Fatty Acid Oxidation Activity Research Articles

The implications of substituting dietary fish oil with vegetable oils on the growth performance, fillet fatty acid profile and modulation of the fatty acid elongase, desaturase and oxidation activities of red hybrid tilapia, Oreochromis sp.

A 75-day feeding trial was carried out to examine the effects of fish oil (FO) replacement with various alternative vegetable oil (VO) on the growth performance and feed utilization efficiency of red hybrid tilapia (Oreochromis sp.). The VO evaluated included canola oil (high in monoenes), perilla oil (high in n−3 polyunsaturated fatty acids, PUFA), sunflower oil (high in n−6 PUFA) and refined, bleached, deodorized palm olein (high in saturates) added as the sole lipid source in semi-purified experimental diets. The ability of hybrid tilapia to biosynthesize long chain-polyunsaturated fatty acids (LC-PUFA) from dietary 18:2n−6 and 18:3n−3 was also determined. With the exception of sunflower oil, results showed that total FO replacement by various VO did not compromise (P>0.05) tilapia growth performance. However, dietary VO significantly (P<0.05) influenced the fatty acid composition of tilapia fillet and whole-body lipids through changes in the overall fatty acid metabolism. The total Δ5 and Δ6 fatty acid desaturase activities were exclusively triggered in tilapia fed VO-based diets. Tilapia fed the VO diets, which contained no PUFA longer than C18, recorded significant amounts of both n−6 and n−3 LC-PUFA in their tissue lipids. Dietary canola oil (CO) increased Δ-6 desaturation of 18:3n−3 to the greatest extent despite the dietary 18:3n−3 content being comparatively lower than dietary perilla oil (PeO). Beta-oxidation of 18:3n−3 was observed to be highest in fish fed the PeO diet, indicating that the 18:3n−3 content in the PeO diet exceeded the optimum substrate level for Δ-6 desaturase. Omega-3 LC-PUFA biosynthetic activities were highest in fish fed the CO diet and resulted in a comparatively higher eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA) content than in fish fed other VO diets. However, endogenous LC-PUFA synthesis induced by the VO diets was insufficient to rival the n−3 LC-PUFA content of fish fed the FO diet. It was also observed that β-oxidation of 18:3n−3 and 18:2n−6 was regulated by tissue n−3/n−6 PUFA ratio and proportional to their dietary level. This study provided the first comprehensive evidence of the significant impact of dietary oils with different fatty acid class profile on the in vivo fatty acid metabolism of tilapia. Statement of relevanceMarine fish oil (FO) was traditionally used as the major lipid source in commercial tilapia feeds to provide energy and essential fatty acids as well as to impart palability to the feed. With the rising costs of FO and limited global supplies, the use of alternative lipid resources in aquafeeds, including tilapia feeds, has become more critical to enable the continued sustainability and scalability of the global aquaculture industry. Terrestrial vegetable oil (VO) are viable alternatives since their production is more sustainable and cost-effective. However, unlike FO, which is rich in n−3 long chain-polyunsaturated fatty acids (LC-PUFA), VO are deficient in these essential fatty acids and but high in other fatty acid classes. The present study evaluated perilla oil (high levels of n−3 polyunsaturated fatty acids), sunflower oil (rich in n−6 PUFA), canola oil (high amounts of monounsaturated fatty acids) and palm oil (rich in saturated fatty acids) together with fish oil. By understanding how dietary lipid source with different major fatty acid classes modulates in vivo fatty acid metabolism, we can better formulate fish feeds that can optimize not only growth performance but also maintaining the health benefits of eating seafood for the human consumer in regards to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels in farmed fish fillets. As far as we know, there is currently no information comparing the fatty acid metabolism of red hybrid tilapia fed diets with lipids containing different major fatty acid classes.

Gemigliptin ameliorates Western-diet-induced metabolic syndrome in mice.

Dipeptidyl peptidase 4 (DPP-4) inhibitors are widely used antihyperglycemic agents for type 2 diabetes mellitus. Recently, increasing attention has been focused on the pleiotropic actions of DPP-4 inhibitors. The aim of the present study was to examine whether gemigliptin, a recently developed DPP-4 inhibitor, could ameliorate features of metabolic syndrome. Mice were fed a Western diet (WD) for 12 weeks and were subsequently divided into 2 groups: mice fed a WD diet alone or mice fed a WD diet supplemented with gemigliptin for an additional 4 weeks. Gemigliptin treatment attenuated WD-induced body mass gain, hypercholesterolemia, adipocyte hypertrophy, and macrophage infiltration into adipose tissue, which were accompanied by an increased expression of uncoupling protein 1 in subcutaneous fat. These events contributed to improved insulin sensitivity, as assessed by the homeostasis model assessment of insulin resistance and intraperitoneal insulin tolerance test. Furthermore, gemigliptin reduced WD-induced hepatic triglyceride accumulation via inhibition of de novo lipogenesis and activation of fatty acid oxidation, which was accompanied by AMP-dependent protein kinase activation. Gemigliptin ameliorated WD-induced hepatic inflammation and fibrosis through suppression of oxidative stress. These results suggest that DPP-4 inhibitors may represent promising therapeutic agents for metabolic syndrome beyond their current role as antihyperglycemic agents.

Mitochondrial and Metabolic Gene Expression in the Aged Rat Heart.

Aging is associated with a decline in cardiac function. Exercise intervention has been suggested as a way to improve this decrement. Age-related decline in cardiac function is associated with decreases in fatty acid oxidation, mitochondrial function, and AMP-activated protein kinase (AMPK) activity. The molecular mechanisms involved with age-related changes in mitochondrial function and substrate metabolism are poorly understood. We determined gene expression differences in hearts of Young (6 mo), Old (33 mo), and old exercise trained (Old + EXE) (34 mo) FBN rats, using Qiagen PCR arrays for Glucose, Fatty acid, and Mitochondrial metabolism. Old rats demonstrated decreased (p < 0.05) expression for key genes in fatty acid oxidation, mitochondrial function, and AMPK signaling. There were no differences in the expression of genes involved in glucose metabolism with age. These gene expression changes occurred prior to altered protein translation as we found no differences in the protein content of peroxisome proliferator activated receptor gamma, coactivators 1 alpha (PGC-1α), peroxisome proliferator activated receptor alpha (PPARα), and AMPKα2 between young and old hearts. Four months of exercise training did not attenuate the decline in the gene expression in aged hearts. Despite this lack of change in gene expression, exercise-trained rats demonstrated increased exercise capacity compared to their sedentary counterparts. Taken together, our results show that differential expression of genes associated with fatty acid metabolism, AMPK signaling and mitochondrial function decrease in the aging heart which may play a role in age-related declines in fatty acid oxidation, AMPK activity, and mitochondrial function in the heart.

FNDC5 Alleviates Hepatosteatosis by Restoring AMPK/mTOR-Mediated Autophagy, Fatty Acid Oxidation, and Lipogenesis in Mice.

Fibronectin type III domain-containing 5 (FNDC5) protein induces browning of subcutaneous fat and mediates the beneficial effects of exercise on metabolism. However, whether FNDC5 is associated with hepatic steatosis, autophagy, fatty acid oxidation (FAO), and lipogenesis remains unknown. Herein, we show the roles and mechanisms of FNDC5 in hepatic steatosis, autophagy, and lipid metabolism. Fasted FNDC5-/- mice exhibited severe steatosis, reduced autophagy, and FAO, and enhanced lipogenesis in the liver compared with wild-type mice. Energy deprivation-induced autophagy, FAO, and AMPK activity were attenuated in FNDC5-/- hepatocytes, which were restored by activating AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Inhibition of mammalian target of rapamycin (mTOR) complex 1 with rapamycin enhanced autophagy and FAO and attenuated lipogenesis and steatosis in FNDC5-/- livers. FNDC5 deficiency exacerbated hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. Exogenous FNDC5 stimulated autophagy and FAO gene expression in hepatocytes and repaired the attenuated autophagy and palmitate-induced steatosis in FNDC5-/- hepatocytes. FNDC5 overexpression prevented hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. These results indicate that FNDC5 deficiency impairs autophagy and FAO and enhances lipogenesis via the AMPK/mTOR pathway. FNDC5 deficiency aggravates whereas FNDC5 overexpression prevents the HFD-induced hyperlipemia, hepatic lipid accumulation, and impaired FAO and autophagy in the liver.

Abstract 4657: Selective inhibitors epigenetically modify and eradicate tumor-initiating stem-like cells through downregulating microRNA 22-mediated TET induction and apoptosis

Abstract Tumor-initiating stem-like cells (TICs) are a minor population in bulk tumors that play a critical role in tumor recurrence and therapy-resistance. We previously showed that a key stemness marker Nanog is upregulated that regulates self-renewal and pluripotency of embryonic stem cells and TICs. We demonstrated that alcohol-mediated activation of Toll-like receptor 4 (TLR4) by endotoxin resulted in increased expression of pluripotency stem cell transcription factor NANOG that metabolically reprograms tumor-initiating stem-like cells (TICs: cancer stem cells) via inhibition of oxidative phosphorylation and activation of fatty acid oxidation in our recently accepted Cell Metabolism paper. Discovery of a drug that specifically targets TICs would be a vital goal for cancer therapy. To identify selective TIC inhibitors, we conducted a high throughput screening of an FDA-approved drug library using combination of Nanog promoter-GFP-based screening with viability-based screening and their combination screening of each hit compound. Our high-throughput screening identified the best combination of repurposed FDA-approved drugs: all-trans retinoic acid (ATRA) and the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) that repressed NANOG and induces apoptosis of TICs. Treatment of ATRA inhibits self-renewal ability in vitro. To specifically target the CD133 (+) population, we encapsulated ATRA into nanoparticles conjugated with CD133 antibody using biodegradable poly(D,L-lactide-co-glycolide) (PLGA) polymer. The combination treatment significantly inhibited tumor growth compared to single drug treatments while the ATRA or SAHA monotherapy groups did not reduce or even promoted tumor growth. The combination of ATRA with SAHA specifically induces apoptosis of TICs. Targeting of TICs by NANOG antagonism increases drug susceptibility in tumor-bearing mice and achieves ∼90% tumor growth suppression. RNA-seq analysis identified that ATRA+SAHA treatment reduced expression of MicroRNA-22 (miR-22), leading to induction of PTEN and ten-eleven translocation enzymes (TET2), which demethylates DNA. PTEN induction promoted P-FOXO3A and induced BIM, leading to induction of apoptosis. The combined treatment Induced TET1/2 to demethylate p53-binding sites of NANOG promoter, leading to downregulation of NANOG. Taken together, combination treatment of ATRA with SAHA may serve as a novel avenue for HCC treatment. Novel combination of repurposed drugs is cost-effective therapeutic strategy to target microRNA for eradication of TICs, leading to inhibition of metastasis and recurrence. Citation Format: Chia-Lin Chen, Suresh Swaminathan, Ameya Kirtane, Jayanth Panyam, Keigo Machida. Selective inhibitors epigenetically modify and eradicate tumor-initiating stem-like cells through downregulating microRNA 22-mediated TET induction and apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4657.

Hepatic ATF6 Increases Fatty Acid Oxidation to Attenuate Hepatic Steatosis in Mice Through Peroxisome Proliferator-Activated Receptor α.

The endoplasmic reticulum quality control protein activating transcription factor 6 (ATF6) has emerged as a novel metabolic regulator. Here, we show that adenovirus-mediated overexpression of the dominant-negative form of ATF6 (dnATF6) increases susceptibility to develop hepatic steatosis in diet-induced insulin-resistant mice and fasted mice. Overexpression of dnATF6 or small interfering RNA-mediated knockdown of ATF6 decreases the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα)/retinoid X receptor complex, and inhibits oxygen consumption rates in hepatocytes, possibly through inhibition of the binding of PPARα to the promoter of its target gene. Intriguingly, ATF6 physically interacts with PPARα, enhances the transcriptional activity of PPARα, and triggers activation of PPARα downstream targets, such as CPT1α and MCAD, in hepatocytes. Furthermore, hepatic overexpression of the active form of ATF6 promotes hepatic fatty acid oxidation and protects against hepatic steatosis in diet-induced insulin-resistant mice. These data delineate the mechanism by which ATF6 controls the activity of PPARα and hepatic mitochondria fatty acid oxidation. Therefore, strategies to activate ATF6 could be used as an alternative avenue to improve liver function and treat hepatic steatosis in obesity.

NANOG Metabolically Reprograms Tumor-Initiating Stem-like Cells through Tumorigenic Changes in Oxidative Phosphorylation and Fatty Acid Metabolism

Stem cell markers, including NANOG, have been implicated in various cancers; however, the functional contribution of NANOG to cancer pathogenesis has remained unclear. Here, we show that NANOG is induced by Toll-like receptor 4 (TLR4) signaling via phosphorylation of E2F1 and that downregulation of Nanog slows down hepatocellular carcinoma (HCC) progression induced by alcohol western diet and hepatitis C virus protein in mice. NANOG ChIP-seq analyses reveal that NANOG regulates the expression of genes involved in mitochondrial metabolic pathways required to maintain tumor-initiating stem-like cells (TICs). NANOG represses mitochondrial oxidative phosphorylation (OXPHOS) genes, as well as ROS generation, and activates fatty acid oxidation (FAO) to support TIC self-renewal and drug resistance. Restoration of OXPHOS activity and inhibition of FAO renders TICs susceptible to a standard care chemotherapy drug for HCC, sorafenib. This study provides insights into the mechanisms of NANOG-mediated generation of TICs, tumorigenesis, and chemoresistance through reprogramming of mitochondrial metabolism.

Perturbation of energy metabolism by fatty-acid derivative AIC-47 and imatinib in BCR-ABL-harboring leukemic cells

In Ph-positive leukemia, imatinib brought marked clinical improvement; however, further improvement is needed to prevent relapse. Cancer cells efficiently use limited energy sources, and drugs targeting cellular metabolism improve the efficacy of therapy. In this study, we characterized the effects of novel anti-cancer fatty-acid derivative AIC-47 and imatinib, focusing on cancer-specific energy metabolism in chronic myeloid leukemia cells. AIC-47 and imatinib in combination exhibited a significant synergic cytotoxicity. Imatinib inhibited only the phosphorylation of BCR-ABL; whereas AIC-47 suppressed the expression of the protein itself. Both AIC-47 and imatinib modulated the expression of pyruvate kinase M (PKM) isoforms from PKM2 to PKM1 through the down-regulation of polypyrimidine tract-binding protein 1 (PTBP1). PTBP1 functions as alternative splicing repressor of PKM1, resulting in expression of PKM2, which is an inactive form of pyruvate kinase for the last step of glycolysis. Although inactivation of BCR-ABL by imatinib strongly suppressed glycolysis, compensatory fatty-acid oxidation (FAO) activation supported glucose-independent cell survival by up-regulating CPT1C, the rate-limiting FAO enzyme. In contrast, AIC-47 inhibited the expression of CPT1C and directly fatty-acid metabolism. These findings were also observed in the CD34+ fraction of Ph-positive acute lymphoblastic leukemia cells. These results suggest that AIC-47 in combination with imatinib strengthened the attack on cancer energy metabolism, in terms of both glycolysis and compensatory activation of FAO.

FABP4 reversed the regulation of leptin on mitochondrial fatty acid oxidation in mice adipocytes.

Fatty acid binding protein 4 (FABP4), plays key role in fatty acid transportation and oxidation, and increases with leptin synergistically during adipose inflammation process. However, the regulation mechanism between FABP4 and leptin on mitochondrial fatty acid oxidation remains unclear. In this study, we found that FABP4 reduced the expression of leptin, CPT-1 and AOX1 in mice adipocytes. Conversely, FABP4 was down-regulated in a time-dependent manner by leptin treatment. Additionally, forced expression of FABP4 attenuated the expression of PGC1-α, UCP2, CPT-1, AOX1 and COX2 compared with leptin incubation. Moreover, mitochondrial membrane potential, fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and Cyt C levels were reduced in response to the overexpression of FABP4. These reductions correspond well with the reduced release of free fatty acid and the inactivation of mitochondrial complexes I and III by FABP4 overexpression. Furthermore, addition of the Akt/mTOR pathway-specific inhibitor (MK2206) blocked the mitochondrial fatty acid oxidation and respiration factors, whereas interference of FABP4 overcame these effects. Taken together, FABP4 could reverse the activation of the leptin-induced mitochondrial fatty acid oxidation, and the inhibition of Akt/mTOR signal pathway played a key role in this process.

Abstract 3040: NANOG metabolically reprograms tumor-initiating stem-like cells in oxidative phosphorylation and fatty acid metabolism

Abstract Stem cell markers such as NANOG have been implicated in various cancers; however, the functional contribution of pluripotency transcription factor NANOG to cancer pathogenesis has remained unclear. Gene profiling, proteomics and metabolomics approaches were combined to identify the pathway(s) altered in tumors. Here, we show that Toll-like receptor 4 (TLR4) signaling phosphorylates E2F1 to transactivate NANOG via TAK1/TBK1. CD133+/CD49f+ TICs isolated from human HCC have tumorigenic activities dependent on TLR4 and NANOG. Down-regulation of Nanog significantly reduces tumor development in obesity-associated HCC mouse model. In the search for the NANOG-dependent mechanisms underlying growth of tumor-initiating stem-like cells (TICs), NANOG ChIP-seq identifies the genes associated with mitochondrial metabolic pathways. The specific pathways, which were examined: oxidative phosphorylation (OXPHOS) and fatty acid oxidation were identified as novel NANOG-mediated oncogenic pathways by NANOG ChIP-seq analysis and metabolomics. The causal roles of NANOG in mitochondrial metabolic reprogramming occurs through the inhibition of oxidative phosphorylation (OXPHOS) and levels of mitochondrial ROS and activation of fatty acid oxidation (FAO), which are required for both the self-renewal of TICs and their drug resistance. PPARδ physically interacts with NANOG. Restoration of OXPHOS and inhibition of FAO rendered TICs susceptible to drug. Our data showed that NANOG reprogramming of mitochondrial metabolism was responsible for human TIC oncogenicity and chemo-resistance. Together, NANOG-mediated metabolic reprogramming through dysregulation of mitochondrial functions generates TICs and drives tumorigenesis, providing insight into mechanisms of NANOG-mediated metabolic reprogramming and its roles in TIC oncogenic activity and chemo-resistance. NANOG suppresses OXPHOS and activates FAO, thus inhibiting OCR and ROS production, conferring a tumor chemoresistant state. These novel pathways are potential drug targets for the highly malignant TICs found in cancer patients. Reversal of NANOG-dependent effects on OXPHOS and FAO gene may offer a noteworthy strategy of countering therapeutic drug resistance associated with NANOG activation. Citation Format: Chia-Lin Chen, Dinesh Babu Uthaya Kumar, Vasu Punj, Jun Xu, Linda Sher, Sonja Hess, Keigo Machida. NANOG metabolically reprograms tumor-initiating stem-like cells in oxidative phosphorylation and fatty acid metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3040. doi:10.1158/1538-7445.AM2015-3040

Deletion of nuclear factor-κB p50 upregulates fatty acid utilization and contributes to an anti-obesity and high-endurance phenotype in mice.

The transcription factor nuclear factor-κB (NF-κB) plays an important role in regulating physiological processes such as immunity and inflammation. In addition to this primary role, NF-κB interacts physically with peroxisome proliferator-activated receptors regulating lipid metabolism-related gene expression and inhibits their transcriptional activity. Therefore, inhibition of NF-κB may promote fatty acid utilization, which could ameliorate obesity and improve endurance capacity. To test this hypothesis, we attempted to elucidate the energy metabolic status of mice lacking the p50 subunit of NF-κB (p50 KO mice) from the tissue to whole body level. p50 KO mice showed a significantly lower respiratory quotient throughout the day than did wild-type (WT) mice; this decrease was associated with increased fatty acid oxidation activity in liver and gastrocnemius muscle of p50 KO mice. p50 KO mice that were fed a high-fat diet were also resistant to fat accumulation and adipose tissue inflammation. Furthermore, p50 KO mice showed a significantly longer maximum running time compared with WT mice, with a lower respiratory exchange ratio during exercise as well as higher residual muscle glycogen content and lower blood lactate levels after exercise. These results suggest that p50 deletion facilitates fatty acid catabolism, leading to an anti-obesity and high-endurance phenotype of mice and supporting the idea that NF-κB is an important regulator of energy metabolism.

High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells.

Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation.

SG-04 * THE eEF2 KINASE, A MEDIATOR OF MEDULLOBLASTOMA ADAPTATION TO METABOLIC STRESS, SUPPORTS FATTY ACID OXIDATION

During tumour progression, brain tumour cells are exposed to nutrient deprivation due to defective tumour vasculature. The ability of brain tumour cells to adapt to reduced nutrient availability favours selection for aggressive clones. The molecular pathways supporting such metabolic adaptation are still poorly defined. We previously reported that the translation elongation factor 2 kinase (eEF2K), which controls mRNA translation at the step of elongation, is an evolutionarily conserved mediator of the response to nutrient deprivation. Gene expression analysis show that eEF2K expression is upregulated in the most aggressive subgroup of medulloblastoma (MB), namely group 3, and that high eEF2K expression is strongly associated with poor survival in both MB. In addition, genetic targeting of eEF2K significantly increases sensitivity of MB cells to nutrient deprivation, supporting a role for eEF2K in facilitating adaptation of MB to nutrient stress. To decipher the basis for eEF2K protective functions under nutrient deprivation, we performed gene expression analysis of eEF2K proficient versus deficient cells under nutrient-deprived conditions. Gene set enrichment analysis (GSEA) highlighted fatty acid oxidation (FAO) as one of the biological pathways most dramatically deregulated upon eEF2K loss. Remarkably, many genes involved in FAO are downregulated in eEF2K deficient cells under starvation, including genes encoding acyl-coA synthetases and acyl-coA dehydrogenases. Furthermore, cells lacking eEF2K are deficient in metabolizing exogenous palmitate to produce ATP, strongly supporting a role for eEF2K in stimulating FAO activity. Finally, our data show that pharmacological induction of FAO in eEF2K deficient cells using bezafibrate was sufficient to rescue cell death occurring under nutrient depleted conditions. The molecular basis for eEF2K-mediated regulation of the FAO transcriptional program is under investigation. Together, our work supports a critical role for eEF2K in driving FAO activity under nutrient deprivation, which could profoundly impact medulloblastoma adaptation to nutrient stress and therefore emergence of aggressive phenotypes.

Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration.

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

Effect of Heat Stress on Pig Skeletal Muscle Metabolism

Hyperthermic pigs differ metabolically, including altered insulin profiles, reduced lipolysis, and compromised intestinal integrity, leading to lower lean tissue and elevated adipose tissue mass. This is distinct from thermal neutral pigs on a similar plane of nutrition. To delineate between direct and indirect (via feed intake changes) HS effects, skeletal muscle substrate metabolism was assessed. Pigs (35 ± 0.8 kg) were assigned to three treatments: thermal neutral fed ad libitum (TN, 21°C, n=8), heat stress fed ad libitum (HS, 35°C, n=8), and thermal neutral, pair-fed to HS intake (PF, n=8) for 7 days. Body temperature (TB) and feed intake (FI) were recorded daily. Longissimus dorsi muscle was biopsied on day -2, 3, and 7 relative to start of treatments for metabolic assays. Heat stress increased TB and decreased FI (P < 0.05). Heat stress tended to decrease acid soluble metabolites (P = 0.07), and did not alter glucose oxidation. Metabolic flexibility decreased in HS versus TN and PF (P < 0.05). Total fatty acid oxidation and pyruvate dehydrogenase (PDH) activity increased in PF (P < 0.05), however, TN and HS did not differ. Heat stress inhibited citrate synthase and beta hydroxyacyl-CoA dehydrogenase (BHAD) activities (P < 0.05). Heat stress did not alter PDH phosphorylation or carnitine palmitoyltransferase 1 abundances, but reduced acetyl-CoA carboxylase (ACC) protein abundance (P < 0.05). In conclusion, HS altered skeletal muscle fatty acid oxidation and metabolic flexibility, likely involving BHAD and ACC regulation.

Krill Oil Ameliorates Mitochondrial Dysfunctions in Rats Treated with High-Fat Diet.

In recent years, several studies focused their attention on the role of dietary fats in the pathogenesis of hepatic steatosis. It has been demonstrated that a high-fat diet is able to induce hyperglycemia, hyperinsulinemia, obesity, and nonalcoholic fatty liver disease. On the other hand, krill oil, a novel dietary supplement of n-3 PUFAs, has the ability to improve lipid and glucose metabolism, exerting possible protective effects against hepatic steatosis. In this study we have investigated the effects of krill oil on mitochondrial energetic metabolism in animals fed a high-fat diet. To this end, male Sprague-Dawley rats were divided into three groups and fed for 4 weeks with a standard diet (control group), a diet with 35% fat (HF group), or a high-fat diet supplemented with 2.5% krill oil (HF+KO group). The obtained results suggest that krill oil promotes the burning of fat excess introduced by the high-fat diet. This effect is obtained by stimulating mitochondrial metabolic pathways such as fatty acid oxidation, Krebs cycle, and respiratory chain complexes activity. Modulation of the expression of carrier proteins involved in mitochondrial uncoupling was also observed. Overall, krill oil counteracts the negative effects of a high-fat diet on mitochondrial energetic metabolism.

L-Asparaginase Causes Metabolic Reprogramming in ALL Cells

Acute lymphoblastic leukemia (ALL) is the most frequent type of childhood cancer. The key component in the therapy, L-asparaginase (ASNase), hydrolyzes plasma asparagine and glutamine. Leukemic cells are sensitive to the depletion due to low activity of asparagine synthetase. Although the treatment is very effective, resistance and side effects remain a serious problem in some cases and its mechanism of action is not well understood. Our aim is to clarify the intracellular consequences of the amino acid depletion to define the reason of different patients´ response.We have generated ASNase-resistant subclones through chronic exposure to the enzyme. Pathway analysis of gene expression profiles of the cell lines (REH;TEL/AML1-positive, NALM-6; TEL/PDGFRB1-positive and their resistant counterparts) and primary samples (sensitive and resistant to ASNase; Holleman et al. (NEJM, 2004)) revealed that ASNase affects the translation machinery and metabolism of leukemic cells. The key nutrient sensor positively regulating protein synthesis, pyrimidine synthesis, glycolysis and lipid synthesis is mTORC1. Since ASNase depletes glutamine that is essential for mTORC1 activity, we hypothesized that the effect of ASNase is driven through mTORC1 signaling. Main aim of the study was to explore the effect of ASNase on downstream mTORC1 targets in leukemic cells.ASNase treatment inhibited protein synthesis, displayed by dephosphorylation of p-P70SK6 and p-S6. ASNase also decreased de-novo DNA synthesis as shown by dephosphorylation of p-CAD. This result was confirmed by analysis of de-novo pyrimidine synthesis intermediates, uridine monophosphate and uridine, measured by UPLC-ToF-MS. Both were decreased upon ASNase treatment. Except experiments done on ALL cell lines we also detected dephosphorylation of p-S6, p-CAD in primary samples.Regarding the effect on glycolysis we observed inhibition of glucose uptake and decrease of lactate production in cells treated with ASNase. We also detected the decrease of protein levels of c-Myc, the activator of glucose and glutamine catabolism, and glucose transporter 1 (Glut-1). On the contrary, ASNase increased fatty acid oxidation (FAO) followed by the elevation of the capacity of cell respiration and NAD+/NADH ratio in both cell lines.Next, we wanted to elucidate whether ASNase inhibits mTORC1 targets through RagB, the key protein mediating response to general amino acid deprivation. We established a RagB mutant leukemic cell line with constitutive activation of mTORC1 pathway despite deprivation of amino acids. ASNase treatment did not inhibit p-S6 and p-CAD in this cell line. Similarly, ASNase failed to increase FAO in cells with active mTORC1. These results suggest that the effect of ASNase on protein translation, de novo pyrimidine synthesis and FAO is mediated through RagB-mTORC1 pathway. By contrast, c-Myc expression was decreased in both RagB wild type and mutant cells, indicating that ASNase inhibits glycolysis in RagB-mTORC1 independent manner.The activation of FAO has been suggested to have a pro-survival function in leukemic cells under nutrient stress conditions. We tested whether the increase of FAO in ALL cells treated with ASNase also serves to cope with the metabolic stress. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, cells with the inability to increase FAO (RagB mutant) were more sensitive to ASNase compared to RagB wild type cells.Our results show that the inhibitory effect of ASNase on mTORC1 leads not only to apoptosis but also to metabolic reprogramming. We propose that inhibition of protein translation and pyrimidine synthesis are part of apoptotic processes whereas increased FAO and cell respiration represent pro-survival pathway. Altogether, our study suggests that targeting of FAO in leukemic cells resistant to ASNase is a promising new therapeutic strategy.IGA-NT1249, GAUK-632513 DisclosuresNo relevant conflicts of interest to declare.

CTRP5 ameliorates palmitate-induced apoptosis and insulin resistance through activation of AMPK and fatty acid oxidation

Lipotoxicity resulting from a high concentration of saturated fatty acids is closely linked to development of insulin resistance, as well as apoptosis in skeletal muscle. CTRP5, an adiponectin paralog, is known to activate AMPK and fatty acid oxidation; however, the effects of CTRP5 on palmitate-induced lipotoxicity in myocytes have not been investigated. We found that globular domain of CTRP5 (gCTRP5) prevented palmitate-induced apoptosis and insulin resistance in myocytes by inhibiting the activation of caspase-3, reactive oxygen species accumulation, and IRS-1 reduction. These beneficial effects of gCTRP5 are mainly attributed to an increase in fatty acid oxidation through phosphorylation of AMPK. These results provide a novel function of CTRP5, which may have preventive and therapeutic potential in management of obesity, insulin resistance, and type 2 diabetes mellitus.

PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells

Although non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease there is no pharmacological agent approved for its treatment. Since peroxisome proliferator-activated receptors (PPARs) are closely associated with hepatic lipid metabolism, they seem to play important roles in NAFLD. However, the effects of PPAR agonists on steatosis that is a common pathology associated with NAFLD, remain largely controversial. In this study, the effects of various PPAR agonists, i.e. fenofibrate, bezafibrate, troglitazone, rosiglitazone, muraglitazar and tesaglitazar on oleic acid-induced steatotic HepaRG cells were investigated after a single 24-hour or 2-week repeat treatment. Lipid vesicles stained by Oil-Red O and triglycerides accumulation caused by oleic acid overload, were decreased, by up to 50%, while fatty acid oxidation was induced after 2-week co-treatment with PPAR agonists. The greatest effects on reduction of steatosis were obtained with the dual PPARα/γ agonist muraglitazar. Such improvement of steatosis was associated with up-regulation of genes related to fatty acid oxidation activity and down-regulation of many genes involved in lipogenesis. Moreover, modulation of expression of some nuclear receptor genes, such as FXR, LXRα and CAR, which are potent actors in the control of lipogenesis, was observed and might explain repression of de novo lipogenesis. ConclusionAltogether, our in vitro data on steatotic HepaRG cells treated with PPAR agonists correlated well with clinical investigations, bringing a proof of concept that drug-induced reversal of steatosis in human can be evaluated in in vitro before conducting long-term and costly in vivo studies in animals and patients.

Fenofibrate, a peroxisome proliferator-activated receptor α ligand, prevents abnormal liver function induced by a fasting–refeeding process

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, is an anti-hyperlipidemic agent that has been widely used in the treatment of dyslipidemia. In this study, we examined the effect of fenofibrate on liver damage caused by refeeding a high-fat diet (HFD) in mice after 24h fasting. Here, we showed that refeeding HFD after fasting causes liver damage in mice determined by liver morphology and liver cell death. A detailed analysis revealed that hepatic lipid droplet formation is enhanced and triglyceride levels in liver are increased by refeeding HFD after starvation for 24h. Also, NF-κB is activated and consequently induces the expression of TNF-α, IL1-β, COX-2, and NOS2. However, treating with fenofibrate attenuates the liver damage and triglyceride accumulation caused by the fasting–refeeding HFD process. Fenofibrate reduces the expression of NF-κB target genes but induces genes for peroxisomal fatty acid oxidation, peroxisome biogenesis and mitochondrial fatty acid oxidation. These results strongly suggest that the treatment of fenofibrate ameliorates the liver damage induced by fasting–refeeding HFD, possibly through the activation of fatty acid oxidation.