid species (i.e., palmitic acid) seems to be associated with both hypertrophic fat cells and IR/T2D [36,37]. It is thus conceivable that the compartmentalization into adipocytes of specific fatty acids may either protect nonadipose organs from lipotoxicity or modulate insulin signaling at the target tissues, thus preserving metabolic homeostasis. Recent evidence points to dietary polyunsaturated fatty acids (PUFAs) as signaling molecules that induce antiadipogenic actions targeting regulatory transcription factors [38,39]. At the same time the fatty acid composition of the AT environment is of importance for adipogenesis [38–40]. Notably, higher proportions of monounsaturated (MUFAs) to saturated (SFAs) fatty acids are present in subcutaneous (sc) adipose depots than in intra-abdominal sites [40]. Because SFAs and the MUFA oleic acid stimulate adipocyte differentiation, and PUFAs seem to be antiadipogenic, the heterogeneity of fatty acid composition between and within different adipose depots may contribute to explaining the occurrence of unhealthy obese phenotypes according to the reduced ability to recruit and differentiate new preadipocytes in sc fat at the expenses of visceral fat expansion. Second, the enlargement of preexisting adipocytes is responsible of macrophage and lymphocyte infiltration. The inflamed AT secretes a pattern of prototypical adipose-derived hormones (i.e., adipokines) that promote detrimental cardiovascular and metabolic consequences [6,29,41]. Accordingly, increased macrophage infiltration and reduced circulating levels of adiponectin characterize the dysfunctional and hypertrophic fat of morbidly obese, but insulin-resistant, individuals (unhealthy obesity), compared with similarly obese, but insulin-sensitive, counterparts (healthy obesity) [8]. Third, the inflammatory microenvironment in inappropriately expanded fat organ negatively affects preadipocyte differentiation [42]. Locally released inflammatory molecules (i.e., tumor necrosis factor-α, TNF-α) can either sustain a macrophage-like phenotype in undifferentiated precursor cells [43] or impair the ability of mature adipose cells to safely store triglycerides [44], thereby perpetuating a vicious cycle. Together, these findings provide a robust mechanistic platform for linking impaired preadipocyte differentiation with the cues of the adipose dysregulation, which ultimately initiates IR and/or favors progression toward frank diabetes.

Adipose precursor cell differentiation: the Wnt signaling and MAPK pathways Currently, a great deal of interest has been directed toward the characterization of the adipogenic precursor cells in the context of healthy or unhealthy obesity. Adipose cell formation begins from undifferentiated mesenchymal precursor cells (ASCs). Whereas the molecular aspects of adipogenesis are beyond the purpose of this review, adipogenesis is a complex and well-organized multistep process that mainly comprises a “commitment” step of the undifferentiated precursors into (pre)adipocytes and a “terminal differentiation” to mature fat cells [45,46]. The molecular regulation of terminal differentiation relies on sequential activation of transcription factors, whereby induction of specific transcription factors, namely the peroxisome proliferator-activated receptor γ (PPARγ) and CCAT/enhancerbinding protein α (C/EBPα) operate synergistically to generate and maintain the phenotype of the mature fat cell [45,46]. In contrast, the molecular events regulating the commitment of ASCs to the adipogenic lineage seem more complex than previously thought and dependent upon different, and yet not fully characterized, pathways.

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The wingless-type MMTV integration site (Wnt) family signaling pathway Growing data show Wnt signaling as a critical regulator of adipogenic precursor cell fate [16,26,41,42,47–54]. Basically, the Wnt′s are a family of secreted glycoproteins that, acting in autocrine or paracrine mode, regulate several developmental processes influencing adult tissue remodeling [16,54–56]. Wnt′s practice their effects through canonical and noncanonical signaling pathways that are differentiated by the involvement of the transcriptional regulator β-catenin (recently reviewed in [57]). A central feature of the canonical Wnt/β-catenin pathway, which seems to have a major role in the biology of the AT, is the regulation of cytosolic β-catenin protein levels (Fig. 1A). Briefly, in the absence of Wnt′s, cytoplasmic β-catenin is recruited to a degradation complex containing axin and adenomatosus polyposis coli, which cooperate in inducing sequential phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β) to sustain the subsequent proteasomal degradation of β-catenin. Binding of Wnt ligands to a receptor complex, which includes Frizzled and the low-density lipoprotein-receptor-related protein5 or -6 (LRP5/6) coreceptor, promotes activation of Disheveled (Dvl) and recruitment of axin to LRP5/6. Such a signaling cascade inactivates the degradation complex, preventing ubiquitination by βTrCP and proteasomal degradation of β-catenin. The stabilization of cytosolic β-catenin promotes its translocation to the nucleus. Nuclear events include the heterodimerization of β-catenin with the lymphoid enhancer-binding factor/T-cell-specific transcription factors (LEF/TCF) and the interaction of β-catenin with the histone acetyltransferase CBP, the chromatin-remodeling SWI/SNF complex, and Bcl9 bound to pygopus, thus leading to Wnt-target gene transcription [16].To date, more than 80 target genes are known to be regulated by the Wnt/β-catenin pathway, and among others they include those that promote cell cycle progression, cellular differentiation, and metabolism [50]. Remarkably, AT has several Wnt-producing cells, and the canonical Wnt/β-catenin pathway seems to be highly active in undifferentiated ASCs where it may direct the cell fate toward osteogenic/ myogenic and away from adipogenic differentiation [47–50]. The failure to suppress Wnt activation in ASCs has thus been suggested as a possible mechanism underlying the perturbed adipogenesis of hypertrophic/dysregulated fat. Indeed, the inhibition of Wnt signaling represents a prerequisite either for ASC commitment toward the adipogenic lineage or for appropriate (pre)adipocyte differentiation and induction of PPARγ and C/EBPα [26,41,42,51–53,58]. At the same time, recent studies highlighted the concept that to maintain the physiological adipogenic function of the adipose organ Wnt activation is essential to raise the number of (pre)adipocytes recruited for differentiation, but this signal must be terminated before terminal differentiation [48]. Notably, in the sc fat of nonobese but insulinresistant individuals, the gene expression of β-catenin and GSK3β is upregulated and the reduced expression/activity of essential adipogenic transcription factors (i.e., C/EBPα and PPAR-γ) is inversely correlated with the size of adipocytes [59]. Thus, it can be speculated that “inappropriate” activation of Wnt/β-catenin signaling in AT might be an early and adiposity-independent instigator of impaired adipogenesis and systemic IR. Moreover, even if Wnt′s are molecules mainly secreted by cells that have not undergone full terminal differentiation, such as the same (pre)adipocytes, mature fat cells also apparently release factors that modulate Wnt signaling, suggesting a paracrine regulation and engagement of early precursor cells in AT homeostasis as the mature adipocytes increase [26,51]. These findings support the view that Wnt/β-catenin signaling could be targeted by means of either pharmacological or dietary interventions aimed at preventing unhealthy AT expansion and the resulting development of metabolic complications. Underlying

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Fig. 1. (A) The canonical Wnt/β-catenin signaling and (B) putative redox regulation by oxysterols and reactive aldehydes. Wnt binding to Frizzled and LRP5/6 coreceptors results in hypophosphorylation of β-catenin that translocates to the nucleus, where it activates a number of Wnt target genes involved in the cell cycle and metabolism, such as cyclin D1 and MyC. Oxysterols and 4-hydroxynonenal (4-HNE) may modulate Wnt signaling at different steps.

mechanisms of the impairment of Wnt/β-catenin signaling in adipose precursor cells remain, however, largely unexplored and this may limit the efficacy of such prevention strategies. Whereas genetic factors are likely to play a role [60,61], a dysregulated secretion of endogenous Wnt antagonists was recently postulated [26,51,62,63]. Interestingly, the finding that the antiadipogenic effects of TNF-α can involve Wnt activation is suggestive of a role for adipose inflammation as a contributor to deregulation of Wnt/ β-catenin signaling [41–43]. On the other hand, this signaling cascade is likely to represent a converging pathway for various modulators of (pre)adipocyte recruitment and differentiation. Redundancy of stimuli engaging the Wnt/β-catenin route and transregulation mechanisms by other pathways are important points awaiting further investigation.

MAPK involvement in normal and pathological adipogenesis The MAPKs, which comprise the extracellular signal-regulated kinases (ERKs), p38, and the c-Jun N-terminal kinases (JNKs), are involved in intracellular signaling pathways with pivotal roles in many cellular processes including proliferation, differentiation, and apoptotic death. MAPKs are activated by a large variety of stimuli such as hormonal substances and stressors [64], and one of their major functions is to connect cell surface receptors to transcription factors in the nucleus, which consequently triggers long-term cellular responses. Several in vitro studies have assessed the role of MAPKs in (pre) adipocyte differentiation [65]. In the case of ERK, after a first set of conflicting data, a consensus scenario has arisen: ERK would be necessary to initiate the (pre)adipocyte into the differentiation process and, thereafter, this signal transduction pathway needs to be shut off to proceed with adipocyte maturation. However, the limitation of the cellular models used to obtain this evidence is that only terminal adipocyte differentiation can be analyzed, eluding the early proliferative steps of adipogenesis.

Fig. 2. Time-regulated involvement of MAPK pathways during various stages of adipogenesis.

New insights into the role of MAPKs in adipobiology have been provided by recent investigations carried out in knockout model systems (reviewed in [65]). These studies confirmed and refined the description of MAPK functions in adipogenesis, also revealing previously unrecognized roles. For example, a role for JNK has been described in obesity and ERK activity has been confirmed in adipogenesis (summarized in Fig. 2). These studies have also assigned specific functions to each isoform of the MAPK pathways. It appears now that a fine-tuning of the MAPKs regulates both normal and pathological adipogenesis. A precise understanding of the cascade of such molecular events and the way to regulate them

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will certainly be crucial to better understand obesity and its associated metabolic dysfunctions. In this scenario, reactive oxygen species (ROS) seem to be of importance because changes in intracellular redox status have been consistently involved in regulating adipogenesis (see below), clearly influencing the balance of activity between ERK and stress kinases [64]. Oxidative stress and its effects on MAPK regulation may thus represent a novel, and so far not fully explored, molecular cue at the interface between adipose dysregulation and obesity-linked metabolic complications.

Oxidative stress as a modulator of adipose biology Nearly a century ago, the observation that animals with higher metabolic rate often have shorter life spans led to the formulation of the rate-of-living hypothesis, which states that the metabolic rate of a species ultimately determines its life expectancy [66]. Further insights into the mechanistic link between metabolism and aging were provided by the so-called free radical theory of aging, which postulated that endogenous ROS generated in cells result in a pattern of cumulative damage, thereby working as a pacesetter of life expectancy. Unhealthy AT expansion is reported as a condition of increased ROS production [67] and, at the same time, caloric restriction and body fat control in primates and humans improve oxidative stress (OS) markers and have a favorable impact on overall morbidity and survival projections (reviewed in [68,69]). As a consequence, the pathophysiology of ROS and the occurrence of OS attracted a great deal of interest in experimental and clinical research aimed at revealing determinants of longevity and explaining the linkage between dysfunctional AT and metabolic syndrome. Oxidative stress in dysfunctional fat Oxidative stress can be defined as the perturbation of the steadystate condition in which the free radical/ROS flux is balanced by antioxidant defenses [70,71]. Conditions of increased flux of free radicals/ROS and/or reduced antioxidant levels are now accepted as playing a critical role in the pathogenesis of obesity, atherosclerosis, T2D, and IR [72–74]. AT itself has been suggested as a major source of endogenous ROS [73–75], leading to the postulation that increased OS in accumulated fat might be an early instigator of obesity-associated metabolic complications [73,75,76]. However, the inherent mechanisms linking oxidative damage to adipose dysfunction in humans remain largely unknown. ROS are generated either during physiological cellular processes or under various stress conditions [77]. In past decades, an extensive number of studies described the function of ROS as signaling molecules engaging a number of redox-sensitive pathways with pathophysiological relevance (reviewed in [71,78,79]). Of note, ROS are clearly involved in the control of tissue homeostasis [80], and a redox regulation of signal transduction has been described in adipogenesis [81–83]. In line with the paradigm of the expandability hypothesis (see Adipose (dys)function and type 2 diabetes: two facets of the same coin? and [13]), OS in fat tissue may well represent a suitable candidate for 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 the model and experimental settings, OS has been associated with both pro- and antiadipogenic attitudes [75,81,82,84,85]. These conflicting observations may partly be explained by differentiating the effects of physiological and tightly regulated intracellular redox changes from the noxious actions of increased and uncontrolled production of ROS. The adipose stem cells are very sensitive to

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redox changes. Under physiological conditions, when ROS flux is balanced, redox signaling works to maintain long-term regenerative potential and survival of the stem cell [82,86]. Because ROS are generally assumed to promote cell proliferation, an initial burst of ROS production may be required for the very early stages of adipogenic differentiation, to induce signaling events that switch committed (pre)adipocytes from proliferation to differentiation. On the other hand, an excessive load of redox stimuli, and the occurrence of oxidative damage, may detrimentally affect precursor cell recruitment/differentiation, thereby promoting the development of unhealthy fat expansion [73,81,85,87]. Accordingly, ROS production was found to be markedly increased during differentiation of 3T3-L1 cells into adipocytes [73]. However, in contrast with mature fat cells, (pre)adipocytes may spontaneously produce ROS at a low rate [87]. In a mouse model of obesity-linked IR, the activation of the NADPH oxidase (Nox) complex has indeed been indicated as the major source of ROS in adipocytes [73]. However, although decreased Nox4 mRNA content was reportedly shown to be a hallmark of (pre)adipocyte differentiation [87], the expression of Nox4 was elsewhere proposed as a molecular switch promoting insulin-induced differentiation of the (pre)adipocyte [85]. It is thus plausible that Nox4mediated ROS production may either trigger adipogenesis, when released intracellularly, or provide a cellular cross talk between various cell types within fat tissue, when secreted into the extracellular space [87]. In this regard, in 3T3-L1 (pre)adipocytes, ROS downregulate adiponectin and PPARγ, while increasing mRNA expression of IL-6 and monocyte chemoattractant protein-1, a well-known regulator of monocyte recruitment to sites of inflammation [73]. In addition, macrophages are also a leading source of ROS, and by-products of lipid peroxidation (see below) are themselves potent chemoattractants that may perpetuate OS in the microinflamed AT [88]. Thus, as previously postulated in [29], the cross talk between (pre)adipocyte and macrophages may concur to sustain OS in accumulated fat. Moreover, in AT of obese rodents, decreased expression of selenoprotein P, a selenium transporter with proposed antioxidant function, was linked with AT inflammation, impaired preadipocyte differentiation, and development of IR [89]. Overall, this evidence supports the assumption that ROS in accumulated fat may impair (pre)adipocyte differentiation and induce macrophage infiltration, providing a self-sustaining loop by which inflammation and oxidative stress conspire to induce adipose dysregulation and metabolic dysfunctions. As a consequence, the fine-tuning of the redox balance in AT is expected to represent a key prerequisite for maintaining ROS-mediated cross talk between adipose cell types by the effect of local and systemic stimuli that include auto/paracrine and endocrine factors, inflammatory mediators, etc. Redox regulation of Wnt and MAPKs at the interface between oxidative stress and insulin resistance The mechanisms underlying the effects of adipose stress on recruitment and differentiation of the precursor cells remain basically unknown. Novel insights into this topic were recently gained by the demonstration that the Wnt signaling pathway may undergo a redox-dependent regulation [83]. In detail, the ROSstimulated Wnt cascade seems to be orchestrated in a temporal manner by the thioredoxin-related protein nucleoredoxin (NRX). NRX usually blocks Wnt pathway activation by interacting with Dvl, an essential adaptor protein for Wnt signaling. In various experimental settings, Funato and Miki [83] demonstrated that OS induces the dissociation of NRX from Dvl, thus transactivating the Wnt signaling pathway downstream. It is thus tantalizing to speculate that aberrant ROS production in the adipose micromilieu may engage Wnt (Fig. 1B) or other stress-sensing components,

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such as MAPK pathways, which transactivation hampers (pre) adipocyte recruitment and differentiation [65]. In humans, evidence that supports such a hypothesis is still lacking. However, recent experimental data in mice support the assumption that increased OS in fat may inhibit (pre)adipocyte differentiation through glutathione depletion [81]. The molecular mechanisms underlying the effects of such a redox imbalance in adipose precursor cells rely on transcriptional repression of target genes required for the S phase of cell cycle progression (i.e., E2F, cyclin A), which ultimately blocks adipogenesis at the early stage of mitotic clonal expansion [81]. These findings therefore provide a previously unrecognized role for adipose OS in the regulation of fat cell formation through inhibition of the cell cycle, which may contribute to explaining adipose dysfunction. Interestingly, in mesenchymal osteoblast progenitor cells, an acute ROS challenge antagonizes the osteogenic-favoring adipogenic differentiation by diverting the pool of β-catenin away from Wnt-related (LEF/TCF) transcription factors [86]. Such a Wnt antagonism fits well with the decrease in bone formation and the increase in 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 (see below), have also importantly been implicated as endogenous signals modulating lineage-specific differentiation in favor of osteogenesis and against adipogenic differentiation [90,91]. Even more, the oxysterol-associated antiadipogenic actions were consistently linked with selective activation of Wnt target genes [92]. Finally, a major oxidation product of membrane lipids containing polyunsaturated n-6 acyl groups, namely 4-hydroxynonenal (4-HNE), has recently emerged as a potent activator of the canonical Wnt pathway in retinal cell lines and in a rat model of diabetic retinopathy [93]. Collectively, these data highlight the biological importance of OS in regulating the biology of AT through effects on precursor cell commitment and differentiation that are mediated, at least in part, by Wnt or MAPK signaling pathways. In this context, lipid peroxidation by-products such as oxysterols and 4-HNE may serve as reliable molecular signatures of adipose OS and, in addition to potential toxic effects, these by-products may well act as signaling molecules engaging critical transduction pathways involved in adipogenesis and metabolic homeostasis.

Lipid peroxidation at the crossroads of adipose dysfunction and insulin resistance A number of oxidants are produced as by-products of normal aerobic cell metabolism, and there is growing evidence that they play key roles in the pathogenesis of metabolic disorders. Oxidative stress can lead to lipid peroxidation and to the formation of secondary by-products. Lipid peroxidation refers to the oxidative damage and eventual degradation of lipids, a process initiated on PUFAs by ROS escaping the antioxidant system. By-products of lipid peroxidation, although initially identified as toxic end products, at nontoxic physiological levels may serve as signaling molecules regulating various cell functions [94–97]. Such signaling effects primarily result from the adduct-forming capacity of some of these by-products with various macromolecules and particularly with unsaturated lipids, proteins, and nucleic acids, which ultimately may cause structural damage and impaired biological activity [96–98]. Among lipid peroxidation products, the nonenzymatic oxysterols and the reactive aldehyde 4-HNE have received the most attention because increasing data indicate their mechanistic implication in the pathophysiology of obesity-linked metabolic diseases [95,96,99–106].

Accordingly, these lipid peroxidation species are discussed in detail in the next sections.

Oxysterols Oxysterols are 27-carbon oxygenated products of cholesterol that arise through enzymatic or nonenzymatic oxidation processes or are absorbed from the diet [70]. As ligands of sterol-regulated transcription factors and intermediates in the biosynthesis of bile acids and steroid hormones, oxysterols control gene expression in lipid metabolism, regulate immune and inflammatory responses, and modify cellular calcium signaling by acting at the transcriptional, translational, and posttranslational levels. A number of key proteins implicated in the control of metabolic homeostasis are recognized targets of oxysterol signaling [107–111]. AT plays a key role in regulating the trafficking of lipids and peroxidation by-products. Indeed, fat stores over half of total body cholesterol [112], the progenitor of oxysterols, and this proportion increases when fat cells become enlarged [113]. Moreover, adipocytes remove serum oxidized low-density lipoproteins (oxLDL′s), an action that seems to be beneficial for preserving glucose homeostasis [112–114]. As far as AT acts as a sink to safely store harmful cholesterol metabolites, the loss of these protective qualities would reasonably be expected to induce local and systemic oxysterol “spillover” that, through paracrine/autocrine or endocrine actions, may well affect (pre)adipocyte differentiation and systemic insulin sensitivity [104]. In line with such a hypothesis, serum concentrations of 7-ketocholesterol (7κ-C) and 7β-hydroxycholesterol (7β OH-C), the most abundant OS-derived oxysterols that are carried on oxLDL [115], have been consistently associated with the occurrence of T2D and correlated with coronary multiple risks [103,116]. Moreover, oxLDL and 7κ-C were also reported to impair (pre) adipocyte differentiation and induce IR in mature adipocytes [104,117,118]. On the other hand, as previously mentioned, oxysterols can also behave as antiadipogenic signals by diverting pluripotent mesenchymal cells′ fate away from adipogenic and in favor of the osteogenic lineage through transactivation of Wntmediated signaling pathway [90,104,119]. New insights into the role of oxysterols in adipogenesis were recently provided by the characterization of oxysterol-binding protein homologues (ORPs) [120]. Basically, ORPs are lipid-binding proteins that regulate cellular lipid homeostasis by accommodating lipids such as oxysterols, also acting as sterol transporters and signaling sensors [120]. Several members of the ORP family have been putatively linked with metabolic diseases: ORP11 was found to be abundantly expressed in visceral AT and its increased expression has been associated with cardiovascular risk factors in obese subjects with metabolic syndrome [121]. Furthermore, the AT expression of OSBPL11 was reportedly shown to be significantly different between high and low responders to caloric restriction [122]. Of note, by analyzing the expression patterns of ORP mRNAs in human sc and visceral adipose depots, Zhou et al. [120] recently identified a functional impact of ORPs on adipocyte differentiation and metabolic phenotype. Although oxysterols are present at low concentrations in tissues, available information on oxysterol production/secretion in human AT in vivo is still lacking. To measure oxysterols as components of the human AT “secretome,” we assessed by gas chromatography/mass spectrometry analysis samples of interstitial fluid collected in the abdominal sc fat by means of the microdialysis technique [123,124]. Preliminary data in healthy volunteers indicate the presence of 7κ-C and 7β OH-C in AT interstitial fluid at concentrations r1 μM. Moreover, in a currently ongoing study, obese individuals with T2D show higher levels of these oxysterols in isolated mature fat

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cells compared with nondiabetic controls (G. Murdolo et al., unpublished data). Therefore we speculate that, although under physiological conditions adipocytes provide a protective mechanism for removing and trapping harmful oxysterols, hypertrophic/dysfunctional fat cells induce unfavorable metabolic outcomes partly by increasing the formation rate and release of such lipid peroxidation by-products. These may cause paracrine/autocrine loop effects feeding microinflammation, local oxidative stress, and impaired recruitment/differentiation of new precursor cells, further sustaining the adipose dysfunction. 4-Hydroxynonenal In addition to cholesterol, PUFAs may also undergo free radicalmediated oxidation generating a series of reactive carbonyls [97]. The role of such second-generation reactive molecules on IR comorbidity has recently been outlined [95]. 4-HNE is one of the most investigated reactive molecules in this group of lipid peroxidation products, which is formed during the oxidation of lipid species containing n-6 polyunsaturated acyl groups. The theoretical likelihood that 4-HNE may act as a key diabetogenic signal at various levels is circumstantiated by observations showing that 4-HNE may: (1) impair insulin signaling/ action in skeletal muscle by inactivating critical components of the insulin signaling pathway, (2) blunt glucose-induced insulin secretion in pancreatic β-cells, and (3) disrupt the insulin biological activity through its direct adduction [101,102,125–127]. Although the involvement of muscle lipid peroxidation in the development of human IR has been postulated [125], AT is by far the major organ equipped to store lipids. Thus, peroxidation would be expected to occur first and, more likely, at the adipose level. In line with the adipocentric view of IR, recent data failed to find differences in skeletal muscle protein–HNE content between similarly obese insulin-sensitive and insulin-resistant patients [126]. Furthermore, the inverse relationship between muscle protein–HNE content and glucose uptake seems to be independent of BMI [125], implying that muscle lipid peroxidation characterizes IR phenotype above and beyond fat mass expansion. This paradigm extends the previous concept that fat dysregulation initiates systemic IR [30,128], suggesting a role for AT lipoxidation and 4-HNE production/reactivity as instigators of muscle IR. Recent scientific interest has thus been focused on the characterization of adipose lipid peroxidation in the setting of obesity and IR. From a chemical standpoint, 4-HNE is highly reactive because of its aldehyde function, which covalently modifies lysines and other selected residues of proteins that are abundantly expressed as posttranslational modifications of serum proteins in chronic kidney disease and diabetic patients (reviewed in [71,129]). Protein–HNE adducts are long-lived “footprints” of lipid peroxidation and thus might represent a suitable and metastable biomarker of adipose oxidative stress in vivo [125,130]. Accordingly, in animal 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 adipocyte proteins involved in lipid metabolism [105,131]. Moreover, exposure of adipose cells to increasing 4-HNE concentrations causes biological events associated with the development of IR. In this context, 4-HNE may cause a selective impairment of insulin signaling in adipocytes by the downregulation of IRS-1 and upregulation of p38 MAPK activity, thereby leading to increased production of lactate and impaired adiponectin secretion [132–134]. Nonetheless, in differentiating (pre)adipocytes, oxidative stress can induce intracellular 4-HNE production, which in turn activates p38 MAPK [135]. Along with the previously reported effects of Wnt signal and oxysterols (see Oxysterols), p38 activity is also known to inhibit

Fig. 3. Proposed model of unhealthy adipose expansion and impairment of insulin sensitivity: central role of lipokines and lipid peroxidation.

(pre)adipocyte differentiation, fostering the noxious effects of 4-HNE on adipose homeostasis [65]. Finally, 4-HNE may also stimulate ROS production in mature adipose cells, thus establishing a selffeeding oxidative loop between (pre)adipocyte and differentiated fat cells that is further propelled by other cell types such as activated monocytes and macrophages within the adipose organ [132]. Although the occurrence of lipid peroxidation as well as its putative association with the development of IR in human AT needs to be investigated, 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 IR. Collectively, we suggest a possible scenario whereby adipose oxidative stress emerges as an important instigator linking impaired precursor cell differentiation and unhealthy adipose tissue expansion with metabolic perturbations (Fig. 3).

Conclusions and perspectives The mechanisms of OS-induced IR are complex and still not fully clarified. Notwithstanding, attempts to crudely use antioxidant supplementation as a means of alleviating oxidative stress and thereby improving metabolic dysfunction in obesity and T2D have been carried out in clinical trials, but findings have been disappointing.

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The theoretical framework for such a straightforward approach came from several epidemiological and observational studies that, in individuals with a reduced plasma antioxidant status, found an increased risk for cardiovascular events and development of T2D. Moreover, observational trials in humans showed that antioxidants such as α-lipoic acid, glutathione, vitamin E, vitamin C, and flavonols ameliorate insulin sensitivity in patients with IR, T2D, and/or atherosclerotic cardiovascular disease [136]. However, the encouraging results obtained in these studies were affected by methodological biases (i.e., small sample size, short duration of treatment), hindering their interpretation. Thus, appropriate double-blind, randomized, placebocontrolled studies, with a comprehensive evaluation of different markers of oxidative stress and careful assessment of individual insulin sensitivity/resistance, are warranted. Nonetheless, a better definition of biologically relevant reactive species, oxidation mechanisms, and targets of cellular modification are also required to design more efficient interventions aimed at alleviating OS-derived and/or obesitylinked IR. The available literature suggests a scientific rationale for targeting “adipose oxidation” with novel therapeutic strategies. In vitro findings, circumstantial data from experimental animal models, as well as anecdotic observations in humans, seem to support the assumption that a selective control of cellular redox in AT may have a favorable impact on organ dysfunction and glucose homeostasis [73,100,132,137,138]. The detoxification of toxic products of lipid peroxidation in adipose precursor cells may also provide further insights into the adipose-specific mechanisms that influence the redox balance, which may ultimately improve (pre)adipocyte recruitment and differentiation through Wnt/β-catenin and MAPKmediated pathways. Lipid peroxidation by-products such as the nonenzymatic oxysterols and 4-HNE, in addition to representing hallmarks of adipose oxidative damage, may behave as active players in adipose dysfunction and development of insulin resistance/type 2 diabetes. Clearly, unlocking the adipose-specific mechanisms underlying the impairment of redox balance and its consequence on hampered precursor cell differentiation is attractive for targeted therapeutics.

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