Rosa M. Sánchez

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.

Impairment of hepatic Stat‐3 activation and reduction of PPARα activity in fructose‐fed rats

Fructose makes up a significant proportion of energy intake in westernized diets; its increased consumption has paralleled the growing prevalence of obesity and metabolic syndrome over the past two decades. In the current study, we demonstrate that fructose administration (10% wt/vol) in the drinking water of rats reduces the trans‐activating and trans‐repressing activity of the hepatic peroxisome proliferator‐activated receptor α (PPARα). As a consequence, fructose decreases hepatic fatty oxidation and increases pro‐inflammatory transcription factor nuclear factor κB (NF‐κB) activity. These changes were not observed in glucose‐administered rats (10% wt/vol), although both carbohydrates produced similar changes in plasma adiponectin and in the hepatic expression of transcription factors and enzymes involved in fatty acid synthesis. Fructose‐fed, but not glucose‐fed, rats were hyperleptinemic and exhibited increased tyrosine phosphorylation of the signal transducer and activator of transcription‐3 (STAT‐3) transcription factor, although they did not present a similar increase in the serine phosphorylation of nuclear STAT3. Thus, an impairment in the hepatic transduction of the leptin signal could be responsible for the observed alterations in PPARα activity in fructose‐fed rats. Because PPARα activity is lower in human than in rodent liver, fructose ingestion in humans should cause even worse effects, which would partly explain the link between increased consumption of fructose and widening epidemics of obesity and metabolic syndrome. Conclusion: Hypertriglyceridemia and hepatic steatosis induced by fructose ingestion result from a reduction in the hepatic catabolism of fatty acids driven by a state of leptin resistance. (HEPATOLOGY 2007;45:778–788.)

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.

Suppressor of cytokine signaling‐3 (SOCS‐3) and a deficit of serine/threonine (Ser/Thr) phosphoproteins involved in leptin transduction mediate the effect of fructose on rat liver lipid metabolism

There is controversy regarding whether fructose in liquid beverages constitutes another dietary ingredient of high caloric density or introduces qualitative changes in energy metabolism that further facilitate the appearance of metabolic diseases. Central to this issue is the elucidation of the molecular mechanism responsible for the metabolic alterations induced by fructose ingestion. Fructose administration (10% wt/vol) in the drinking water of Sprague‐Dawley male rats for 14 days induced hyperleptinemia and hepatic leptin resistance. This was caused by impairment of the leptin‐signal transduction mediated by both janus‐activated kinase‐2 and the mitogen‐activated protein kinase pathway. The subsequent increase in activity in the liver of the unphosphorylated and active form of the forkhead box O1 nuclear factor, which transrepresses peroxisome proliferator‐activated receptor α activity, and a lack of activation of the adenosine monophosphate‐activated protein kinase, led to hypertriglyceridemia and hepatic steatosis. These alterations are attributable to two key events: (1) an increase in the amount of suppressor of cytokine signaling‐3 protein, which blocks the phosphorylation and activation of janus‐activated kinase‐2 and Tyr985 on the long form of the leptin receptor; and (2) a common deficit of phosphorylation in serine/threonine residues of key proteins in leptin‐signal transduction pathways. The latter is probably produced by the early activation of protein phosphatase 2A, and further sustained by the accumulation in liver tissue of ceramide, an activator of protein phosphatase 2A, due to incomplete oxidation of fatty acids. Conclusion: Our data indicate that fructose ingestion as a liquid solution induces qualitative changes in liver metabolism that lead to metabolic diseases. (HEPATOLOGY 2008.)

Increased reactive oxygen species production down-regulates peroxisome proliferator-activated alpha pathway in C2C12 skeletal muscle cells.

Generation of reactive oxygen species may contribute to the pathogenesis of diseases involving intracellular lipid accumulation. To explore the mechanisms leading to these pathologies we tested the effects of etomoxir, an inhibitor of carnitine palmitoyltransferase I which contains a fatty acid-derived structure, in C2C12 skeletal muscle cells. Etomoxir treatment for 24 h resulted in a down-regulation of peroxisome proliferator-activated receptor α (PPARα) mRNA expression, achieving an 87% reduction at 80 μm etomoxir. The mRNA levels of most of the PPARα target genes studied were reduced at 100 μm etomoxir. By using several inhibitors of de novo ceramide synthesis and C2-ceramide we showed that they were not involved in the effects of etomoxir. Interestingly, the addition of triacsin C, a potent inhibitor of acyl-CoA synthetase, to etomoxir-treated C2C12 skeletal muscle cells did not prevent the down-regulation in PPARα mRNA levels, suggesting that the active form of the drug, etomoxir-CoA, was not involved. Given that saturated fatty acids may generate reactive oxygen species (ROS), we determined whether the addition of etomoxir resulted in ROS generation. Etomoxir increased ROS production and the activity of the well known redox transcription factor NF-κB. In the presence of the pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of NF-κB activity, etomoxir did not down-regulate PPARα mRNA in C2C12 skeletal muscle cells. These results indicate that ROS generation and NF-κB activation are responsible for the down-regulation of PPARα and may provide a new mechanism by which intracellular lipid accumulation occurs in skeletal muscle cells.

Different effect of simvastatin and atorvastatin on key enzymes involved in VLDL synthesis and catabolism in high fat/cholesterol fed rabbits

The effects of atorvastatin (3 mg kg−1) and simvastatin (3 mg kg−1) on hepatic enzyme activities involved in very low density lipoprotein metabolism were studied in coconut oil/cholesterol fed rabbits. Plasma cholesterol and triglyceride levels increased 19 and 4 fold, respectively, after 7 weeks of feeding. Treatment with statins during the last 4 weeks of feeding abolished the progression of hypercholesterolaemia and reduced plasma triglyceride levels. 3‐Hydroxy‐3‐methyl‐glutaryl Coenzyme A reductase, acyl‐coenzyme A:cholesterol acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities were not affected by drug treatment. Accordingly, hepatic free cholesterol, cholesteryl ester and triglyceride content were not modified. Simvastatin treatment caused an increase (72%) in lipoprotein lipase activity without affecting hepatic lipase activity. Atorvastatin caused a reduction in hepatic phospholipid content and a compensatory increase in CTP:phosphocholine cytidylyl transferase activity. The results presented in this study suggest that, besides the inhibitory effect on 3‐hydroxy‐3‐methyl‐glutaryl Coenzyme A reductase, simvastatin and atorvastatin may have additional effects that contribute to their triglyceride‐lowering ability.

Enhancing privacy and dynamic federation in IdM for consumer cloud computing

Consumer cloud computing paradigm has emerged as the natural evolution and integration of advances in several areas including distributed computing, service oriented architecture and consumer electronics. In this complex ecosystem, security and identity management challenges have cropped up, given their dynamism and heterogeneity. As a direct consequence, dynamic federated identity management with privacy improvements has arisen as an indispensable mechanism to enable the global scalability and usability that are required for the successful implantation of Cloud technologies. With these requirements in mind, we present an IdM architecture based on privacy and reputation extensions compliance with the SAMLv2/ID-FF standards1.

Atorvastatin reverses age-related reduction in rat hepatic PPARα and HNF-4

1 Old rats are resistant to fibrate‐induced hypolipidemia owing to a reduction in hepatic peroxisome proliferator‐activated receptor α (PPARα). We tested whether the age‐related decrease in PPARα is prevented by atorvastatin (ATV), a hypolipidemic statin. 2 We determined the activity and expression of Liver X receptor α (LXRα) and PPARα in the liver of 18‐month‐old rats treated with 10 mg kg−1 of ATV for 21 days. We measured fatty acid oxidation (FAO), the expression of PPARα‐target genes, liver triglyceride (TG) and cholesteryl ester (CE) contents and plasma concentrations of TG, cholesterol, glucose, nonesterified fatty acids (NEFA), insulin and leptin. While old female rats were practically unresponsive, ATV‐treated old males showed lower liver TG (−41%) and CE (−48%), and plasma TG (−35%), glucose (−18%) and NEFA (−39%). Age‐related alterations in LXRα expression and binding activity were reverted in ATV‐treated old males. These changes were related to an increase in hepatic FAO (1.2‐fold), and PPARα mRNA (2.2‐fold), PPARα protein (1.6‐fold), and PPARα‐binding activity. 3 Hepatic nuclear factor‐4 (HNF‐4) and chicken ovalbumin upstream‐transcription factor‐II participate in the transcriptional regulation of the PPARα gene, while peroxisome proliferator‐activated receptor gamma coactivator 1 (PGC‐1) behaves as a PPAR coactivator. Ageing reduced the hepatic content of HNF‐4 (74%) and PGC‐1 (77%) exclusively in male rats. ATV administration to old males enhanced the hepatic expression and binding activity (two‐fold) of HNF‐4. 4 ATV‐induced changes in hepatic HNF‐4 and PPARα may be responsible for the improvement of the lipid metabolic phenotype produced by ATV administration to senescent male rats.

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.)