Marta Alegret

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.)

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.

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.

Reduction of liver fructokinase expression and improved hepatic inflammation and metabolism in liquid fructose-fed rats after atorvastatin treatment

Consumption of beverages that contain fructose favors the increasing prevalence of metabolic syndrome alterations in humans, including non-alcoholic fatty liver disease (NAFLD). Although the only effective treatment for NAFLD is caloric restriction and weight loss, existing data show that atorvastatin, a hydroxymethyl-glutaryl-CoA reductase inhibitor, can be used safely in patients with NAFLD and improves hepatic histology. To gain further insight into the molecular mechanisms of atorvastatins therapeutic effect on NAFLD, we used an experimental model that mimics human consumption of fructose-sweetened beverages. Control, fructose (10% w/v solution) and fructose+atorvastatin (30 mg/kg/day) Sprague-Dawley rats were sacrificed after 14 days. Plasma and liver tissue samples were obtained to determine plasma analytes, liver histology, and the expression of liver proteins that are related to fatty acid synthesis and catabolism, and inflammatory processes. Fructose supplementation induced hypertriglyceridemia and hyperleptinemia, hepatic steatosis and necroinflammation, increased the expression of genes related to fatty acid synthesis and decreased fatty acid β-oxidation activity. Atorvastatin treatment completely abolished histological signs of necroinflammation, reducing the hepatic expression of metallothionein-1 and nuclear factor kappa B binding. Furthermore, atorvastatin reduced plasma (x 0.74) and liver triglyceride (x 0.62) concentrations, decreased the liver expression of carbohydrate response element binding protein transcription factor (x 0.45) and its target genes, and increased the hepatic activity of the fatty acid β-oxidation system (x 1.15). These effects may be related to the fact that atorvastatin decreased the expression of fructokinase (x 0.6) in livers of fructose-supplemented rats, reducing the metabolic burden on the liver that is imposed by continuous fructose ingestion.

High doses of atorvastatin and simvastatin induce key enzymes involved in VLDL production

Treatments with high doses of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors may induce the expression of sterol regulatory element binding protein (SREBP)-target genes, causing different effects from those attributed to the reduction of hepatic cholesterol content. The aim of this study was to investigate the effects of high doses of statins on the key enzymes involved in VLDL production in normolipidemic rats. To examine whether the effects caused by statin treatment are a consequence of HMG-CoA reductase inhibition, we tested the effect of atorvastation on these enzymes in mevalonatefed rats. Atorvastatin and simvastatin enhanced not only HMG-CoA reductase but also the expression of the SREBP-2 gene itself. As a result of the overexpression of SREBP-2 caused by the statin treatment, genes regulated basically by SREBP-1, as FA synthase and acetyl-coenzyme A carboxylase, were also induced and their mRNA levels increased. DAG acyltransferase and microsomal IG transfer protein mRNA levels as well as phosphatidate phosphohydrolase activity were increased by both statins. Simvastatin raised liver cholesterol content, ACAT mRNA levles, and CTP:phosphocholine cytidylyltransferase activity, whereas it reduced liver DAG and phospholipid content. Mevalonate feeding reversed all changes induced by the atorvastatin treatment. These results show that treatment with high doses of statins induces key enzymes controlling rat liver lipid synthesis and VLDL assembly, probably as a result of SREBP-2 overexpression. Despite the induction of the key enzymes involved in VLDL production, both statins markedly reduced plasma TG levels, suggesting that different mechanisms may be involved in the hypotriglyceridemic effect of statins at high or low doses.

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.

Way back for fructose and liver metabolism: Bench side to molecular insights

The World Health Organization recommends that the daily intake of added sugars should make up no more than 10% of total energy. The consumption of sugar-sweetened beverages is the main source of added sugars. Fructose, together with glucose, as a component of high fructose corn syrups or as a component of the sucrose molecule, is one of the main sweeteners present in this kind of beverages. Data from prospective and intervention studies clearly point to high fructose consumption, mainly in the form of sweetened beverages, as a risk factor for several metabolic diseases in humans. The incidence of hypertension, nonalcoholic fatty liver disease (NAFLD), dyslipidemia (mainly hypertriglyceridemia), insulin resistance, type 2 diabetes mellitus, obesity, and the cluster of many of these pathologies in the form of metabolic syndrome is higher in human population segments that show high intake of fructose. Adolescent and young adults from low-income families are especially at risk. We recently reviewed evidence from experimental animals and human data that confirms the deleterious effect of fructose on lipid and glucose metabolism. In this present review we update the information generated in the past 2 years about high consumption of fructose-enriched beverages and the occurrence of metabolic disturbances, especially NAFLD, type 2 diabetes mellitus, and metabolic syndrome. We have explored recent data from observational and experimental human studies, as well as experimental data from animal and cell models. Finally, using information generated in our laboratory and others, we provide a view of the molecular mechanisms that may be specifically involved in the development of liver lipid and glucose metabolic alterations after fructose consumption in liquid form.

Benzafibrate induces acyl-CoA oxidase mRNA levels and fatty acid peroxisomal β-oxidation in rat white adipose tissue

Rats treated with bezafibrate, a PPAR activator, gain less body weight and increase daily food intake. Previously, we have related these changes to a shift of thermogenesis from brown adipose tissue to white adipose tissue attributable to bezafibrate, which induces uncoupling proteins (UCP), UCP-1 and UCP-3, in rat white adipocytes. Nevertheless, UCP induction was weak, implying additional mechanisms in the change of energy homeostasis produced by bezafibrate. Here we show that bezafibrate, in addition to inducing UCPs, modifies energy homeostasis by directly inducing aco gene expression and peroxisomal fatty acid β-oxidation in white adipose tissue. Further, bezafibrate significantly reduced plasma triglyceride and leptin concentrations, without modifying the levels of PPARγ or ob gene in white adipose tissue. These results indicate that bezafibrate reduces the amount of fatty acids available for triglyceride synthesis in white adipose tissue.