Teresa Coll

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.

TNF-α reduces PGC-1α expression through NF-κB and p38 MAPK leading to increased glucose oxidation in a human cardiac cell model

AIMS Inflammatory responses in the heart that are driven by sustained increases in cytokines have been associated with several pathological processes, including cardiac hypertrophy and heart failure. Emerging data suggest a link between cardiomyopathy and myocardial metabolism dysregulation. To further elucidate the relationship between a pro-inflammatory profile and cardiac metabolism dysregulation, a human cell line of cardiac origin, AC16, was treated with tumour necrosis factor-alpha (TNF-alpha). METHODS AND RESULTS Exposure of AC16 cells to TNF-alpha inhibited the expression of peroxisome proliferator-activated receptor coactivator 1alpha (PGC-1alpha), an upstream regulator of lipid and glucose oxidative metabolism. Studies performed with cardiac-specific transgenic mice (Mus musculus) overexpressing TNF-alpha, which have been well characterized as a model of cytokine-induced cardiomyopathy, also displayed reduced PGC-1alpha expression in the heart compared with that of control mice. The mechanism by which TNF-alpha reduced PGC-1alpha expression in vitro appeared to be largely mediated via both p38 mitogen-activated protein kinase and nuclear factor-kappaB pathways. PGC-1alpha downregulation resulted in an increase in glucose oxidation rate, which involved a reduction in pyruvate dehydrogenase kinase 4 expression and depended on the DNA-binding activity of both peroxisome proliferator-activated receptor beta/delta and estrogen-related receptor alpha transcription factors. CONCLUSION These results point to PGC-1alpha downregulation as a potential contributor to cardiac dysfunction and heart failure in metabolic disorders with an inflammatory background.

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.

The p65 subunit of NF-κB binds to PGC-1α, linking inflammation and metabolic disturbances in cardiac cells

AIMS Nuclear factor-kappaB (NF-kappaB) is a transcription factor induced by a wide range of stimuli, including hyperglycaemia and pro-inflammatory cytokines. It is associated with cardiac hypertrophy and heart failure. It was previously reported that the NF-kappaB-mediated inhibition of proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) might explain the shift in glucose metabolism during cardiac pathological processes induced by pro-inflammatory stimuli, although the specific mechanisms remain to be elucidated. We addressed the specific mechanisms by which exposure to tumour necrosis factor-alpha (TNF-alpha) results in PGC-1alpha down-regulation in cardiac cells and, as a consequence, in the metabolic dysregulation that underlies heart dysfunction and failure. METHODS AND RESULTS By using coimmunoprecipitation studies, we report for the first time that the p65 subunit of NF-kappaB is constitutively bound to PGC-1alpha in human cardiac cells and also in mouse heart, and that NF-kappaB activation by TNF-alpha exposure increases this binding. Overexpression and gene silencing analyses demonstrated that the main factor limiting the degree of this association is p65, because only the modulation of this protein modified the physical interaction. Our data show that the increased physical interaction between p65 and PGC-1alpha after NF-kappaB activation is responsible for the reduction in PGC-1alpha expression and subsequent dysregulation of glucose oxidation. CONCLUSION On the basis of these data, we propose that p65 directly represses PGC-1alpha activity in cardiac cells, thereby leading to a reduction in pyruvate dehydrogenase kinase 4 (PDK4) expression and the subsequent increase in glucose oxidation observed during the proinflammatory state.

Activation of Peroxisome Proliferator-Activated Receptor-δ by GW501516 Prevents Fatty Acid-Induced Nuclear Factor-κB Activation and Insulin Resistance in Skeletal Muscle Cells

Elevated plasma free fatty acids cause insulin resistance in skeletal muscle through the activation of a chronic inflammatory process. This process involves nuclear factor (NF)-kappaB activation as a result of diacylglycerol (DAG) accumulation and subsequent protein kinase Ctheta (PKCtheta) phosphorylation. At present, it is unknown whether peroxisome proliferator-activated receptor-delta (PPARdelta) activation prevents fatty acid-induced inflammation and insulin resistance in skeletal muscle cells. In C2C12 skeletal muscle cells, the PPARdelta agonist GW501516 prevented phosphorylation of insulin receptor substrate-1 at Ser(307) and the inhibition of insulin-stimulated Akt phosphorylation caused by exposure to the saturated fatty acid palmitate. This latter effect was reversed by the PPARdelta antagonist GSK0660. Treatment with the PPARdelta agonist enhanced the expression of two well known PPARdelta target genes involved in fatty acid oxidation, carnitine palmitoyltransferase-1 and pyruvate dehydrogenase kinase 4 and increased the phosphorylation of AMP-activated protein kinase, preventing the reduction in fatty acid oxidation caused by palmitate exposure. In agreement with these changes, GW501516 treatment reversed the increase in DAG and PKCtheta activation caused by palmitate. These effects were abolished in the presence of the carnitine palmitoyltransferase-1 inhibitor etomoxir, thereby indicating that increased fatty acid oxidation was involved in the changes observed. Consistent with these findings, PPARdelta activation by GW501516 blocked palmitate-induced NF-kappaB DNA-binding activity. Likewise, drug treatment inhibited the increase in IL-6 expression caused by palmitate in C2C12 and human skeletal muscle cells as well as the protein secretion of this cytokine. These findings indicate that PPARdelta attenuates fatty acid-induced NF-kappaB activation and the subsequent development of insulin resistance in skeletal muscle cells by reducing DAG accumulation. Our results point to PPARdelta activation as a pharmacological target to prevent insulin resistance.

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.

Peroxisome Proliferator-Activated Receptor (PPAR)β /δ: A New Potential Therapeutic Target for the Treatment of Metabolic Syndrome

Metabolic syndrome is defined as the clustering of multiple metabolic abnormalities, including abdominal obesity, dyslipidemia (high serum triglycerides and low serum HDL-cholesterol levels), glucose intolerance and hypertension. The pathophysiology underlying metabolic syndrome involves a complex interaction of crucial factors, but two of these, insulin resistance and obesity (especially visceral obesity), play a major role. The nuclear receptors Peroxisome Proliferator-Activated Receptors (PPAR)α and PPARγ are therapeutic targets for hypertriglyceridemia and insulin resistance, respectively. Evidence is now emerging that the PPARβ/δ isotype is a potential pharmacological target for the treatment of disorders associated with metabolic syndrome. PPARβ/δ activation increases lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in several animal models. In addition, PPARβ/δ ligands prevent weight gain and suppress macrophage-derived inflammation. These data are promising and indicate that PPARβ/δ ligands may become a therapeutic option for the treatment of metabolic syndrome. However, clinical trials in humans assessing the efficacy and safety of these drugs should confirm these promising perspectives in the treatment of the metabolic syndrome.

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.)