Fenofibrate prevents orotic acid--induced hepatic steatosis in rats

The experiments performed in this report were designed to investigate the mechanisms involved in the metabolic alterations associated with orotic acid-induced hepatic steatosis and the effect of fenofibrate, a stimulant of peroxisome proliferators-activated receptor alpha (PPARalpha), on these alterations. Male Wistar rats were divided into three experimental groups: 1) fed a balanced diet (C); 2) fed a balanced diet supplemented with 1% orotic acid (OA); 3) fed OA diet containing 100 mg.kg(-1) bw.day(-1) fenofibrate (OA+F), for 9 days. Administration of OA to rats induced significant increase in the hepatic total lipids content, marked microvesicular steatosis and decrease in plasma lipids concentrations compared to control group. Fenofibrate treatment prevented fatty liver induction, caused an additional reduction on plasma lipids concentrations and caused a 40% decrease in the lipogenic rate in adipose tissue. The results also showed a 40% increase in lipoprotein lipase (LPL) activity in adipose tissue from OA treated group and fenofibrate administration induced a 50% decrease in LPL activity. The liver mRNA expression of PPARalpha and ACO (acyl CoA oxidase) were 85% and 68% decreased in OA group when compared to control, respectively. Fenofibrate treatment increased the PPARalpha and ACO expressions whereas the CPT-1 (carnitine palmitoyl transferase-1) expression was not altered. Our results have shown that fenofibrate treatment decreases the hepatic lipid content induced by OA which is mediated by an important increase in fatty acid oxidation consequent to an increase in hepatic mRNA expression of PPARalpha and ACO.     

The absorption, distribution and biological effects of a modified fatty acid in its free form and as an ethyl ester in rats

It has been demonstrated that the absorption of EPA and DHA is significantly lower for ethyl esters than for the corresponding free fatty acids. Since these fatty acids exist in nature and are catabolized by beta-oxidation, we instead wanted to investigate the absorption, distribution and biological effects of a non-beta-oxidizable modified fatty acid. The modified fatty acid tetradecylthioacetic acid (TTA) and the ethyl ester of TTA (EtTTA) were administered to rats for 10 days, in doses corresponding to 150 mg TTA/kg BW/day. No significant differences were found between the accumulated amounts of TTA or its Delta9 desaturated metabolite in plasma, liver, heart and epididymal white adipose tissue between EtTTA and TTA treated rats. EtTTA and TTA increased the activities of carnitine palmitoyltransferase-II and fatty acyl-CoA oxidase in liver, with no differences between the two treatment groups, but did not affect these activities in heart. EtTTA and TTA treatment decreased the plasma levels of triacylglycerols, cholesterol and phospholipids to similar extents, but no significant effects were seen in hepatic and cardiac lipid levels. EtTTA and TTA had similar effects on the fatty acid composition in plasma, liver, heart and epididymal white adipose tissue. Based on changes in fatty acid indexes it seems that these drugs had comparable stimulating effects on stearoyl-CoA desaturase and Delta6 desaturase, and reduced the Delta5 desaturase activity in liver. From the presented results we conclude that the absorption and distribution of the ethyl ester and the free form of TTA are not significantly different, and that the two administered forms of TTA have similar effects on lipid metabolism in rats.     

Human skeletal muscle PPARalpha expression correlates with fat metabolism gene expression but not BMI or insulin sensitivity

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a key regulator of fatty acid oxidation in skeletal muscle, but few data exist from humans in vivo. To investigate whether insulin sensitivity in skeletal muscle and body mass index (BMI) were associated with skeletal muscle expression of PPARalpha and with important genes regulating lipid metabolism in humans in vivo, we undertook hyperinsulinemic-euglycemic clamps and measured PPARalpha mRNA levels and mRNA levels of lipid regulating PPARalpha response genes in skeletal muscle biopsies. mRNA levels were measured in 16 men, using a novel highly sensitive and specific medium throughput quantitative competitive PCR that allows reproducible measurement of multiple candidate mRNAs simultaneously. mRNA levels of PPARalpha were positively correlated with mRNA levels of CD36 (r = 0.77, P = 0.001), lipoprotein lipase (r = 0.54, P = 0.024), muscle-type carnitine palmitoyltransferase-I (r = 0.54, P = 0.024), uncoupling protein-2 (r = 0.63, P = 0.008), and uncoupling protein-3 (r = 0.53, P = 0.026), but not with measures of insulin sensitivity, BMI, or GLUT4, which plays an important role in insulin-mediated glucose uptake. Thus our data suggest that in humans skeletal muscle PPARalpha expression and genes regulating lipid metabolism are tightly linked, but there was no association between both insulin sensitivity and BMI with PPARalpha expression in skeletal muscle.     

Tetradecylthioacetic acid prevents high fat diet induced adiposity and insulin resistance

Tetradecylthioacetic acid (TTA) is a non-beta-oxidizable fatty acid analog, which potently regulates lipid homeostasis. Here we evaluate the ability of TTA to prevent diet-induced and genetically determined adiposity and insulin resistance. In Wistar rats fed a high fat diet, TTA administration completely prevented diet-induced insulin resistance and adiposity. In genetically obese Zucker (fa/fa) rats TTA treatment reduced the epididymal adipose tissue mass and improved insulin sensitivity. All three rodent peroxisome proliferator-activated receptor (PPAR) subtypes were activated by TTA in the ranking order PPARalpha > PPARdelta > PPARgamma. Expression of PPARgamma target genes in adipose tissue was unaffected by TTA treatment, whereas the hepatic expression of PPARalpha-responsive genes encoding enzymes involved in fatty acid uptake, transport, and oxidation was induced. This was accompanied by increased hepatic mitochondrial beta-oxidation and a decreased fatty acid/ketone body ratio in plasma. These findings indicate that PPARalpha-dependent mechanisms play a pivotal role, but additionally, the involvement of PPARalpha-independent pathways is conceivable. Taken together, our results suggest that a TTA-induced increase in hepatic fatty acid oxidation and ketogenesis drains fatty acids from blood and extrahepatic tissues and that this contributes significantly to the beneficial effects of TTA on fat mass accumulation and peripheral insulin sensitivity.     

Changed energy state and increased mitochondrial beta-oxidation rate in liver of rats associated with lowered proton electrochemical potential and stimulated uncoupling protein 2 (UCP-2) expression: evidence for peroxisome proliferator-activated receptor-alpha independent induction of UCP-2 expression

Lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver cellular metabolism, which become poised toward peroxisome proliferator-activated receptor (PPAR) alpha-regulated fatty acid catabolism in mitochondria. After dietary treatment of rats with the hypolipidemic, modified fatty acid, tetradecylthioacetic acid (TTA), the energy state parameters of the liver were altered at the tissue, cell, and mitochondrial levels. Thus, the hepatic phosphate potential, energy charge, and respiratory control coefficients were lowered, whereas rates of oxygen uptake, oxidation of pyridine nucleotide redox pairs, beta-oxidation, and ketogenesis were elevated. Moderate uncoupling of mitochondria from TTA-treated rats was confirmed, as the proton electrochemical potential (Delta(p)) was 15% lower than controls. The change affected the Delta(Psi) component only, leaving the (Delta)pH component unaltered, suggesting that TTA causes induction of electrogenic ion transport rather than electrophoretic fatty acid activity. TTA treatment induced expression of hepatic uncoupling protein 2 (UCP-2) in rats as well as in wild type and PPARalpha-deficient mice, accompanied by a decreased double bond index of the mitochondrial membrane lipids. However, changes of mitochondrial fatty acid composition did not seem to be related to the effects on mitochondrial energy conductance. As TTA activates PPARdelta, we discuss how this subtype might compensate for deficiency of PPARalpha. The overall changes recorded were moderate, making it likely that liver metabolism can maintain its function within the confines of its physiological regulatory framework where challenged by a hypolipemic agent such as TTA, as well as others.     

Differential action of 13-HPODE on PPARalpha downstream genes in rat Fao and human HepG2 hepatoma cell lines

In rats, oxidized fats activate the peroxisome proliferator-activated receptor alpha (PPARalpha), leading to reduced triglyceride concentrations in liver, plasma and very low density lipoproteins. Oxidation products of linoleic acid constitute an important portion of oxidized dietary fats. This study was conducted to check whether the primary lipid peroxidation product of linoleic acid, 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), might be involved in the PPARalpha-activating effect of oxidized fats. Therefore, we examined the effect of 13-HPODE on the expression of PPARalpha target genes in the rat Fao and the human HepG2 hepatoma cell lines. In Fao cells, 13-HPODE increased the mRNA concentration of the PPARalpha target genes acyl-CoA oxidase (ACO), cytochrome P450 4A1 and carnitine-palmitoyltransferase 1A (CPT1A). Furthermore, the concentration of cellular and secreted triglycerides was reduced in Fao cells treated with 13-HPODE. Because PPARalpha mRNA was not influenced, we conclude that these effects are due to an activation of PPARalpha by 13-HPODE. In contrast, HepG2 cells seemed to be resistant to PPARalpha activation by 13-HPODE because no remarkable induction of the PPARalpha target genes ACO, CPT1A, mitochondrial HMG-CoA synthase and delta9-desaturase was observed. Consequently, cellular and secreted triglyceride levels were not changed after incubation of HepG2 cells with 13-HPODE. In conclusion, this study shows that 13-HPODE activates PPARalpha in rat Fao but not in human HepG2 hepatoma cells.     

Concomitant suppression of hyperlipidemia and intestinal polyp formation by increasing lipoprotein lipase activity in Apc-deficient mice

Epidemiologically, a high-fat diet is associated with the risk of colon cancer. In addition, serum levels of triglycerides (TGs) and cholesterol have been demonstrated to be positively associated with colon carcinogenesis. We recently found that an age-dependent hyperlipidemic state (high serum TG levels) exists in Apc-deficient mice, an animal model for human familial adenomatous polyposis. The mRNA levels of lipoprotein lipase (LPL), which catalyzes TG hydrolysis, were shown to be downregulated in the liver and intestines of mice. Moreover, treatment with a peroxisome proliferator-activated receptor (PPAR) alpha agonist, bezafibrate, or a PPARgamma agonist, pioglitazone, suppressed both hyperlipidemia and intestinal polyp formation in the mice, with induction of LPL mRNA. PPARalpha and PPARgamma agonists are reported to exert anti-proliferative and pro-apoptotic effects in cancer cells. One compound that also increases LPL expression levels but does not possess PPAR agnostic activity is NO-1886. When given at 400 or 800 ppm in the diet, it suppresses both hyperlipidemia and intestinal polyp formation in Apc-deficient mice, with elevation of LPL mRNA. In conclusion, a decrease in serum lipid levels by increasing LPL activity may contribute to a reduction in intestinal polyp formation with Apc deficiency. PPARalpha and PPARgamma agonists, as well as NO-1886, could be useful as chemopreventive agents for colon cancer.     

Lipid-Lowering Effects of Tetradecylthioacetic Acid in Antipsychotic-Exposed,Female Rats: Challenges with Long-Term Treatment

Background

Psychiatric patients often require chronic treatment with antipsychotic drugs, and while rats are frequently used to study antipsychotic-induced metabolic adverse effects, long-term exposure has only partially mimicked the appetite-stimulating and weight-inducing effects found in the clinical setting. Antipsychotic-induced effects on serum lipids are also inconsistent in rats, but in a recent study we demonstrated that subchronic treatment with the orexigenic antipsychotic olanzapine resulted in weight-independent increase in serum triglycerides and activation of lipogenic gene expression in female rats. In addition, a recent long-term study in male rats showed that chronic treatment with antipsychotic drugs induced dyslipidemic effects, despite the lack of weight gain.

Aims

In the current study, we sought to examine long-term effects of antipsychotic drugs on weight gain, lipid levels and lipid composition after twice-daily administration of antipsychotics to female rats, and to investigate potential beneficial effects of the lipid-lowering agent tetradecylthioacetic acid (TTA), a modified fatty acid.

Methods

Female rats were exposed to orexigenic antipsychotics (olanzapine or clozapine), metabolically neutral antipsychotics (aripiprazole or ziprasidone), or TTA for 8 weeks. Separate groups received a combination of clozapine and TTA or olanzapine and TTA. The effects of TTA and the combination of olanzapine and TTA after 2 weeks were also investigated.

Results

The antipsychotic-induced weight gain and serum triglyceride increase observed in the subchronic setting was not present after 8 weeks of treatment with antipsychotics, while lipid-lowering effect of TTA was much more pronounced in the chronic than in the subchronic setting, with concomitant upregulation of key oxidative enzymes in the liver. Unexpectedly, TTA potentiated weight gain in rats treated with antipsychotics.

Conclusion

TTA is a promising candidate for prophylactic treatment of antipsychotic-induced dyslipidemic effects, but a more valid long-term rat model for antipsychotic-induced metabolic adverse effects is required.     

WY14,643, a peroxisome proliferator-activated receptor alpha (PPARalpha ) agonist, improves hepatic and muscle steatosis and reverses insulin resistance in lipoatrophic A-ZIP/F-1 mice

WY14,643 is a specific peroxisome proliferator-activated receptor alpha (PPARalpha) agonist with strong hypolipidemic effects. Here we have examined the effect of WY14,643 in the A-ZIP/F-1 mouse, a model of severe lipoatrophic diabetes. With 1 week of treatment, all doses of WY14,643 that were tested normalized serum triglyceride and fatty acid levels. Glucose and insulin levels also improved but only with high doses and longer treatment duration. WY14,643 reduced liver and muscle triglyceride content and increased levels of mRNA encoding fatty acid oxidation enzymes. In liver, the elevated lipogenic mRNA profile (including PPARgamma) in A-ZIP/F-1 mice remained unchanged. These results suggest that WY14,643 acts by increasing beta-oxidation rather by than decreasing lipogenesis or lipid uptake. Hyperinsulinemic euglycemic clamp studies indicated that WY14,643 treatment improved liver more than muscle insulin sensitivity and that hepatic mRNA levels of gluconeogenic enzymes were reduced. Combination treatment with both WY14,643 and a PPARgamma ligand, rosiglitazone, did not lower glucose levels more effectively than did treatment with WY14,643 alone. These data support the hypothesis that reducing intracellular triglycerides in non-adipose tissues improves insulin sensitivity and suggest that further investigation of the role of PPARalpha agonists in the treatment of lipoatrophic diabetes is warranted.     

Fish Oil and the Pan-PPAR Agonist Tetradecylthioacetic Acid Affect the Amino Acid and Carnitine Metabolism in Rats

Peroxisome proliferator-activated receptors (PPARs) are important in the regulation of lipid and glucose metabolism. Recent studies have shown that PPARα-activation by WY 14,643 regulates the metabolism of amino acids. We investigated the effect of PPAR activation on plasma amino acid levels using two PPARα activators with different ligand binding properties, tetradecylthioacetic acid (TTA) and fish oil, where the pan-PPAR agonist TTA is a more potent ligand than omega-3 polyunsaturated fatty acids. In addition, plasma L-carnitine esters were investigated to reflect cellular fatty acid catabolism. Male Wistar rats (Rattus norvegicus) were fed a high-fat (25% w/w) diet including TTA (0.375%, w/w), fish oil (10%, w/w) or a combination of both. The rats were fed for 50 weeks, and although TTA and fish oil had hypotriglyceridemic effects in these animals, only TTA lowered the body weight gain compared to high fat control animals. Distinct dietary effects of fish oil and TTA were observed on plasma amino acid composition. Administration of TTA led to increased plasma levels of the majority of amino acids, except arginine and lysine, which were reduced. Fish oil however, increased plasma levels of only a few amino acids, and the combination showed an intermediate or TTA-dominated effect. On the other hand, TTA and fish oil additively reduced plasma levels of the L-carnitine precursor γ-butyrobetaine, as well as the carnitine esters acetylcarnitine, propionylcarnitine, valeryl/isovalerylcarnitine, and octanoylcarnitine. These data suggest that while both fish oil and TTA affect lipid metabolism, strong PPARα activation is required to obtain effects on amino acid plasma levels. TTA and fish oil may influence amino acid metabolism through different metabolic mechanisms.     

Effects of fatty acids and growth hormone on liver fatty acid binding protein and PPARalpha in rat liver

The aim of this study was to investigate the interaction between long-chain fatty acids (LCFA) and growth hormone (GH) in the regulation of liver fatty acid binding protein (LFABP) and peroxisome proliferator-activated receptor-alpha (PPARalpha). Cultured rat hepatocytes were given oleic acid (OA; 500 microM) and GH (100 ng/ml) for 3 days. LFABP mRNA increased 3.6-fold by GH and 5.7-fold by OA, and combined incubation with GH and OA increased LFABP mRNA 17.6-fold. PPARalpha mRNA was decreased 50% by GH, but OA had no effect. Hypophysectomized (Hx) female rats were treated with L-thyroxine, cortisol, GH, and dietary fat for 7 days. PPARalpha mRNA levels were three- to fourfold higher in Hx than in normal female rats. GH decreased PPARalpha mRNA 50% in Hx rats. Dietary triglycerides (10% corn oil) increased LFABP mRNA and cytosolic LFABP about twofold but had no effect on PPARalpha mRNA in Hx rats. GH and dietary triglycerides had an additive effect on LFABP expression. Dietary triglycerides increased mitochondrial hydroxymethylglutaryl-CoA synthase mRNA only in the presence of GH. The diet increased serum triglycerides in Hx rats, and GH treatment prevented this increase. Addition of cholesterol to the diet did not influence LFABP levels but mitigated increased hepatic triglyceride content. In summary, these studies show that GH regulates LFABP expression independently of PPARalpha. Moreover, GH has different effects on PPARalpha-responsive genes and does not counteract the effect of LCFA on the expression of these gene products.     

The establishment of a novel non-alcoholic steatohepatitis model accompanied with obesity and insulin resistance in mice

Non-alcoholic steatohepatitis (NASH) is a hepatic manifestation of the metabolic syndrome that can progress to liver cirrhosis. The major aim of this study was to establish a novel NASH mouse model accompanied by obesity and insulin resistance, then explore the molecular mechanisms of NASH and evaluate the effects of both the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist fenofibrate and the PPARgamma agonist rosiglitazone in this established NASH model. The novel model was induced in C57BL/6 mice by 23 weeks of ad libitum feeding of a modified high-fat diet (mHFD), with lower methinione and choline and higher fat content. In comparison to the controls, the model animals developed pronounced obesity, dyslipidemia and insulin resistance. Marked liver lesions characterized by severe steatosis, inflammation, fibrosis, increased hepatic triglyceride content, and elevated serum alanine aminotransferase (ALT) levels were observed in the models. In this novel model, treatment with fenofibrate or rosiglitazone significantly improved insulin sensitivity and corrected dyslipidemia; however, fenofibrate was more effective than rosiglitazone in improving hepatic morphology and ALT levels. Further study showed that long-term feeding of mHFD significantly increased expression of mRNA for hepatic PPARgamma, adipose fatty acid binding protein (ap2) and CD36 and suppressed expression of mRNA for hepatic PPARalpha and carnitine palmitoyl transferase-1a (CPT-1a). These results showed the successful establishment of the combined NASH and obese-insulin resistance mouse model. Additionally, aberrant expressions of hepatic PPARalpha and PPARgamma may play a major role in the pathogenesis of NASH by affecting hepatic lipogenesis and fatty acid oxidation in this novel model.     

Novel form of lipolysis induced by leptin.

Hyperleptinemia causes disappearance of body fat without a rise in free fatty acids (FFA) or ketones, suggesting that leptin can deplete adipocytes of fat without releasing FFA. To test this, we measured FFA and glycerol released from adipocytes obtained from normal lean Zucker diabetic fatty rats (+/+) and incubated for 0, 3, 6, or 24 h in either 20 ng/ml recombinant leptin or 100 nM norepinephrine (NE). Whereas NE increased both FFA and glycerol release from adipocytes of +/+ rats, leptin increased glycerol release in +/+ adipocytes without a parallel increase in FFA release. In adipocytes of obese Zucker diabetic fatty rats (fa/fa) with defective leptin receptors, NE increased both FFA and glycerol release, but leptin had no effect on either. Leptin significantly lowered the mRNA of leptin and fatty acid synthase of adipocytes (FAS) (p < 0.05), and up-regulated the mRNA of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyl transferase-1, (CPT-1), and acyl CoA oxidase (ACO) (p < 0.05). NE (100 nM) also lowered leptin mRNA (p < 0.05) but did not affect FAS, PPARalpha, ACO, or CPT-1 expression. We conclude that in normal adipocytes leptin directly decreases FAS expression, increases PPARalpha and the enzymes of FFA oxidation, and stimulates a novel form of lipolysis in which glycerol is released without a proportional release of FFA.     

Causes and prevention of tamoxifen-induced accumulation of triacylglycerol in rat liver

Tamoxifen can induce hepatic steatosis in women. In this study, we wanted to elucidate the mechanism behind the tamoxifen-induced accumulation of triacylglycerol in liver in female rats, and we hoped to prevent this development by combination treatment with the modified fatty acid tetradecylthioacetic acid (TTA). The increased hepatic triacylglycerol level after tamoxifen treatment was accompanied by decreased acetyl-coenzyme A carboxylase (ACC) and FAS activities, increased glycerol-3-phosphate acyltransferase (GPAT) activity, and a tendency to increased diacylglycerol acyltransferase (DGAT) activity. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids from liver were unaffected by tamoxifen, whereas the uptake of lipoproteins was unchanged and the uptake of fatty acids was decreased. Combination treatment with tamoxifen and TTA (Tam+TTA) normalized the hepatic triacylglycerol level and increased the activities of ACC, FAS, GPAT, and DGAT compared with tamoxifen-treated rats. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids were increased after Tam+TTA treatment. In conclusion, tamoxifen increased the hepatic triacylglycerol level, probably as a result of increased triacylglycerol biosynthesis combined with unchanged beta-oxidation. The tamoxifen-induced accumulation of triacylglycerol was prevented by cotreatment with TTA, through mechanisms of increased mitochondrial and peroxisomal beta-oxidation.     

Fish oil and 3-thia fatty acid have additive effects on lipid metabolism but antagonistic effects on oxidative damage when fed to rats for 50 weeks

The 3-thia fatty acid tetradecylthioacetic acid (TTA) is a synthetic modified fatty acid, which, similar with dietary fish oil (FO), influences the regulation of lipid metabolism, the inflammatory response and redox status. This study was aimed to penetrate the difference in TTA's mode of action compared to FO in a long-term experiment (50 weeks of feeding). Male Wistar rats were fed a control, high-fat (25% w/v) diet or a high-fat diet supplemented with either TTA (0.375% w/v) or FO (10% w/v) or their combination. Plasma fatty acid composition, hepatic lipids and expression of relevant genes in the liver and biomarkers of oxidative damage to protein were assessed at the end point of the experiment. Both supplements given in combination demonstrated an additive effect on the decrease in plasma cholesterol levels. The FO diet alone led to removal of plasma cholesterol and a concurrent cholesterol accumulation in liver; however, with TTA cotreatment, the hepatic cholesterol level was significantly reduced. Dietary FO supplementation led to an increased oxidative damage, as seen by biomarkers of protein oxidation and lipoxidation. Tetradecylthioacetic acid administration reduced the levels of these biomarkers confirming its protective role against lipoxidation and protein oxidative damage. Our findings explore the lipid reducing effects of TTA and FO and demonstrate that these bioactive dietary compounds might act in a different manner. The experiment confirms the antioxidant capacity of TTA, showing an improvement in FO-induced oxidative stress.