The PPARβ/δ-AMPK Connection in the Treatment of Insulin Resistance

The current treatment options for type 2 diabetes mellitus do not adequately control the disease in many patients. Consequently, there is a need for new drugs to prevent and treat type 2 diabetes mellitus. Among the new potential pharmacological strategies, activators of peroxisome proliferator-activated receptor (PPAR)β/δ show promise. Remarkably, most of the antidiabetic effects of PPARβ/δ agonists involve AMP-activated protein kinase (AMPK) activation. This review summarizes the recent mechanistic insights into the antidiabetic effects of the PPARβ/δ-AMPK pathway, including the upregulation of glucose uptake, muscle remodeling, enhanced fatty acid oxidation, and autophagy, as well as the inhibition of endoplasmic reticulum stress and inflammation. A better understanding of the mechanisms underlying the effects resulting from the PPARβ/δ-AMPK pathway may provide the basis for the development of new therapies in the prevention and treatment of insulin resistance and type 2 diabetes mellitus.     

Co-Incubation with PPARβ/δ Agonists and Antagonists Modeled Using Computational Chemistry: Effect on LPS Induced Inflammatory Markers in Pulmonary Artery

Peroxisome proliferator activated receptor beta/delta (PPARβ/δ) is a nuclear receptor ubiquitously expressed in cells, whose signaling controls inflammation. There are large discrepancies in understanding the complex role of PPARβ/δ in disease, having both anti- and pro-effects on inflammation. After ligand activation, PPARβ/δ regulates genes by two different mechanisms; induction and transrepression, the effects of which are difficult to differentiate directly. We studied the PPARβ/δ-regulation of lipopolysaccharide (LPS) induced inflammation (indicated by release of nitrite and IL-6) of rat pulmonary artery, using different combinations of agonists (GW0742 or L−165402) and antagonists (GSK3787 or GSK0660). LPS induced release of NO and IL-6 is not significantly reduced by incubation with PPARβ/δ ligands (either agonist or antagonist), however, co-incubation with an agonist and antagonist significantly reduces LPS-induced nitrite production and Nos2 mRNA expression. In contrast, incubation with LPS and PPARβ/δ agonists leads to a significant increase in Pdk−4 and Angptl−4 mRNA expression, which is significantly decreased in the presence of PPARβ/δ antagonists. Docking using computational chemistry methods indicates that PPARβ/δ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. Dual binding in the PPARβ/δ binding pocket indicates the ligands retain similar binding energies, which suggests that co-incubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARβ/δ binding pocket. To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARβ/δ binding pocket has been explored. Agonist binding followed by antagonist simultaneously switches the PPARβ/δ mode of action from induction to transrepression, which is linked with an increase in Nos2 mRNA expression and nitrite production.     

Cardioprotective Effects of PPARβ/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation,Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production

Accumulating evidence support the cardioprotective properties of the nuclear receptor peroxisome proliferator activated receptor β/δ (PPARβ/δ); however, the underlying mechanisms are not yet fully elucidated. The aim of the study was to further investigate the mechanisms underlying PPARβ/δ-mediated cardioprotection in the setting of myocardial ischemia/reperfusion (I/R). For this purpose, rats were treated with PPARβ/δ agonist GW0742 and/or antagonist GSK0660 in vivo and hearts were subjected to ex vivo global ischemia followed by reperfusion. PPARβ/δ activation improved left ventricular developed pressure recovery, reduced infarct size (IS) and incidence of reperfusion-induced ventricular arrhythmias while it also up-regulated superoxide dismutase 2, catalase and uncoupling protein 3 resulting in attenuation of oxidative stress as evidenced by the reduction in 4-hydroxy-2-nonenal protein adducts and protein carbonyl formation. PPARβ/δ activation also increased both mRNA expression and enzymatic activity of aldehyde dehydrogenase 2 (ALDH2); inhibition of ALDH2 abrogated the IS limiting effect of PPARβ/δ activation. Furthermore, upregulation of PGC-1α and isocitrate dehydrogenase 2 mRNA expression, increased citrate synthase activity as well as mitochondrial ATP content indicated improvement in mitochondrial content and energy production. These data provide new mechanistic insight into the cardioprotective properties of PPARβ/δ in I/R pointing to ALDH2 as a direct downstream target and suggesting that PPARβ/δ activation alleviates myocardial I/R injury through coordinated stimulation of the antioxidant defense of the heart and preservation of mitochondrial function.     

Leptin,Acting at Central Level,Increases FGF21 Expression in White Adipose Tissue via PPARβ/δ

The altered function of adipose tissue can result in obesity, insulin resistance, and its metabolic complications. Leptin, acting on the central nervous system, modifies the composition and function of adipose tissue. To date, the molecular changes that occur in epididymal white adipose tissue (eWAT) during chronic leptin treatment are not fully understood. Herein we aimed to address whether PPARβ/δ could mediate the metabolic actions induced by leptin in eWAT. To this end, male 3-month-old Wistar rats, infused intracerebroventricularly (icv) with leptin (0.2 μg/day) for 7 days, were daily co-treated intraperitoneally (ip) without or with the specific PPARβ/δ receptor antagonist GSK0660 (1 mg/kg/day). In parallel, we also administered GSK0660 to control rats fed ad libitum without leptin infusion. Leptin, acting at central level, prevented the starvation-induced increase in circulating levels of FGF21, while induced markedly the endogenous expression of FGF21 and browning markers of eWAT. Interestingly, GSK0660 abolished the anorectic effects induced by icv leptin leading to increased visceral fat mass and reduced browning capacity. In addition, the pharmacological inhibition of PPARβ/δ alters the immunomodulatory actions of central leptin on eWAT. In summary, our results demonstrate that PPARβ/δ is involved in the up-regulation of FGF21 expression induced by leptin in visceral adipose tissue.     

PPARdelta in Affected Atopic Dermatitis and Psoriasis: A Possible Role in Metabolic Reprograming

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors expressed in the skin. Three PPAR isotypes, α (NRC1C1), β or δ (NRC1C2) and γ (NRC1C3), have been identified. After activation through ligand binding, PPARs heterodimerize with the 9-cis-retinoic acid receptor (RXR), another nuclear hormone receptor, to bind to specific PPAR-responsive elements in regulatory regions of target genes mainly involved in organogenesis, cell proliferation, cell differentiation, inflammation and metabolism of lipids or carbohydrates. Endogenous PPAR ligands are fatty acids and fatty acid metabolites. In past years, much emphasis has been given to PPARα and γ in skin diseases. PPARβ/δ is the least studied PPAR family member in the skin despite its key role in several important pathways regulating inflammation, keratinocyte proliferation and differentiation, metabolism and the oxidative stress response. This review focuses on the role of PPARβ/δ in keratinocytes and its involvement in psoriasis and atopic dermatitis. Moreover, the relevance of targeting PPARβ/δ to alleviate skin inflammation is discussed.     

Candesartan Does Not Activate PPARγ and Its Target Genes in Early Gestation Trophoblasts

Angiotensin II receptor 1 blockers are commonly used to treat hypertension in women of childbearing age. While the fetotoxic effects of these drugs in the second and third trimesters of pregnancy are well documented, their possible impacts on placenta development in early gestation are unknown. Candesartan, a member of this group, also acts as a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, a key regulator shown to be important for placental development. We have previously shown that trophoblasts do not express the candesartan target–receptor angiotensin II type 1 receptor AGTR1. This study investigated the possible role of candesartan on trophoblastic PPARγ and its hallmark target genes in early gestation. Candesartan did not affect the PPARγ protein expression or nuclear translocation of PPARγ. To mimic extravillous trophoblasts (EVTs) and cytotrophoblast/syncytiotrophoblast (CTB/SCT) responses to candesartan, we used trophoblast cell models BeWo (for CTB/SCT) and SGHPL-4 (EVT) cells as well as placental explants. In vitro, the RT-qPCR analysis showed no effect of candesartan treatment on PPARγ target genes in BeWo or SGHPL-4 cells. Treatment with positive control rosiglitazone, another PPARγ agonist, led to decreased expressions of LEP and PPARG1 in BeWo cells and an increased expression of PPARG1 in SGHPL-4 cells. Our previous data showed early gestation–placental AGTR1 expression in fetal myofibroblasts only. In a CAM assay, AGTR1 was stimulated with angiotensin II and showed increased on-plant vessel outgrowth. These results suggest candesartan does not negatively affect PPARγ or its target genes in human trophoblasts. More likely, candesartan from maternal serum may first act on fetal-placental AGTR1 and influence angiogenesis in the placenta, warranting further research.     

Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay

Peroxisome proliferator-activated receptor γ (PPARγ) is a molecular target of metabolic syndrome and inflammatory disease. PPARγ is an important nuclear receptor and numerous PPARγ ligands were developed to date; thus, efficient assay methods are important. Here, we investigated the incorporation of 7-diethylamino coumarin into the PPARγ agonist rosiglitazone and used the compound in a binding assay for PPARγ. PPARγ-ligand-incorporated 7-methoxycoumarin, 1, showed weak fluorescence intensity in a previous report. We synthesized PPARγ-ligand-incorporating coumarin, 2, in this report, and it enhanced the fluorescence intensity. The PPARγ ligand 2 maintained the rosiglitazone activity. The obtained partial agonist 6 appeared to act through a novel mechanism. The fluorescence intensity of 2 and 6 increased by binding to the ligand binding domain (LBD) of PPARγ and the affinity of reported PPARγ ligands were evaluated using the probe.     

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.     

MAO-A Inhibition by Metaxalone Reverts IL-1β-Induced Inflammatory Phenotype in Microglial Cells

Experimental and clinical studies have suggested that several neurological disorders are associated with the occurrence of central nervous system neuroinflammation. Metaxalone is an FDA-approved muscle relaxant that has been reported to inhibit monoamine oxidase A (MAO-A). The aim of this study was to investigate whether metaxalone might exert antioxidant and anti-inflammatory effects in HMC3 microglial cells. An inflammatory phenotype was induced in HMC3 microglial cells through stimulation with interleukin-1β (IL-1β). Control cells and IL-1β-stimulated cells were subsequently treated with metaxalone (10, 20, and 40 µM) for six hours. IL-1β stimulated the release of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), but reduced the anti-inflammatory cytokine interleukin-13 (IL-13). The upstream signal consisted of an increased priming of nuclear factor-kB (NF-kB), blunted peroxisome proliferator-activated receptor gamma (PPARγ), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression. IL-1β also augmented MAO-A expression/activity and malondialdehyde levels and decreased Nrf2 mRNA expression and protein levels. Metaxalone decreased MAO-A activity and expression, reduced NF-kB, TNF-α, and IL-6, enhanced IL-13, and also increased PPARγ, PGC-1α, and Nrf2 expression. The present experimental study suggests that metaxalone has potential for the treatment of several neurological disorders associated with neuroinflammation.     

Ethyl Gallate Dual-Targeting PTPN6 and PPARγ Shows Anti-Diabetic and Anti-Obese Effects

The emergence of the high correlation between type 2 diabetes and obesity with complicated conditions has led to the coinage of the term “diabesity”. AMP-activated protein kinase (AMPK) activators and peroxisome proliferator-activated receptor (PPARγ) antagonists have shown therapeutic activity for diabesity, respectively. Hence, the discovery of compounds that activate AMPK as well as antagonize PPARγ may lead to the discovery of novel therapeutic agents for diabesity. In this study, the knockdown of PTPN6 activated AMPK and suppressed adipogenesis in 3T3-L1 cells. By screening a library of 1033 natural products against PTPN6, we found ethyl gallate to be the most selective inhibitor of PTPN6 (K_i = 3.4 μM). Subsequent assay identified ethyl gallate as the best PPARγ antagonist (IC₅₀ = 5.4 μM) among the hit compounds inhibiting PTPN6. Ethyl gallate upregulated glucose uptake and downregulated adipogenesis in 3T3-L1 cells as anticipated. These results strongly suggest that ethyl gallate, which targets both PTPN6 and PPARγ, is a potent therapeutic candidate to combat diabesity.     

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.     

α2δ-4 and Cachd1 Proteins Are Regulators of Presynaptic Functions

The α₂δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α₂δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α₂δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α₂δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α₂δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α₂δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α₂δ-1-3 isoforms (α₂δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α₂δ proteins. In contrast to this redundant presynaptic function, α₂δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α₁-α₂δ protein–protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α₂δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α₂δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α₂δ and Cachd1.     

Gene Doping with Peroxisome-Proliferator-Activated Receptor Beta/Delta Agonists Alters Immunity but Exercise Training Mitigates the Detection of Effects in Blood Samples

Synthetic ligands of peroxisome-proliferator-activated receptor beta/delta (PPARβ/δ) are being used as performance-enhancing drugs by athletes. Since we previously showed that PPARβ/δ activation affects T cell biology, we wanted to investigate whether a specific blood T cell signature could be employed as a method to detect the use of PPARβ/δ agonists. We analyzed in primary human T cells the in vitro effect of PPARβ/δ activation on fatty acid oxidation (FAO) and on their differentiation into regulatory T cells (Tregs). Furthermore, we conducted studies in mice assigned to groups according to an 8-week exercise training program and/or a 6-week treatment with 3 mg/kg/day of GW0742, a PPARβ/δ agonist, in order to (1) determine the immune impact of the treatment on secondary lymphoid organs and to (2) validate a blood signature. Our results show that PPARβ/δ activation increases FAO potential in human and mouse T cells and mouse secondary lymphoid organs. This was accompanied by increased Treg polarization of human primary T cells. Moreover, Treg prevalence in mouse lymph nodes was increased when PPARβ/δ activation was combined with exercise training. Lastly, PPARβ/δ activation increased FAO potential in mouse blood T cells. Unfortunately, this signature was masked by training in mice. In conclusion, beyond the fact that it is unlikely that this signature could be used as a doping-control strategy, our results suggest that the use of PPARβ/δ agonists could have potential detrimental immune effects that may not be detectable in blood samples.     

Altered PPARγ Expression Promotes Myelin-Induced Foam Cell Formation in Macrophages in Multiple Sclerosis

Macrophages play a crucial role during the pathogenesis of multiple sclerosis (MS), a neuroinflammatory autoimmune disorder of the central nervous system. Important regulators of the metabolic and inflammatory phenotype of macrophages are liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs). Previously, it has been reported that PPARγ expression is decreased in peripheral blood mononuclear cells of MS patients. The goal of the present study was to determine to what extent PPARγ, as well as the closely related nuclear receptors PPARα and β and LXRα and β, are differentially expressed in monocytes from MS patients and how this change in expression affects the function of monocyte-derived macrophages. We demonstrate that monocytes of relapsing-remitting MS patients display a marked decrease in PPARγ expression, while the expression of PPARα and LXRα/β is not altered. Interestingly, exposure of monocyte-derived macrophages from healthy donors to MS-associated proinflammatory cytokines mimicked this reduction in PPARγ expression. While a reduced PPARγ expression did not affect the inflammatory and phagocytic properties of myelin-loaded macrophages, it did impact myelin processing by increasing the intracellular cholesterol load of myelin-phagocytosing macrophages. Collectively, our findings indicate that an inflammation-induced reduction in PPARγ expression promotes myelin-induced foam cell formation in macrophages in MS.