Manuel Merlos

Oleate Reverses Palmitate-induced Insulin Resistance and Inflammation in Skeletal Muscle Cells

Here we report that in skeletal muscle cells the contribution to insulin resistance and inflammation of two common dietary long-chain fatty acids depends on the channeling of these lipids to distinct cellular metabolic fates. Exposure of cells to the saturated fatty acid palmitate led to enhanced diacylglycerol levels and the consequent activation of the protein kinase Cθ/nuclear factor κB pathway, finally resulting in enhanced interleukin 6 secretion and down-regulation of the expression of genes involved in the control of the oxidative capacity of skeletal muscle (peroxisome proliferator-activated receptor (PPAR)γ-coactivator 1α) and triglyceride synthesis (acyl-coenzyme A: diacylglycerol acyltransferase 2). In contrast, exposure to the monounsaturated fatty acid oleate did not lead to these changes. Interestingly, co-incubation of cells with palmitate and oleate reversed both inflammation and impairment of insulin signaling by channeling palmitate into triglycerides and by up-regulating the expression of genes involved in mitochondrial β-oxidation, thus reducing its incorporation into diacylglycerol. Our findings support a model of cellular lipid metabolism in which oleate protects against palmitate-induced inflammation and insulin resistance in skeletal muscle cells by promoting triglyceride accumulation and mitochondrial β-oxidation through PPARα- and protein kinase A-dependent mechanisms.

Palmitate-Mediated Downregulation of Peroxisome Proliferator–Activated Receptor-γ Coactivator 1α in Skeletal Muscle Cells Involves MEK1/2 and Nuclear Factor-κB Activation

The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. Previous studies have reported that insulin-resistant states are characterized by a reduction in the expression of peroxisome proliferator–activated receptor-γ coactivator (PGC)-1, a transcriptional activator that promotes oxidative capacity in skeletal muscle cells. However, little is known about the factors responsible for reduced PGC-1 expression. The expression of PGC-1 mRNA levels was assessed in C2C12 skeletal muscle cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. We report that exposure of C2C12 skeletal muscle cells to 0.75 mmol/l palmitate, but not oleate, reduced PGC-1α mRNA levels (66%; P < 0.001), whereas PGC-1β expression was not affected. Palmitate led to mitogen-activated protein kinase (MAPK)–extracellular signal–related kinase (ERK) 1/2 (MEK1/2) activation. In addition, pharmacological inhibition of this pathway by coincubation of the palmitate-exposed cells with the MEK1/2 inhibitors PD98059 and U0126 prevented the downregulation of PGC-1α. Furthermore, nuclear factor-κB (NF-κB) activation was also involved in palmitate-mediated PGC-1α downregulation, since the NF-κB inhibitor parthenolide prevented a decrease in PGC-1α expression. These findings indicate that palmitate reduces PGC-1α expression in skeletal muscle cells through a mechanism involving MAPK-ERK and NF-κB activation.

Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2.

OBJECTIVE—Chronic activation of the nuclear factor-κB (NF-κB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator–activated receptor (PPAR) β/δ activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS—First, we examined whether the PPARβ/δ agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)–Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-κB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARβ/δ expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-κB DNA-binding activity. Furthermore, IL-6 expression and NF-κB DNA-binding activity was higher in white adipose tissue from PPARβ/δ-null mice than in wild-type mice. Because mitogen-activated protein kinase–extracellular signal–related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-κB activation in adipocytes, we explored whether PPARβ/δ prevented NF-κB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-κB activity, such as ZDF rats and PPARβ/δ-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS—These findings indicate that activation of PPARβ/δ inhibits enhanced cytokine production in adipocytes by preventing NF-κB activation via ERK1/2, an effect that may help prevent insulin resistance.

Synthesis and Biological Evaluation of the 1-Arylpyrazole Class of σ1 Receptor Antagonists: Identification of 4-{2-[5-Methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862)

The synthesis and pharmacological activity of a new series of 1-arylpyrazoles as potent σ(1) receptor (σ(1)R) antagonists are reported. The new compounds were evaluated in vitro in human σ(1)R and guinea pig σ(2) receptor (σ(2)R) binding assays. The nature of the pyrazole substituents was crucial for activity, and a basic amine was shown to be necessary, in accordance with known receptor pharmacophores. A wide variety of amines and spacer lengths between the amino and pyrazole groups were tolerated, but only the ethylenoxy spacer and small cyclic amines provided compounds with sufficient selectivity for σ(1)R vs σ(2)R. The most selective compounds were further profiled, and compound 28, 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862), which showed high activity in the mouse capsaicin model of neurogenic pain, emerged as the most interesting candidate. In addition, compound 28 exerted dose-dependent antinociceptive effects in several neuropathic pain models. This, together with its good physicochemical, safety, and ADME properties, led compound 28 to be selected as clinical candidate.

Sigma 1 receptor: a new therapeutic target for pain.

Sigma 1 receptor (σ₁ receptor) is a unique ligand-regulated molecular chaperone located mainly in the endoplasmic reticulum and the plasma membrane. σ₁ receptor is activated under stress or pathological conditions and interacts with several neurotransmitter receptors and ion channels to modulate their function. The effects reported preclinically with σ₁ receptor ligands are consistent with a role for σ₁ receptor in central sensitization and pain hypersensitivity and suggest a potential therapeutic use of σ₁ receptor antagonists for the management of neuropathic pain as monotherapy. Moreover, data support their use in opioid adjuvant therapy: combination of σ₁ receptor antagonists and opioids results in potentiation of opioid analgesia, without significant increases in opioid-related unwanted effects. Results from clinical trials using selective σ₁ receptor antagonists in several pain conditions are eagerly awaited to ascertain the potential of σ₁ receptor modulation in pain therapy.

PPARβ/δ activation blocks lipid-induced inflammatory pathways in mouse heart and human cardiac cells.

Owing to its high fat content, the classical Western diet has a range of adverse effects on the heart, including enhanced inflammation, hypertrophy, and contractile dysfunction. Proinflammatory factors secreted by cardiac cells, which are under the transcriptional control of nuclear factor-κB (NF-κB), may contribute to heart failure and dilated cardiomyopathy. The underlying mechanisms are complex, since they are linked to systemic metabolic abnormalities and changes in cardiomyocyte phenotype. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate metabolism and are capable of limiting myocardial inflammation and hypertrophy via inhibition of NF-κB. Since PPARβ/δ is the most prevalent PPAR isoform in the heart, we analyzed the effects of the PPARβ/δ agonist GW501516 on inflammatory parameters. A high-fat diet induced the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6, and enhanced the activity of NF-κB in the heart of mice. GW501516 abrogated this enhanced proinflammatory profile. Similar results were obtained when human cardiac AC16 cells exposed to palmitate were coincubated with GW501516. PPARβ/δ activation by GW501516 enhanced the physical interaction between PPARβ/δ and p65, which suggests that this mechanism may also interfere NF-κB transactivation capacity in the heart. GW501516-induced PPARβ/δ activation can attenuate the inflammatory response induced in human cardiac AC16 cells exposed to the saturated fatty acid palmitate and in mice fed a high-fat diet. This is relevant, especially taking into account that PPARβ/δ has been postulated as a potential target in the treatment of obesity and the insulin resistance state.

Atorvastatin prevents carbohydrate response element binding protein activation in the fructose‐fed rat by activating protein kinase A

High fructose intake contributes to the overall epidemic of obesity and metabolic disease. Here we examined whether atorvastatin treatment blocks the activation of the carbohydrate response element binding protein (ChREBP) in the fructose‐fed rat. Fructose feeding increased blood pressure (21%, P < 0.05), plasma free fatty acids (59%, P < 0.01), and plasma triglyceride levels (129%, P < 0.001) compared with control rats fed standard chow. These increases were prevented by atorvastatin. Rats fed the fructose‐rich diet showed enhanced hepatic messenger RNA (mRNA) levels of glycerol‐3‐phosphate acyltransferase (Gpat1) (1.45‐fold induction, P < 0.05), which is the rate‐limiting enzyme for the synthesis of triglycerides, and liver triglyceride content (2.35‐fold induction, P < 0.001). Drug treatment inhibited the induction of Gpat1 and increased the expression of liver‐type carnitine palmitoyltransferase 1 (L‐Cpt‐1) (128%, P < 0.01). These observations indicate that atorvastatin diverts fatty acids from triglyceride synthesis to fatty acid oxidation, which is consistent with the reduction in liver triglyceride levels (28%, P < 0.01) observed after atorvastatin treatment. The expression of Gpat1 is regulated by ChREBP and sterol regulatory element binding protein‐1c (SREBP‐1c). Atorvastatin treatment prevented fructose‐induced ChREBP translocation and the increase in ChREBP DNA‐binding activity while reducing SREBP‐1c DNA‐binding activity. Statin treatment increased phospho‐protein kinase A (PKA), which promotes nuclear exclusion of ChREBP and reduces its DNA‐binding activity. Human HepG2 cells exposed to fructose showed enhanced ChREBP DNA‐binding activity, which was not observed in the presence of atorvastatin. Furthermore, atorvastatin treatment increased the CPT‐I mRNA levels in these cells. Interestingly, both effects of this drug were abolished in the presence of the PKA inhibitor H89. Conclusion: These findings indicate that atorvastatin inhibits fructose‐induced ChREBP activity and increases CPT‐I expression by activating PKA. (HEPATOLOGY > 2009;49:106‐115.)

Intestinal anti-inflammatory activity of UR-12746, a novel 5-ASA conjugate, on acute and chronic experimental colitis in the rat

The present study was undertaken to investigate the intestinal anti‐inflammatory effects of UR‐12746 on the acute and chronic stages of a trinitrobenzene sulphonic acid (TNBS) experimental model of inflammatory bowel disease (IBD) in the rat. UR‐12746 is a novel, locally‐acting compound which combines, through an azo bond, 5‐aminosalicylic (5‐ASA) and UR‐12715, a potent platelet activating factor (PAF)‐antagonist. UR‐12746 oral pretreatment of colitic rats (50 and 100 mg kg−1) reduced acute colonic damage when evaluated 2 days after colonic insult. Postreatment for 4 weeks with UR‐12746 (50 and 100 mg kg−1) resulted in a faster recovery of the damaged colonic mucosa, which was macroscopically significant from the third week. The intestinal anti‐inflammatory effect of UR‐12746 was associated with a decrease in leukocyte infiltration in the colonic mucosa, which was evidenced both biochemically, by a reduction in myeloperoxidase activity, and histologically, by a lower leukocyte count after morphometric analysis. This effect was higher than that seen with sulphasalazine, when assayed at the same doses and in the same experimental conditions. Several mechanisms can be involved in the beneficial effects showed by UR‐12746: inhibition of leukotriene B4 synthesis in the inflamed colon, improvement of the altered colonic oxidative status, and reduction of colonic interleukin‐1β production. The results suggest that the intestinal anti‐inflammatory activity of UR‐12746 can be attributed to the additive effects exerted by 5‐ASA and UR‐12715, the PAF antagonist compound, that are released in the colonic lumen after reduction of the azo bond by the intestinal bacteria.

Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates

1 The time‐course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl‐CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague‐Dawley rats. 2 The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3 Drug treatment produced gross hepatomegaly and increase in peroxisomal β‐oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB ≥ GFB. 4 Both plasma cholesterol (BFB ∼ CFB > GFB) and triglyceride (BFB ∼ GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal β‐oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5 Cytosolic and microsomal (but not mitochondrial) palmitoyl‐CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal β‐oxidation. 6 Palmitoyl‐CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB >GFB), probably as a consequence of the enhancement of hydrolase activities. 7 Some of the effects of fibrate treatment can be explained, at least in part, in terms of peroxisomal induction and caution should be exercised in the extrapolation of these results to species, such as man, that are insensitive to peroxisomal proliferation.

Effects of PAF-antagonists in mouse ear oedema induced by several inflammatory agents

1 Several platelet activating factor (PAF)‐antagonists of different chemical structures were tested in the arachidonic acid‐, tetradecanoylphorbol acetate‐, dithranol‐, and benzoic acid‐induced mouse ear oedema models. 2 Topical application of UR‐10324, UR‐11353, CV‐6209 and WEB‐2086 markedly inhibited ear oedema induced by the four irritants tested, mimicking the profile obtained with dexamethasone. YM‐461 was highly effective only in the dithranol‐induced ear oedema, while BN‐52021 failed to inhibit ear oedema in all models tested. 3 Leukocyte recruitment into the inflamed ears was prevented by PAF‐antagonists, as measured by myeloperoxidase activity in the supernatants of ear homogenates. 4 A relationship between PAF‐antagonist and anti‐inflammatory activities was found in some cases, but other mechanisms cannot be excluded to explain the topical anti‐inflammatory effect of these compounds. 5 Our results suggest that topical formulations containing PAF‐antagonists could be useful in the treatment of some inflammatory skin diseases and provide evidence on the involvement of PAF in these inflammatory processes.