Expression Of CPT1A Research Articles

Somatic cell count–based selection reduces susceptibility to energy shortage during early lactation in a sheep model

During the transition from late gestation to early lactation ruminants experience a negative energy balance (NEB), which is considered to increase susceptibility to mammary infections. Our previous study in 2 divergent lines of sheep selected for high and low somatic cell score (SCS) suggested an association between the response to NEB and genetic susceptibility to mastitis. Forty-eight early-lactation primiparous dairy ewes from the 2 SCS genetic lines were allocated to 2 homogeneous subgroups-an NEB group, which was energy restricted and received 60% of the energy requirements for 15 d, and a control-fed group-to obtain 4 balanced groups of 12 ewes: high-SCS positive energy balance, low-SCS positive energy balance, high-SCS NEB, and low-SCS NEB. High-SCS ewes showed greater weight loss and increased plasmatic concentrations of β-hydroxybutyrate and nonesterified fatty acids than low-SCS ewes when confronted with an induced NEB. The aim of this study was to further characterize this interaction by combining transcriptomic and phenotypic data with a generalized partial least squares discriminant analysis using mixOmics package framework. A preliminary analysis using 3 blocks of phenotypes (fatty acids, weight and production, blood metabolites) revealed a high correlation between fat-to-protein ratio, β-hydroxybutyrate, and nonesterified fatty acids concentrations with milk long-chain fatty acid yields. These phenotypes allowed good discrimination of the energy-restricted high-SCS ewes and confirmed a high level of adipose tissue mobilization in this group. A second analysis, which included RNA-seq data, revealed high correlations between the long-chain fatty acid yields in milk and PDK4, CPT1A, SLC25A20, KLF10, and KLF11 expression, highlighting the relationship between mobilization of body reserves and enhanced fatty acids utilization for energy production in blood cells. Finally, analysis of milk composition measured in 1,025 ewes from the 2 genetic lines over 10 yr confirmed significant higher fat-to-protein ratio in high-SCS ewes in early lactation. Altogether, our results strongly confirmed a genetic link between susceptibility to mastitis and metabolic adaptation to energy shortage. Improving genetic resistance to mastitis using SCS should be accompanied by a favorable effect on the response to metabolic stress, especially in highly stressful early lactation. Moreover, this study suggests that the fat-to-protein ratio could be used as a low-cost tool for monitoring energy balance and ketosis during this critical phase of lactation.

Effect of dietary arachidonic acid levels on growth performance, fatty acid profiles and lipid metabolism of juvenile yellow catfish (Pelteobagrus fulvidraco)

An 8-week feeding trial was conducted to evaluate the effects of different dietary arachidonic acid (ARA) levels on growth performance, fatty acid composition, hepatic intermediary metabolism and antioxidant responses for juvenile yellow catfish. An ARA-enriched oil was supplemented into the basal diet to formulate six iso-nitrogenous and iso-energetic practical diets containing 0.03% (the control diet), 0.30%, 0.60%, 0.90%, 1.20% and 1.50% of dry weight. Dietary ARA levels were 0.39%, 2.47%, 4.96%, 7.49%, 9.79% and 12.64% total fatty acids, respectively. Triplicate groups of 20 fish (average initial weight is about 1.55±0.01g) were fed to apparent satiation twice daily. The results indicated that fish fed the diets containing 4.96% and 7.49% ARA of total fatty acids have higher final body weight and percent weight gain (PWG) than those fed the other diets. Survival, feed conversion ratio, hepatosomatic index and viscerosomatic index were not significantly influenced by the dietary ARA levels. Fatty acid compositions both in liver and muscle reflected closely those of fatty acid profiles in diets, ARA proportion in liver and muscle significantly increased with dietary ARA levels increasing from 0.39% to 12.64% of total fatty acids, while, EPA proportion in liver significantly decreased with increase of dietary ARA levels. Fish fed the control diet had the highest aspartate aminotransferase (AST) and glucose content in serum among all treatments, and fish fed the 9.79% and 12.64% ARA of total fatty acids had higher malondialdehyde (MDA) content in liver than those fed the other diets. However, the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) in liver were not significantly influenced by dietary ARA levels. The relative gene expression of accα, g6pd, 6pgd and evovl5 in liver down-regulated significantly with the dietary ARA levels increasing from 0.39% to 12.64% of total fatty acids. The relative gene expression of atgl and hslb in liver down-regulated significantly with the dietary ARA levels increasing from 0.39% to 7.49% of total fatty acids, and then up-regulated as dietary ARA levels increasing from 9.79% to 12.64%. However, relative gene expression of cpt1a and ppara in liver are down-regulated in ARA supplemental groups. These results suggested that dietary moderate ARA levels (4.96% and 7.49% of total fatty acids) could improve growth performance and antioxidant response, and up-regulate or down-regulate relative expression levels of some lipid metabolism-related gene in liver. Two slope broken-line regression analysis of PWG against dietary ARA levels indicated that optimal dietary ARA requirement for juvenile yellow catfish was 6.45% of total fatty acids.

Baicalin attenuates non-alcoholic steatohepatitis by suppressing key regulators of lipid metabolism, inflammation and fibrosis in mice

AimsBaicalin (BA), an active flavonoid compound originating from the herb of Scutellaria baicalensis Georgi, has been previously shown to exert anti-inflammation and anti-oxidant effects in liver diseases. However, the potential role of BA in the regulation of non-alcoholic steatohepatitis (NASH) remains elusive. In this study, we newly explored the hepatoprotective effects of BA in MCD diet-induced NASH by ameliorating hepatic steatosis, inflammation, fibrosis and apoptosis. Main methodsNASH was induced in mice fed a methionine and choline-deficient (MCD) diet for 4weeks. The mice were simultaneously treated with or without BA for 4weeks. Serum liver functional markers and inflammatory indicators were assessed by biochemical and ELISA methods, respectively. The livers were histologically examined using H&E, Oil Red O and Masson's trichrome staining methods. The qRT-PCR, IHC and Western blotting assays were applied to analyze mechanisms underlying BA protection. Key findingsBA treatment significantly attenuated MCD diet-induced hepatic lipid accumulation partly through regulating the expression of SREBP-1c, FASN, PPARα and CPT1a. BA treatment dramatically suppressed MCD diet-induced hepatic inflammation, which was associated with decrease in serum TNF-α, IL-1β and MCP-1 production, macrophage influx and suppression of nuclear factor-κB activation. Additionally, BA was proved to prevent liver fibrosis, which appears to be mediated by inhibition of α-SMA, TGF-β1 and Col1A1. Furthermore, BA markedly inhibited hepatocyte apoptosis and cleaved caspase-3 protein expression in MCD diet-induced mice. SignificanceThese results provide a possible basis of the underlying mechanism for the application of BA in the treatment of NASH.

Effects of dietary lecithin and l-carnitine on fatty acid composition and lipid-metabolic genes expression in subcutaneous fat and longissimus thoracis of growing-finishing pigs

A 2×2 factorial experiment was conducted to investigate the effects of dietary lecithin and l-carnitine on fatty acid composition and lipid-metabolic genes expression in subcutaneous fat and longissimus thoracis of growing-finishing pigs. 160 barrows were assigned to 4 treatments consisting of 8 replicates with 5 pigs in each. The total PUFA, C18:2n-6 and C18:3n-3 in subcutaneous fat were increased by lecithin but the effect of lecithin was dependent of l-carnitine where supplementation of lecithin together with l-carnitine decreased total PUFA, C18:2n-6 and C18:3n-3. l-Carnitine increased the intramuscular fat content when supplemented with lecithin but no effect was observed without lecithin supplementation. l-Carnitine increased the mRNA expression of CPT1A, HSL, FABP4 and CRAT; and reduced the mRNA expression of FAS and ACCα in subcutaneous fat. Lecithin increased the mRNA expression of ACCα and ME1 in longissimus thoracis. l-Carnitine increased the mRNA expression of FAS in longissimus thoracis when supplemented with lecithin but no effect was observed without lecithin supplementation.

Dendrobium nobile Lindl. alkaloids regulate metabolism gene expression in livers of mice.

In our previous studies, Dendrobium nobile Lindl. alkaloids (DNLA) has been shown to have glucose-lowering and antihyperlipidaemia effects in diabetic rats, in rats fed with high-fat diets, and in mice challenged with adrenaline. This study aimed to examine the effects of DNLA on the expression of glucose and lipid metabolism genes in livers of mice. Mice were given DNLA at doses of 10-80mg/kg, po for 8days, and livers were removed for total RNA and protein isolation to perform real-time RT-PCR and Western blot analysis. Dendrobium nobile Lindl. alkaloids increased PGC1α at mRNA and protein levels and increased glucose metabolism gene Glut2 and FoxO1 expression. DNLA also increased the expression of fatty acid β-oxidation genes Acox1 and Cpt1a. The lipid synthesis regulator Srebp1 (sterol regulatory element-binding protein-1) was decreased, while the lipolysis gene ATGL was increased. Interestingly, DNLA increased the expression of antioxidant gene metallothionein-1 and NADPH quinone oxidoreductase-1 (Nqo1) in livers of mice. Western blot on selected proteins confirmed these changes including the increased expression of GLUT4 and PPARα. DNLA has beneficial effects on liver glucose and lipid metabolism gene expressions, and enhances the Nrf2-antioxidant pathway gene expressions, which could play integrated roles in regulating metabolic disorders.

Mitochondrial Priming by CD28

T cell receptor (TCR) signaling without CD28 can elicit primary effector Tcells, but memory Tcells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon Tcell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for Tcells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory Tcells. Ourdata show that initial CD28 signals during Tcell activation prime mitochondria with latent metabolic capacity that is essential for future Tcell responses.

Maresin 1 mitigates liver steatosis in ob/ob and diet-induced obese mice.

The aim of this study was to characterize the effects of Maresin 1 (MaR1) in obesity-related liver steatosis and the mechanisms involved. MaR1 effects on fatty liver disease were tested in ob/ob (2-10 μg kg-1 i.p., 20 days) and in diet-induced obese (DIO) mice (2 μg kg-1, i.p., or 50 μg kg-1, oral gavage for 10 days), as well as in cultured hepatocytes. In ob/ob mice, MaR1 reduced liver triglycerides (TG) content, fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 protein expression, while increased acetyl-CoA carboxylase (ACC) phosphorylation and LC3II protein expression, in parallel with a drop in p62 levels. Similar effects on hepatic TG, ACC phosphorylation, p62 and LC3II were observed in DIO mice after MaR1 i.p. injection. Interestingly, oral gavage of MaR1 also decreased serum transaminases, reduced liver weight and TG content. MaR1-treated mice exhibited reduced hepatic lipogenic enzymes content (FAS) or activation (by phosphorylation of ACC), accompanied by upregulation of carnitine palmitoyltransferase (Cpt1a), acyl-coenzyme A oxidase (Acox1) and autophagy-related proteins 5 and 7 (Atg5-7) gene expression, along with increased number of autophagic vacuoles and reduced p62 protein levels. MaR1 also induced AMP-activated protein kinase (AMPK) phosphorylation in DIO mice and in primary hepatocytes, and AMPK inhibition completely blocked MaR1 effects on Cpt1a, Acox1, Atg5 and Atg7 expression. MaR1 ameliorates liver steatosis by decreasing lipogenic enzymes, while inducing fatty acid oxidation genes and autophagy, which could be related to AMPK activation. Thus, MaR1 may be a new therapeutic candidate for reducing fatty liver in obesity.

Deregulation of MicroRNAs mediated control of carnitine cycle in prostate cancer: molecular basis and pathophysiological consequences.

Cancer cells reprogram their metabolism to maintain both viability and uncontrolled proliferation. Although an interplay between the genetic, epigenetic and metabolic rewiring in cancer is beginning to emerge, it remains unclear how this metabolic plasticity occurs. Here, we report that in prostate cancer cells (PCCs) microRNAs (miRNAs) greatly contribute to deregulation of mitochondrial fatty acid (FA) oxidation via carnitine system modulation. We provide evidence that the downregulation of hsa-miR-124-3p, hsa-miR-129-5p and hsa-miR-378 induced an increase in both expression and activity of CPT1A, CACT and CrAT in malignant prostate cells. Moreover, the analysis of human prostate cancer and prostate control specimens confirmed the aberrant expression of miR-124-3p, miR-129-5p and miR-378 in primary tumors. Forced expression of the miRNAs mentioned above affected tumorigenic properties, such as proliferation, migration and invasion, in PC3 and LNCaP cells regardless of their hormone sensitivity. CPT1A, CACT and CrAT overexpression allow PCCs to be more prone on FA utilization than normal prostate cells, also in the presence of high pyruvate concentration. Finally, the simultaneous increase of CPT1A, CACT and CrAT is fundamental for PCCs to sustain FA oxidation in the presence of heavy lipid load on prostate cancer mitochondria. Indeed, the downregulation of only one of these proteins reduces PCCs metabolic flexibility with the accumulation of FA-intermediate metabolites in the mitochondria. Together, our data implicate carnitine cycle as a primary regulator of adaptive metabolic reprogramming in PCCs and suggest new potential druggable pathways for prevention and treatment of prostate cancer.

Attenuation of hepatic steatosis by purple sweet potato colour is associated with blocking Src/ERK/C/EBPβ signalling in high-fat-diet-treated mice.

Our previous work showed that purple sweet potato colour (PSPC), a class of naturally occurring anthocyanins, effectively improved hepatic glucose metabolic dysfunction in high-fat-diet (HFD)-treated mice. This study investigated the effects of PSPC on HFD-induced hepatic steatosis and the signalling events associated with these effects. Mice were divided into 4 groups: control group, HFD group, HFD+PSPC group, and PSPC group. PSPC was administered daily for 20 weeks at oral doses of 700 mg/(kg·day)-1). Our results showed that PSPC significantly improved obesity and related metabolic parameters, as well as liver injury in HFD-treated mice. Moreover, PSPC dramatically attenuated hepatic steatosis in HFD-treated mice. PSPC markedly prevented oxidative stress-mediated Src activation in HFD-treated mouse livers. Furthermore, PSPC feeding remarkably suppressed mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/ERK) signalling and consequent CCAAT/enhancer binding protein β (C/EBPβ) activation and restored AMPK activation in HFD-treated mouse livers, which was confirmed by U0126 treatment. Ultimately, PSPC feeding dramatically reduced protein expression of FAS and CD36 and the activation of ACC, and increased the protein expression of CPT1A in the livers of HFD-treated mice, indicating decreased lipogenesis and fatty acid uptake and enhanced fatty acid oxidation. In conclusion, PSPC exhibited beneficial effects on hepatic steatosis, which were associated with blocking Src and C/EBPβ activation.

Adiponectin regulates AQP3 via PPARα in human hepatic stellate cells

Aquaporins (AQPs) are trans-membrane proteins which allow the movement of water and glycerol required by hepatic stellate cells (HSC) for triglyceride formation and lipid storage. Adiponectin (ADPQ) is a hormone produced by the adipose tissue, which is known to increase AQP3 expression. Since ADPQ receptor signals via the nuclear receptor PPAR we aimed to explore the role of this pathway in AQP3 regulation by ADPQ in HSC. AQP3 and CPT1α expression increased only after ADPQ but not rosiglitazone stimulation. In LX2 cells co-transfected with plasmids expressing PPARα or PPARγ coupled to a luciferase reporter gene, only PPARα increased luciferase activity after ADPQ stimulation. Collectively, our findings demonstrate that ADPQ increases AQP3 expression through PPARα-mediated signaling in HSC.

Xyloketal B Attenuates Fatty Acid-Induced Lipid Accumulation via the SREBP-1c Pathway in NAFLD Models.

The goal of this study was to examine the effects of xyloketal B on nonalcoholic fatty liver disease (NAFLD) and to explore the molecular mechanisms underlying its effects in both in vivo and in vitro models. We discovered an association between xyloketal B and the sterol regulatory element-binding protein-1c (SREBP-1c) signaling pathway, which is related to lipid metabolism. Mice were dosed with xyloketal B (5, 10 and 20 mg/kg/d) and atorvastatin (15 mg/kg/d) via intraperitoneal injection once daily for 40 days after being fed a high fat diet plus 10% high fructose liquid (HFD+HFL) for 8 weeks. Xyloketal B significantly improved HFD+HFL-induced hepatic histological lesions and attenuated lipid and glucose accumulation in the blood as well as lipid accumulation in the liver. Xyloketal B increased the expression of CPT1A, and decreased the expression of SREBP-1c and its downstream targeting enzymes such as ACC1, ACL, and FAS. Xyloketal B also significantly reduced lipid accumulation in HepG2 cells treated with free fatty acids (FFAs). These data suggested that xyloketal B has lipid-lowering effects via the SREBP-1c pathway that regulate lipid metabolism. Thus, targeting SREBP-1c activation with xyloketal B may be a promising novel approach for NAFLD treatment.

5-ALA ameliorates hepatic steatosis through AMPK signaling pathway.

5-Aminolevulinic acid (5-ALA), the first compound in the porphyrin synthesis pathway, has been reported to ameliorate the diabetic state in Otsuka Long-Evans Tokushima Fatty rats by reducing fat pad weight in the retroperitoneal region. Dietary supplementation with 5-ALA has additionally demonstrated the capacity to lower blood glucose and HbA1c levels among subjects with diabetes. The etiology of nonalcoholic fatty liver disease (NAFLD) is complex and its typical characteristics include obesity and insulin resistance. As 5-ALA supplementation has previously normalized glucose and insulin resistance, we sought to investigate whether 5-ALA had potential therapeutic effects on NAFLD and elucidate the signal pathway mediating these effects. To explore these questions, we fed C57BL/6J mice a high-fat diet (HFD) to induce a fatty liver disease and supplemented the diet-induced obese (DIO) mice with 5-ALA. The mice in the presence of 5-ALA demonstrated a decrease in body weight and hepatic lipid content and moderate improvement in glucose homeostasis compared to untreated controls. Further, we found that 5-ALA activated AMPK signaling pathway, which was correlated with enhanced lipolysis and fatty acid β-oxidation. Human hepatocarcinoma cells (HepG2 cells) treated with 5-ALA were additionally used to investigate the mechanics of 5-ALA. Treated cells had a higher expression of lipolysis-related genes, including PGC-1α. Our data indicated that 5-ALA might represent a novel compound that could be useful for the treatment of nonalcoholic fatty liver disease (NAFLD), likely through the restoration of phosphorylation levels of AMPK (Thr172) and acetyl-CoA (ACC) (Ser79), further enhanced PGC1α and CPT1α expression.

Glucagon-induced extracellular cAMP regulates hepatic lipid metabolism.

Hormonal signals help to maintain glucose and lipid homeostasis in the liver during the periods of fasting. Glucagon, a pancreas-derived hormone induced by fasting, promotes gluconeogenesis through induction of intracellular cAMP production. Glucagon also stimulates hepatic fatty acid oxidation but the underlying mechanism is poorly characterized. Here we report that following the acute induction of gluconeogenic genes Glucose 6 phosphatase (G6Pase) and Phosphoenolpyruvate carboxykinase (Pepck) expression through cAMP-response element-binding protein (CREB), glucagon triggers a second delayed phase of fatty acid oxidation genes Acyl-coenzyme A oxidase (Aox) and Carnitine palmitoyltransferase 1a (Cpt1a) expression via extracellular cAMP. Increase in extracellular cAMP promotes PPARα activity through direct phosphorylation by AMP-activated protein kinase (AMPK), while inhibition of cAMP efflux greatly attenuates Aox and Cpt1a expression. Importantly, cAMP injection improves lipid homeostasis in fasted mice and obese mice, while inhibition of cAMP efflux deteriorates hepatic steatosis in fasted mice. Collectively, our results demonstrate the vital role of glucagon-stimulated extracellular cAMP in the regulation of hepatic lipid metabolism through AMPK-mediated PPARα activation. Therefore, strategies to improve cAMP efflux could serve as potential new tools to prevent obesity-associated hepatic steatosis.

Lipid catabolism inhibition sensitizes prostate cancer cells to antiandrogen blockade.

Prostate cancer (PCa) is the most common malignancy among Western men and the second leading-cause of cancer related deaths. For men who develop metastatic castration resistant PCa (mCRPC), survival is limited, making the identification of novel therapies for mCRPC critical. We have found that deficient lipid oxidation via carnitine palmitoyltransferase (CPT1) results in decreased growth and invasion, underscoring the role of lipid oxidation to fuel PCa growth. Using immunohistochemistry we have found that the CPT1A isoform is abundant in PCa compared to benign tissue (n=39, p<0.001) especially in those with high-grade tumors. Since lipid oxidation is stimulated by androgens, we have evaluated the synergistic effects of combining CPT1A inhibition and anti-androgen therapy. Mechanistically, we have found that decreased CPT1A expression is associated with decreased AKT content and activation, likely driven by a breakdown of membrane phospholipids and activation of the INPP5K phosphatase. This results in increased androgen receptor (AR) action and increased sensitivity to the anti-androgen enzalutamide. To better understand the clinical implications of these findings, we have evaluated fat oxidation inhibitors (etomoxir, ranolazine and perhexiline) in combination with enzalutamide in PCa cell models. We have observed a robust growth inhibitory effect of the combinations, including in enzalutamide-resistant cells and mouse TRAMPC1 cells, a more neuroendocrine PCa model. Lastly, using a xenograft mouse model, we have observed decreased tumor growth with a systemic combination treatment of enzalutamide and ranolazine. In conclusion, our results show that improved anti-cancer efficacy can be achieved by co-targeting the AR axis and fat oxidation via CPT1A, which may have clinical implications, especially in the mCRPC setting.

Anti-steatotic and anti-inflammatory effects of Hovenia dulcis Thunb. extracts in chronic alcohol-fed rats

The anti-steatotic and anti-inflammatory effects of fruit water extract (FW) and seed ethanol extract (SE) of Hovenia dulcis Thunb. in chronic alcohol-fed rats were investigated. Rats were fed a liquid diet containing 36% calories from alcohol and orally administered FW or SE (300 and 500mg/kg/day). Both FW and SE reduced hepatic lipid contents and droplets, serum lipid concentration and inflammatory markers (hs-CRP, TNF-α and IL-6) levels compared with the alcohol control group. Alcohol led to significant decreases in the hepatic fatty acid oxidative gene (Ppargc1a, Cpt1a and Acsl1) levels, while it significantly increased the Myd88 and Tnfa gene levels. However, FW or SE supplementation significantly up-regulated gene expression of Ppargc1a, Ppara, Cpt1a and Acsl1, and down-regulated gene expression of Myd88, Tnfa and Crp compared with the alcohol control group. FW or SE supplementation also significantly decreased hepatic activities of fatty acid synthase and phosphatidate phosphohydrolase in chronic alcohol-fed rats. Plasma alcohol and acetaldehyde levels, hepatic enzyme activity and protein expression of CYP2E1 were lowered by FW or SE supplementation. These results indicate that both FW and SE play an important role in improvement of alcoholic hepatic steatosis and inflammation via regulation of lipid and inflammation metabolism.

Hepatic Fat Accumulation Regulates Carnitine Palmitoyltransferase 1 (Cpt1a) Expression through Coordinated Epigenetic Mechanisms

Carnitine palmitoyltransferase 1 (Cpt1a) is a rate‐limiting enzyme that mediates the transport of fatty acids into the mitochondria for subsequent beta oxidation. Cpt1a is highly regulated at the transcriptional, translational, and substrate level. Epigenetic mechanisms regulating Cpt1a expression were investigated in the liver of rats fed a high fat diet. Hepatic Cpt1a mRNA was increased in response to high fat. High fat diet reduced DNA methylation and increased histone 3 lysine residue 4 dimethylation (H3K4Me2) both upstream of and within the promoter region of Cpt1a. This was accompanied by increased binding of C/EBPβ directly downstream of the Cpt1a transcription start site within the first intron. In addition to the transcriptional regulation of Cpt1a, expression of miR‐499, a predicted repressor of both Cpt1a and C/EBPβ, was also reduced. Treatment of rat hepatoma H4IIEC3 cells with non‐esterified fatty acid (NEFA) confirmed the correlation between the upregulation of Cpt1a gene expression and cellular fat accumulation. While H4IIEC3 cells transfected with non‐specific microRNA upregulated Cpt1a expression at high NEFA concentrations, exogenous overexpression of miR‐499 cells abolished this increase, resulting in suppressed beta oxidation and elevated lipid accumulation. We conclude that hepatic fat increases Cpt1a expression through a highly coordinated epigenetic mechanism including histone tail modifications and reduction of DNA methylation at specific chromatin locations, C/EBPβ transcription factor recruitment, as well as post‐transcriptional regulation by miR‐499. The spatial and temporal coordination of these epigenetic events at the Cpt1a locus will be further investigated to characterize gene regulation in fat metabolism.

High fat and high sucrose diets in mice have independent effects on osteoarthritis pathology and chondrocyte metabolic stress

Purpose: Obesity is a primary risk factor for knee osteoarthritis (OA). However, how chondrocyte metabolism and pro-catabolic mediators change in response to excess consumption of specific macronutrients, such as fat and sugar, are not fully understood. The aim of this study was to better understand how chondrocyte metabolism changes in response to a high-fat diet (HFD) and a low-fat high-sucrose diet (LFHS), and how these changes may be associated with OA pathogenesis in mice. We hypothesized that high-fat and high-sucrose feeding would reduce cartilage proteoglycan content by inducing cellular metabolic stresses and expression of matrix proteases. To test this hypothesis, we examined OA pathology and transcriptional changes in a panel of chondrocyte OA mediators, intracellular stress mediators, and metabolic markers following 20 weeks on HFD, LFHS, or matched control low-fat low-sucrose (LFLS) diets. Methods: Male C57BL/6J mice (n = 8 per diet) were fed either HFD (60% kcal fat, 7% sucrose), LFHS (10% kcal fat, 70% kcal carbohydrate [35% sucrose]), or LFLS (10% kcal fat, 70% kcal carbohydrate [7% sucrose]) from Research Diets for 20 weeks beginning at 6 weeks of age. Knee OA histopathology was examined using a modified Mankin OA scoring system and OARSI scoring system in the left knee. mRNA and protein were isolated from right knee articular cartilage to examine chondrocyte transcriptional and protein expression changes. Gene expression changes were determined using custom qPCR arrays containing a range of metabolism and mitochondrial genes, while protein expression is being determined using selected reaction monitoring (SRM) proteomics. Results: There was no difference in the OARSI OA score among the diet groups. However, modified Mankin OA score was moderately but significantly increased in LFLS compared to HFD and LFHS groups (Fig. 1) due to greater loss of Safranin-O staining. Other sub-component scores differed among groups, with the hypertrophic chondrocyte score being lower in the LFLS group compared to HFD and LFHS and the osteophyte and tidemark duplication scores being greater in the HFD group compared to LFHS and LFLS. mRNA expression of extracellular matrix proteins and extracellular matrix proteases (Aggrecan, Col2, Col10, Adamts5, Mmp9, Mmp13) were not different between groups. Of the metabolic and cellular stress genes examined, there were multiple diet specific differences (Fig. 2). Notably, Cpt1a was significantly greater in the HFD compared to LFHS and LFLS. However, Foxo3, Sod1, and Sod2 were greater in LFHS and HFD compared to LFLS. Ampk was greater in LFLS compared to LFHS, but not HFD, with no difference between LFHS and HFD. Conclusions: Both dietary fat and insulin resistance increase the risk of OA. Therefore, we hypothesized that HFD and LFHS diets would induce OA related histological changes. Further, we hypothesized that 20 weeks of HFD and LFHS diets would induce gene expression changes in OA mediators, cellular stress mediators, and metabolic markers mirroring the histological changes. Surprisingly, the modified Mankin OA score was highest in the LFLS group, primarily due to reduced Safranin-O staining. However, the HFD and LFHS groups were also independently associated with other features of OA histopathology. These findings suggest that the varied macronutrient compositions of pro-obesogenic and control diets have independent effects on joint homeostasis. Although diet composition did not alter the expression of anabolic or catabolic extracellular matrix genes, dietary macronutrients uniquely altered the expression of metabolic and stress-response genes in cartilage. For example, HFD increased expression of Cpt1a, the mitochondrial fatty acid transporter that is the rate-limiting step in fatty acid metabolism, suggesting that a HFD increases the uptake and utilization of fatty acids in chondrocytes. In contrast, high sucrose content in carbohydrate-matched diets reduced Ampk expression, supporting the hypothesis that fat and carbohydrate macronutrients have specific effects on chondrocyte phenotypes. In conclusion, chondrocytes appear metabolically flexible in response to high-fat and high-sucrose diets in independent manners related to OA pathology. These findings illustrate the importance of considering dietary macronutrient composition when designing studies and interpreting the effect of diet-induced obesity on knee OA in mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Examination of carnitine palmitoyltransferase 1 abundance in white adipose tissue: implications in obesity research.

Carnitine palmitoyltransferase 1 (CPT1) is essential for the transport of long-chain fatty acids into the mitochondria for oxidation. Recently, it was reported that decreased CPT1b mRNA in adipose tissue was a contributing factor for obesity in rats. We therefore closely examined the expression level of Cpt1 in adipose tissue from mice, rats, and humans. Cpt1a is the predominate isoform in adipose tissue from all three species. Rat white adipose tissue has a moderate amount of Cpt1b mRNA, but it is very minor compared with Cpt1b expression in muscle. Total CPT1 activity in adipose tissue is also minor relative to other tissues. Both Cpt1a and Cpt1b mRNA were increased in gonadal fat but not inguinal fat by diet-induced obesity in mice. We also measured CPT1a and CPT1b expression in subcutaneous adipose tissue from human subjects with a wide range of body mass indexes (BMIs). Interestingly, CPT1a expression positively correlated with BMI (R = 0.46), but there was no correlation with CPT1b (R = 0.04). Our findings indicate that white adipose tissue fatty acid oxidation capacity is minor compared with that of metabolically active tissues. Furthermore, given the already low abundance of Cpt1b in white adipose tissue, it is unlikely that decreases in its expression can quantitatively decrease whole body energy expenditure enough to contribute to an obese phenotype.

Regulation of growth, antioxidant capacity, fatty acid profiles, hematological characteristics and expression of lipid related genes by different dietary n-3 highly unsaturated fatty acids in juvenile black seabream (Acanthopagrus schlegelii)

An 8-week feeding trial was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acid (n-3HUFA) on growth performance, antioxidant capacity, fatty acid profiles, hematological characteristics and expression of some lipid related genes of juvenile black seabream (Acanthopagrus schlegelii) (initial weight 3.77±0.00g). Five isonitrogenous and isolipidic experimental diets were formulated with graded levels of n-3 HUFA (0.23, 0.87, 1.29, 1.75 and 2.53% of dry weight, DHA/EPA ratio approximately at 1.0). The results revealed that fish fed the diet containing 1.75% n-3 HUFA had higher weight gain (WG) and specific growth rate (SGR) than those fed the control diet. Fish fed diets containing 1.29% and 1.75% n-3 HUFA had higher feed efficiency (FE) than those fed the other diets. Hepatic and muscular fatty acid profiles reflected that of diets. The content of malondialdehyde (MDA) increased both in the serum and liver of fish fed high n-3 HUFA level diets, and the highest hepatic activity of glutathione peroxidase (GSH-PX) was recorded in fish fed the diet containing 1.29% n-3 HUFA. The expression of accα, g6pd, fas, srebp-1, lpl, atgl, hsl, elovl5 and fads2 was down-regulated in fish fed the diets with high n-3 HUFA levels. However, the expression levels of 6pgd and pparα significantly increased when the dietary contents of n-3 HUFA increased from 0.23% to 1.29% and then decreased when dietary n-3 HUFA levels increased from 1.75% to 2.53%. The highest expression of cpt1a was found in fish fed the diet containing 1.75% n-3 HUFA. The content of cholesterol (CHOL) in serum increased along with n-3 HUFA level. Over all, this study indicated that fish fed moderate dietary n-3 HUFA (1.34–1.80% n-3 HUFA) could enhance growth, feed utilization and antioxidant capacity. Different dietary levels of n-3 HUFA could strongly affect expression levels of some lipid metabolism relevant genes of the juvenile black seabream. This may contribute to our understanding of the mechanisms related to lipid metabolism (anabolism and catabolism) effects of different dietary levels of n-3 HUFA.

Combination curcumin and vitamin E treatment attenuates diet-induced steatosis in Hfe-/- mice.

AIMTo investigate the synergistic hepato-protective properties of curcumin and vitamin E in an Hfe-/- high calorie diet model of steatohepatitis.METHODSHfe-/- C57BL/6J mice were fed either a high calorie diet or a high calorie diet with 1 mg/g curcumin; 1.5 mg/g vitamin E; or combination of 1 mg/g curcumin + 1.5 mg/g vitamin E for 20 wk. Serum and liver tissue were collected at the completion of the experiment. Liver histology was graded by a pathologist for steatosis, inflammation and fibrosis. RNA and protein was extracted from liver tissue to examine gene and protein expression associated with fatty acid oxidation, mitochondrial biogenesis and oxidative stress pathways.RESULTSHfe-/- mice fed the high calorie diet developed steatohepatitis and pericentral fibrosis. Combination treatment with curcumin and vitamin E resulted in a greater reduction of percent steatosis than either vitamin E or curcumin therapy alone. Serum alanine aminotransferase and non-alcoholic fatty liver disease (NAFLD) activity score were decreased following combination therapy with curcumin and vitamin E compared with high calorie diet alone. No changes were observed in inflammatory or fibrosis markers following treatment. Epididymal fat pad weights were significantly reduced following combination therapy, however total body weight and liver weight were unchanged. Combination therapy increased the mRNA expression of AdipoR2, Ppar-α, Cpt1a, Nrf-1 and Tfb2m suggesting enhanced fatty acid oxidation and mitochondrial biogenesis. In addition, combination treatment resulted in increased catalase activity in Hfe-/- mice.CONCLUSIONCombination curcumin and vitamin E treatment decreases liver injury in this steatohepatitis model, indicating that combination therapy may be of value in NAFLD.