**6.1 PPARβ/δ, inflammation and insulin resistance in adipose tissue**

The expansion of adipose tissue, mainly in the form of visceral obesity, may contribute to enhanced inflammation in this tissue and insulin resistance through several processes. First, macrophages can infiltrate in adipose tissue, which contributes to the overproduction of

Peroxisome Proliferator-Activated

cytokine-induced insulin resistance in adipocytes.

lean control subjects (Kern et al., 2001).

Receptor β/δ (PPARβ/δ) as a Potential Therapeutic Target for Dyslipidemia 225

this drug treatment abolished IL-6-induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6-induced STAT3 phosphorylation on Tyr705 and Ser727 residues in vitro and in vivo. Moreover, GW501516 prevented IL-6-dependent induction of ERK1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPARβ/δ-null mice, STAT3 phosphorylation (Tyr705 and Ser727), STAT3 DNA-binding activity and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPARβ/δ-null mice compared to wild-type mice. Collectively, our findings indicate that PPARβ/δ activation prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of

**6.2 PPARβ/δ, inflammation and insulin resistance in skeletal muscle cells** 

FFAs may cause insulin resistance in skeletal muscle through several mechanisms, including effects on metabolism (Roden et al., 1996; Haber et al., 2003), signaling (Hirabara et al., 2007; Silveira et al., 2008) and mitochondrial function (Schrauwen et al., 2010; Hirabara et al., 2010). In addition, FFAs activate pro-inflammatory pathways, linking the development of this pathology to a chronic low-grade systemic inflammatory response (Wellen & Hotamisligil, 2005). In addition to FFA-induced inflammation through TLR, an additional pathway leads to FFA-mediated inflammation. This pathway involves intracellular accumulation of fatty acid derivatives. Once fatty acids are taken up by skeletal muscle cells they are either stored as fatty acid derivatives or undergo β-oxidation in the mitochondria. In the presence of high plasma FFA, fatty acid flux in skeletal muscle cells exceeds its oxidation, which leads to the accumulation of fatty acid derivatives, such as diacylglycerol (DAG), which can then activate a number of different serine kinases that negatively regulate insulin action. Thus, DAG is a potent allosteric activator of protein kinase Cθ (PKCθ), which is the most abundant PKC isoform in skeletal muscle (Griffin et al., 1999; Cortright et al., 2000; Itani et al., 2000). This PKC isoform inhibits the action of insulin by phosphorylating certain serine residues on insulin receptor substrate 1 (IRS1), including Ser307 in the rodent IRS-1 protein (reviewed in ref. (Gual et al., 2005)). This phosphorylation impairs insulinreceptor signaling through several distinct mechanisms (Hotamisligil et al., 1996). PKC also impairs insulin sensitivity by activating another serine kinase, IκB kinase β (IKKβ) (Perseghin et al., 2003). In addition to phosphorylating IRS-1 in Ser307, IKKβ phosphorylates IκB. Thus, it activates the pro-inflammatory transcription factor NF-κB, which has been linked to fatty acid-induced impairment of insulin action in skeletal muscle in rodents (Kim et al., 2001; Yuan et al., 2001). Once activated, NF-κB regulates the expression of multiple inflammatory mediators, including IL-6. This cytokine correlates strongly with insulin resistance and type 2 diabetes (Pickup et al., 1997; Kern et al., 2001; Pradhan et al., 2001) and its plasma levels are 2-3 times higher in patients with obesity and type 2 diabetes than in

Accumulation of fatty acid derivatives can be attenuated by mitochondrial β-oxidation. The rate-limiting step for β-oxidation of long-chain fatty acids is their transport into mitochondria via CPT-1. The activity of this enzyme is inhibited by malonyl-CoA, the

inflammatory cytokines, such as tumor necrosis factor α (TNF-α and interleukin 6 (IL-6) (Gustafson et al., 2009). Indeed, the infiltration of macrophages into adipose tissue correlates with the degree of insulin resistance (Mathieu et al., 2010). Second, as visceral fat (which is very sensitive to lipolytic stimuli) increases, so does the rate of lipolysis. This leads to increased free fatty acid (FFA) mobilization and elevated levels of circulating FFA. Several studies have consistently demonstrated that elevations of plasma FFA produce insulin resistance in diabetic patients and in nondiabetic subjects (Boden et al., 1991; Boden, 1997). Saturated FFA are potent activators of the Toll-like receptor-4 (TLR4) (Mathieu et al., 2006) and recent evidence suggests that inflammatory processes induced by obesity and a high-fat diet cause systemic insulin resistance via a mechanism involving this receptor (Shi et al., 2006). TLR-4 is expressed in virtually all human cells and binds a wide spectrum of exogenous and endogenous ligands, including bacterial lipopolysaccharide (LPS) (Akira et al., 2006). In the presence of LPS, the TLR4 complex (including CD-14 and an accessory protein, MD-2), recruits the adaptor protein, myeloid differentiation factor-88 (MyD88), which in turn recruits interleukin-1 receptor-associated kinase (IRAK). This leads to the activation of the pro-inflammatory transcription factor NF-κB (Shoelson et al., 2006) and the subsequent enhanced expression of several inflammatory mediators (including IL-6 and monocyte chemoattractant protein-1 [MCP-1]). These observations indicate that saturated FFA derived from adipocytes and from high-fat diets activate TLR and the inflammatory pathway in adipocytes and macrophages, which contribute to the synthesis and production of cytokines such as TNF-α (Nguyen et al., 2007). In addition, high-fat diets raise plasma LPS to a concentration that is high enough to increase body weight, fasting glycemia and inflammation (Cani et al., 2007). Furthermore, LPS receptor-deleted mice (CD14 mutants) are hypersensitive to insulin, and the development of insulin resistance, obesity and diabetes in this animal model is delayed in response to a high-fat diet (Cani et al., 2007). Experiments performed in our laboratory have demonstrated that the PPARβ/δ agonist GW501516 inhibits LPS-induced cytokine expression and secretion by preventing NF-κB activation in adipocytes (Rodriguez-Calvo et al., 2008). Of note, NF-κB activation by LPS requires mitogen-activated protein kinase (MAPK)–extracellular signal–related kinase (ERK)1/2 (MEK1/2) activation, since inhibition of this pathway reduces LPS-induced cytokine production in adipocytes (Chung et al., 2006). In agreement with this role of ERK1/2 in inflammation in adipocytes, the expression of pro-inflammatory cytokines in these cells drops when they are exposed to LPS in the presence of the MAPK pathway inhibitor U0126. Interestingly, in white adipose tissue from PPARβ/δ-null mice we observed increased ERK1/2 phosphorylation and NF-κB activity and higher expression of IL-6 compared with wild-type mice (Rodriguez-Calvo et al., 2008). Moreover, in the white adipose tissue of a genetic model of obesity and diabetes, the Zucker diabetic fatty (ZDF) rat, the reduction in the expression of PPARβ/δ correlated with an increase in ERK1/2 phosphorylation and NF-κB activity. These findings suggest that PPARβ/δ activation prevents LPS-induced NF-κB activation via ERK1/2, thereby reducing the production of pro-inflammatory cytokines involved in the development of insulin resistance.

In addition, it has been suggested that IL-6 is another of the mediators linking obesityderived chronic inflammation with insulin resistance through activation of signal transducer and activator of transcription 3 (STAT3), with subsequent up-regulation of suppressor of cytokine signaling 3 (SOCS3). Recently we have demonstrated that the PPARβ/δ agonist GW501516 prevents both IL-6-dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes (Serrano-Marco et al., 2011). In addition,

inflammatory cytokines, such as tumor necrosis factor α (TNF-α and interleukin 6 (IL-6) (Gustafson et al., 2009). Indeed, the infiltration of macrophages into adipose tissue correlates with the degree of insulin resistance (Mathieu et al., 2010). Second, as visceral fat (which is very sensitive to lipolytic stimuli) increases, so does the rate of lipolysis. This leads to increased free fatty acid (FFA) mobilization and elevated levels of circulating FFA. Several studies have consistently demonstrated that elevations of plasma FFA produce insulin resistance in diabetic patients and in nondiabetic subjects (Boden et al., 1991; Boden, 1997). Saturated FFA are potent activators of the Toll-like receptor-4 (TLR4) (Mathieu et al., 2006) and recent evidence suggests that inflammatory processes induced by obesity and a high-fat diet cause systemic insulin resistance via a mechanism involving this receptor (Shi et al., 2006). TLR-4 is expressed in virtually all human cells and binds a wide spectrum of exogenous and endogenous ligands, including bacterial lipopolysaccharide (LPS) (Akira et al., 2006). In the presence of LPS, the TLR4 complex (including CD-14 and an accessory protein, MD-2), recruits the adaptor protein, myeloid differentiation factor-88 (MyD88), which in turn recruits interleukin-1 receptor-associated kinase (IRAK). This leads to the activation of the pro-inflammatory transcription factor NF-κB (Shoelson et al., 2006) and the subsequent enhanced expression of several inflammatory mediators (including IL-6 and monocyte chemoattractant protein-1 [MCP-1]). These observations indicate that saturated FFA derived from adipocytes and from high-fat diets activate TLR and the inflammatory pathway in adipocytes and macrophages, which contribute to the synthesis and production of cytokines such as TNF-α (Nguyen et al., 2007). In addition, high-fat diets raise plasma LPS to a concentration that is high enough to increase body weight, fasting glycemia and inflammation (Cani et al., 2007). Furthermore, LPS receptor-deleted mice (CD14 mutants) are hypersensitive to insulin, and the development of insulin resistance, obesity and diabetes in this animal model is delayed in response to a high-fat diet (Cani et al., 2007). Experiments performed in our laboratory have demonstrated that the PPARβ/δ agonist GW501516 inhibits LPS-induced cytokine expression and secretion by preventing NF-κB activation in adipocytes (Rodriguez-Calvo et al., 2008). Of note, NF-κB activation by LPS requires mitogen-activated protein kinase (MAPK)–extracellular signal–related kinase (ERK)1/2 (MEK1/2) activation, since inhibition of this pathway reduces LPS-induced cytokine production in adipocytes (Chung et al., 2006). In agreement with this role of ERK1/2 in inflammation in adipocytes, the expression of pro-inflammatory cytokines in these cells drops when they are exposed to LPS in the presence of the MAPK pathway inhibitor U0126. Interestingly, in white adipose tissue from PPARβ/δ-null mice we observed increased ERK1/2 phosphorylation and NF-κB activity and higher expression of IL-6 compared with wild-type mice (Rodriguez-Calvo et al., 2008). Moreover, in the white adipose tissue of a genetic model of obesity and diabetes, the Zucker diabetic fatty (ZDF) rat, the reduction in the expression of PPARβ/δ correlated with an increase in ERK1/2 phosphorylation and NF-κB activity. These findings suggest that PPARβ/δ activation prevents LPS-induced NF-κB activation via ERK1/2, thereby reducing the production of

pro-inflammatory cytokines involved in the development of insulin resistance.

In addition, it has been suggested that IL-6 is another of the mediators linking obesityderived chronic inflammation with insulin resistance through activation of signal transducer and activator of transcription 3 (STAT3), with subsequent up-regulation of suppressor of cytokine signaling 3 (SOCS3). Recently we have demonstrated that the PPARβ/δ agonist GW501516 prevents both IL-6-dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes (Serrano-Marco et al., 2011). In addition, this drug treatment abolished IL-6-induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6-induced STAT3 phosphorylation on Tyr705 and Ser727 residues in vitro and in vivo. Moreover, GW501516 prevented IL-6-dependent induction of ERK1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPARβ/δ-null mice, STAT3 phosphorylation (Tyr705 and Ser727), STAT3 DNA-binding activity and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPARβ/δ-null mice compared to wild-type mice. Collectively, our findings indicate that PPARβ/δ activation prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes.
