Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity

Reduced mitochondrial fatty acid (FA) β-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA β-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA β-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA β-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA β-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.     

Doxorubicin-induced hepatic toxicity in rats: Mechanistic protective role of Omega-3 fatty acids through Nrf2/HO-1 activation and PI3K/Akt/GSK-3β axis modulation

Doxorubicin (DOX), a common antibiotic used to treat a variety of tumors, has several substantial adverse effects that limit its clinical use. As a result, finding effective protective agents to combat DOX-induced organ damage is a necessity. The current study was set to delineate the hepatoprotective role of omega‐3 fatty acids (ω-3FA) against DOX-mediated acute liver damage in rats and the underlined mechanism of GSK-3β inhibition. Five groups of rats were orally received either saline (groups 1 & 2) or ω-3FA (25, 50 and 100 mg/kg/day; groups 3, 4 & 5, respectively) for 28 consecutive days. Single DOX intraperitoneal injection (20 mg/kg) was used to induce hepatic toxicity in all groups except group 1 (negative control). Blood samples and liver tissues were collected 48-hr after injection. Our results revealed that pre-administration of ω-3FA (25, 50 and 100 mg/kg) to DOX-induced hepatic injured rats showed a significant reduction in serum hepatic injury biomarkers (ALT, AST, total and direct bilirubin) as well as hepatic contents of MDA, GSH, Nrf2 and HO-1. Additionally, hepatic PI3K, pAkt and GSK-3β have been restored significantly in a dose-dependent manner. Furthermore, all the hepatic histopathological features have been retained upon ω-3FA treatment together with the immunostaining intensity of tumor necrosis factor-α and caspase-3. These results suggest that ω-3FA have shown a marked activation of the Nrf2/HO-1 signaling pathway and modulation of the PI3K/pAkt/GSK-3β axis against DOX-induced hepatotoxicity.     

Modulation of antioxidant defence system in brain of rainbow trout (Oncorhynchus mykiss) after chronic carbamazepine treatment

We investigated the effect of long-term exposure to CBZ on the antioxidant system in brain tissue of rainbow trout. Fish were exposed to sublethal concentrations of CBZ (1.0 μg/L, 0.2 mg/L or 2.0 mg/L) for 7, 21, and 42 days. Oxidative stress indices (LPO and CP) and activities of antioxidant enzymes (SOD, CAT, GPx and GR) in fish brain were measured. In addition, non-enzymatic antioxidant (GSH) was determined after 42 days exposure. Carbamazepine exposure at 0.2 mg/L led to significant increases (p < 0.05) of LPO and CP after 42 days and, at 2.0 mg/L, after 21 days. Activities of the antioxidant enzymes SOD, CAT, and GPx in CBZ-treated groups slightly increased during the first period (7 days). However, activities of all measured antioxidant enzymes were significantly inhibited (p < 0.05) at 0.2 mg/L exposure after 42 days and after 21 days at 2.0 mg/L. After 42 days, the content of GSH in fish brain was significantly lower (p < 0.05) in groups exposed to CBZ at 0.2 mg/L and 2.0 mg/L than in other groups. Prolonged exposure to CBZ resulted in excess reactive oxygen species formation, finally resulting in oxidative damage to lipids and proteins and inhibited antioxidant capacities in fish brain. In short, a low level of oxidative stress could induce the adaptive responses of antioxidant enzymes, but long-term exposure to CBZ could lead to serious oxidative damage in fish brain.     

Gene gun-mediated in vivo analysis of tissue-specific repression of gene transcription driven by the chicken ovalbumin promoter in the liver and oviduct of laying Hens

Carnitine palmitoyltransferase-I (CPT-I) plays a crucial role in regulating cardiac fatty acid oxidation which provides the primary source of energy for cardiac muscle contraction. CPT-I catalyzes the transfer of long chain fatty acids into mitochondria and is recognized as the primary rate controlling step in fatty acid oxidation. Molecular cloning techniques have demonstrated that two CPT-I isoforms exist as genes encoding the 'muscle' and 'liver' enzymes. Regulation of fatty acid oxidation rates depends on both short-term regulation of enzyme activity and long-term regulation of enzyme synthesis. Most early investigations into metabolic control of fatty acid oxidation at the CPT-I step concentrated on the hepatic enzyme which can be inhibited by malonyl-CoA and can undergo dramatic amplification or reduction of its sensitivity to inhibition by malonyl-CoA. The muscle CPT-I is inherently more sensitive to malonyl-CoA inhibition but has not been found to undergo any alteration of its sensitivity. Short-term control of activity of muscle CPT-I is apparently regulated by malonyl-CoA concentration in response to fuel supply (glucose, lactate, pyruvate and ketone bodies). The liver isoform is the only CPT-I enzyme present in the mitochondria of liver, kidney, brain and most other tissues while muscle CPT-I is the sole isoform expressed in skeletal muscle as well as white and brown adipocytes. The heart is unique in that it contains both muscle and liver isoforms. Liver CPT-I is highly expressed in the fetal heart, but at birth its activity begins to decline whereas the muscle isoform, which is very low at birth, becomes the predominant enzyme during postnatal development. In this paper, the differential regulation of the two CPT-I isoforms at the protein and the gene level will be discussed.     

Analysis of microRNA expression profile induced by AICAR in mouse hepatocytes

AMP-activated protein kinase (AMPK) has been proposed to act as a key energy sensor mediating the metabolism of glucose and lipids, and pharmacological activation of AMPK may provide a new strategy for the management of type 2 diabetes. MicroRNAs (miRNAs) are a group of endogenous noncoding RNA that play important roles in many biological processes including energy metabolism. Whether miRNAs mediate AMPK action in regulating metabolic process is not clear. In this study, 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) was added to increase activation of AMPK in 8 week old C57BL/6 mice primary hepatocytes. MiRNA microarray was performed to compare the miRNA expression profiles of hepatocytes treated with or without AICAR. We discovered that 41 miRNAs were significantly altered in AICAR-treated sample (fold change: > 2) compared with untreated control sample. Among them, 19 miRNAs were upregulated. MiRNA targets were predicted by TargetScan. Further bioinformatic analysis indicated that these predicted targets might be mainly involved in pathways of cellular metabolism and tumor pathogenesis. FUNDO analysis suggested that these predicted targets were enriched in cancer, diabetes mellitus, hypertension, obesity and heart failure (P < 0.01). A series of miRNAs could be regulated by the activation of AMPK and might mediate the action of AMPK during metabolic processes and tumor pathogenesis. Predicted target genes discovered in this study and pathway analysis provide new insights into hepatic metabolism and tumor pathogenesis regulated by AMPK signaling and clues to the possible molecular mechanism underlying the effect of AMPK.     

Impairment of mitochondrial β-oxidation in rats under cold-hypoxic environment

Mitochondrial ß-oxidation of fatty acid provides a major source of energy in mammals. High altitude (HA), characterized by hypobaric hypoxia and low ambient temperatures, causes alteration in metabolic homeostasis. Several studies have depicted that hypoxic exposure in small mammals causes hypothermia due to hypometabolic state. Moreover, cold exposure along with hypoxia reduces hypoxia tolerance in animals. The present study investigated the rate of β-oxidation and key enzymes, carnitine palmitoyl transferase-I (CPT-I) and hydroxyacyl CoA dehydrogenase (HAD), in rats exposed to cold-hypobaric hypoxic environment. Male Sprague Dawley rats (190–220 g) were randomly divided into eight groups (n?=?6 rats in each group): 1 day hypoxia (H1); 7  days hypoxia (H7); 1 day cold (C1); 7 days cold (C7); 1 day cold-hypoxia (CH1); 7 days cold-hypoxia (CH7) exposed; and unexposed control for 1 and 7 days (UC1 and UC7). After exposure, animals were anaesthetized with ketamine (50 mg/kg body weight) and xylazine (10 mg/kg body weight) intraperitonialy and sacrificed. Mitochondrial CPT-I, HAD, ¹⁴C-palmitate oxidation in gastrocnemius muscle and liver, and plasma leptin were measured. Mitochondrial CPT-I was significantly reduced in muscle and liver in CH1 and CH7 as compared to respective controls. HAD activity was significantly reduced in H1 and CH7, and in H1, H7, CH1, and CH7 as compared to unexposed controls in muscle and liver, respectively. A concomitant decrease in ¹⁴C-palmitate oxidation was found. Significant reduction in plasma leptin in hypoxia and cold-hypoxia suggested hypometabolic state. It can be concluded that ß-oxidation of fatty acids is reduced in rats exposed to cold-hypoxic environment due to the persisting hypometabolic state in cold-hypoxia exposure.     

Nox2-derived ROS in PPARγ signaling and cell-cycle progression of lung alveolar epithelial cells

Reactive oxygen species (ROS) play important roles in peroxisome proliferator-activated receptor γ (PPARγ) signaling and cell-cycle regulation. However, the PPARγ redox-signaling pathways in lung alveolar epithelial cells remain unclear. In this study, we investigated the in vivo and in vitro effects of PPARγ activation on the levels of lung ROS production and cell-cycle progression using C57BL/6J wild-type and Nox2 knockout mice (n = 10) after intraperitoneal injection of a selective PPARγ agonist (GW1929, 5 mg/kg body wt, daily) for 14 days. Compared to vehicle-treated mice, GW1929 increased significantly the levels of ROS production in wild-type lungs, and this was accompanied by significant up-regulation of PPARγ, Nox2, PCNA, and cyclin D1 and phosphorylation of ERK1/2 and p38MAPK. These effects were absent in Nox2 knockout mice. In cultured alveolar epithelial cells, GW1929 (5 μM for 24 h) increased ROS production and promoted cell-cycle progression from G0/G1 into S and G2/M phases, and these effects were abolished by (1) adding a PPARγ antagonist (BADGE, 1 μM), (2) knockdown of PPARγ using siRNA, or (3) knockout of Nox2. In conclusion, PPARγ activation through Nox2-derived ROS promotes cell-cycle progression in normal mouse lungs and in cultured normal alveolar epithelial cells.     

Lipoic acid improves hypertriglyceridemia by stimulating triacylglycerol clearance and downregulating liver triacylglycerol secretion

Elevated blood triacylglycerol (TG) is a significant contributing factor to the current epidemic of obesity-related health disorders, including type-2 diabetes, nonalcoholic fatty liver disease, and cardiovascular disease. The observation that mice lacking the enzyme sn-glycerol-3-phosphate acyltransferase are protected from insulin resistance suggests the possibility that the regulation of TG synthesis be a target for therapy. Five-week-old Zucker Diabetic Fatty (ZDF) rats were fed a diet containing (R)-α-lipoic acid (LA, ∼200 mg/kg body weight per day) for 5 weeks. LA offset the rise in blood and liver TG by inhibiting liver lipogenic gene expression (e.g. sn-glycerol-3-phosphate acyltransferase-1 and diacylglycerol O-acyltransferase-2), lowering hepatic TG secretion, and stimulating clearance of TG-rich lipoproteins. LA-induced TG lowering was not due to the anorectic properties of LA, as pair-fed rats developed hypertriglyceridemia. Livers from LA-treated rats exhibited elevated glycogen content, suggesting dietary carbohydrates were stored as glycogen rather than becoming lipogenic substrate. Although AMP-activated protein kinase (AMPK) reportedly mediates the metabolic effects of LA in rodents, no change in AMPK activity was observed, suggesting LA acted independently of this kinase. The hepatic expression of peroxisome proliferator activated receptor α (PPARα) target genes involved in fatty acid β-oxidation was either unchanged or decreased with LA, indicating a different mode of action than for fibrate drugs. Given its strong safety record, LA may have potential clinical applications for the treatment or prevention of hypertriglyceridemia and diabetic dyslipidemia.     

Association of peroxisome proliferator activated receptor-γ gene with non-alcoholic fatty liver disease in Asian Indians residing in north India

Background

Genetics of non-alcoholic fatty liver (NAFLD) in Asian Indians has been inadequately studied. We investigated the association of polymorphisms C161T and Pro12Ala of peroxisome proliferator-activated receptor gamma (PPARγ) with clinical and biochemical parameters in Asian Indians with NAFLD.

Methods

In this case–control study, 162 NAFLD cases and 173 controls were recruited. Abdominal ultrasound, clinical and biochemical profiles, fasting insulin levels and value of homeostasis model assessment of insulin resistance were determined. Polymerase chain reaction–restriction fragment length polymorphisms of two polymorphisms were performed. The association of these polymorphisms with clinical and biochemical parameters was analysed.

Results

Higher frequency of Ala and T alleles of PPARγ was obtained in cases. Ala/Ala genotype of PPARγ (Pro12Ala) was associated with significantly higher serum triglycerides (TG), alkaline phosphatase (ALK) and waist–hip ratio in cases as compared to controls. In C161T polymorphism, TT genotype was significantly increased TG (p = 0.04), total cholesterol (p = 0.01), ALK (p = 0.04) and gamma-glutamyl transpeptidase (p = 0.007) in cases. The linkage disequilibrium for these two single-nucleotide polymorphisms of PPARγ was differed in cases (D1 = 0.1; p = 0.006) and controls (D1 = 0.07; p = 0.1). Using a multivariate analysis after adjusting for age, sex and body mass index, the presence of NAFLD was linked to these two polymorphisms (odds ratio 1.64 (95% CI: 1.09–2.45, p = 0.05)].

Conclusion

Asian Indians in north India carrying the alleles Ala and T of PPARγ (Pro12Ala and C161T) polymorphisms are predisposed to develop NAFLD.     

Lipoic acid entrains the hepatic circadian clock and lipid metabolic proteins that have been desynchronized with advanced age

It is well established that lipid metabolism is controlled, in part, by circadian clocks. However, circadian clocks lose temporal precision with age and correlates with elevated incidence in dyslipidemia and metabolic syndrome in older adults. Because our lab has shown that lipoic acid (LA) improves lipid homeostasis in aged animals, we hypothesized that LA affects the circadian clock to achieve these results. We fed 24 month old male F344 rats a diet supplemented with 0.2% (w/w) LA for 2 weeks prior to sacrifice and quantified hepatic circadian clock protein levels and clock-controlled lipid metabolic enzymes. LA treatment caused a significant phase-shift in the expression patterns of the circadian clock proteins Period (Per) 2, Brain and Muscle Arnt-Like1 (BMAL1), and Reverse Erythroblastosis virus (Rev-erb) β without altering the amplitude of protein levels during the light phase of the day. LA also significantly altered the oscillatory patterns of clock-controlled proteins associated with lipid metabolism. The level of peroxisome proliferator-activated receptor (PPAR) α was significantly increased and acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were both significantly reduced, suggesting that the LA-supplemented aged animals are in a catabolic state. We conclude that LA remediates some of the dyslipidemic processes associated with advanced age, and this mechanism may be at least partially through entrainment of circadian clocks.     

Estrogen and n-3 polyunsaturated fatty acid supplementation have a synergistic hypotriglyceridemic effect in ovariectomized rats

The n-3 polyunsaturated fatty acids (PUFAs), EPA and DHA, as well as estrogen have been shown to decrease circulating levels of triglyceride (TG), but their underlying mode of action is unclear. The purpose of this study was to determine the effects of n-3 PUFA consumption and estrogen injection on TG metabolism. Rats (n = 48) were fed a modified AIN-93G diet with 0, 1, or 2 % EPA + DHA relative to the total energy intake during 12 weeks. At 8 weeks, rats were ovariectomized (OVX), and after a 1-week recovery, rats were injected with either 17β-estradiol-3-benzoate (E₂) or corn oil for the last 3 weeks. The n-3 PUFA consumption and E₂ injection independently decreased the hepatic expressions of sterol regulatory element-binding protein 1, acetyl-CoA carboxylase 1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2) (P < 0.05). There were interactions between n-3 PUFA consumption and E₂ injection on hepatic expression of FAS and DGAT2. In addition, n-3 PUFA consumption and E₂ injection up-regulated the expression of AMP-activated protein kinase (AMPK), phosphorylated AMPK, peroxisomal proliferator-activated receptor α, and carnitine palmitoyltransferase 1 in liver and skeletal muscle. E₂ injection increased the expression of estrogen receptor α and β in skeletal muscle and liver, but n-3 PUFA consumption increased the expression of both receptors only in skeletal muscle. The present study suggests that the hypotriglyceridemic effects of n-3 PUFA consumption and E₂ injection could be due to the down-regulation of hepatic TG synthesis and up-regulation of TG oxidation in liver and skeletal muscle in OVX rats.     

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n − 9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1–6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n − 9, 20:1n − 9 and 20:2n − 9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n − 9 → (Fads2) → 18:2n − 9 → (Elovl5) → 20:2n − 9 → (Fads1) → 20:3n − 9 and pathway 2) 18:1n − 9 → (Elovl5) → 20:1n − 9 → (Fads2) → 20:2n − 9 → (Fads1) → 20:3n − 9.     

Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal

Background

Activation of adenylyl cyclase (AC) by prolonged exposure of mammalian organism to morphine was demonstrated in previous studies of mechanism of action of this drug. However, expression level of individual AC isoforms was not analyzed in crucial cell structure, plasma membrane (PM).

Methods

Rats were adapted to morphine for 10 days and sacrificed 24 h (group + M10) or 20 days (+ M10/−M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups − M10 and (− M10/−M20)). Percoll®-purified PM were isolated from brain cortex and analyzed by immunoblotting and specific radioligand binding.

Results

ACI (ACII) was increased 8× (2.5×) in morphine-adapted rats (+ M10) when compared with controls (− M10). Increase of ACI and II by long-term adaptation to increasing doses of morphine represented a specific effect as the amount of ACIII–ACIX, of prototypical PM marker, Na, K-ATPase and of trimeric G protein α and β subunits was unchanged. Increase of ACI and II was not detected in PM isolated from group (+ M10/−M20). Thus, the marked increase of ACI and ACII faded away 20 days since the last dose of morphine.

Conclusions

We assume that the specific increase in expression level of ACI and ACII in brain cortex of morphine-adapted rats proceeds as a compensatory, homeostatic response to prolonged exposure to inhibitory drug, morphine.

General significance

Our findings demonstrate that the dramatic and specific change of the crucial component of the opioid receptor cascade in brain cortex, manifested as an increase in PM level of ACI and II, is reversible.     

Electroacupuncture preconditioning-induced neuroprotection may be mediated by glutamate transporter type 2

Electroacupuncture has been shown to induce a preconditioning effect in the brain. The mechanisms for this protection are not fully elucidated. We hypothesize that this protection is mediated by excitatory amino acid transporters (EAATs) that have been shown to be neuroprotective. To test this hypothesis, two-month old male Sprague–Dawley rats and EAAT type 3 (EAAT3) knockout mice received or did not receive 30-min electroacupuncture once a day for five consecutive days. They were subjected to a 120-min middle cerebral arterial occlusion (MCAO) at 24 h after the last electroacupuncture. Neurological outcome was assessed 2 days after the MCAO. Brain tissues were harvested at 24 h after the last electroacupuncture for Western blotting. Rats subjected to electroacupuncture at the Baihui acupoint had smaller brain infarct volumes and better neurological deficit scores than control rats. Electroacupuncture increased EAAT type 2 (EAAT2) in the cerebral cortex, tended to increase EAAT3 in the hippocampus, and had no effect on EAAT type 1 expression. Dihydrokainate, an EAAT2 inhibitor, worsened the neurological outcome of rats with electroacupuncture pretreatment. Electroacupuncture pretreatment at the Baihui acupoint increased EAAT2 in the cerebral cortex and improved the neurological outcome of EAAT3 knockout mice. Together, our results suggest that EAAT2 may mediate the electroacupuncture preconditioning-induced neuroprotection.