Medium-chain acyl-CoA Dehydrogenase Expression Research Articles

A gene therapy targeting medium-chain acyl-CoA dehydrogenase (MCAD) did not protect against diabetes-induced cardiac pathology.

Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. KEY MESSAGES: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications.

Diosgenin alleviates NAFLD induced by a high-fat diet in rats via mTOR/SREBP-1c/HSP60/MCAD/SCAD signaling pathway

This study aims to observe the effects of diosgenin on the expression of mammalian target of rapamycin(mTOR), sterol regulatory element-binding protein-1c(SREBP-1c), heat shock protein 60(HSP60), medium-chain acyl-CoA dehydrogenase(MCAD), and short-chain acyl-CoA dehydrogenase(SCAD) in the liver tissue of the rat model of non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin in alleviating NAFLD. Forty male SD rats were randomized into five groups: a control group, a model group, low-(150 mg·kg~(-1)·d~(-1)) and high-dose(300 mg·kg~(-1)·d~(-1)) diosgenin groups, and a simvastatin(4 mg·kg~(-1)·d~(-1)) group. The rats in the control group were fed with a normal diet, while those in the other four groups were fed with a high-fat diet. After feeding for 8 weeks, the body weight of rats in the high-fat diet groups increased significantly. After that, the rats were administrated with the corresponding dose of diosgenin or simvastatin by gavage every day for 8 weeks. The levels of triglyceride(TG), total cholesterol(TC), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were determined by the biochemical method. The levels of TG and TC in the liver were measured by the enzyme method. Oil-red O staining was employed to detect the lipid accumulation, and hematoxylin-eosin(HE) staining to detect the pathological changes in the liver tissue. The mRNA and protein levels of mTOR, SREBP-1c, HSP60, MCAD, and SCAD in the liver tissue of rats were determined by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. Compared with the control group, the model group showed increased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lipid deposition in the liver, obvious hepatic steatosis, up-regulated mRNA and protein expression levels of mTOR and SREBP-1c, and down-regulated mRNA and protein expression levels of HSP60, MCAD, and SCAD. Compared with the model group, the rats in each treatment group showed obviously decreased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lessened lipid deposition in the liver, ameliorated hepatic steatosis, down-regulated mRNA and protein le-vels of mTOR and SREBP-1c, and up-regulated mRNA and protein levels of HSP60, MCAD, and SCAD. The high-dose diosgenin outperformed the low-dose diosgenin and simvastatin. Diosgenin may prevent and treat NAFLD by inhibiting the expression of mTOR and SREBP-1c and promoting the expression of HSP60, MCAD, and SCAD to reduce lipid synthesis, improving mitochondrial function, and promoting fatty acid β oxidation in the liver.

MCAD activation by empagliflozin promotes fatty acid oxidation and reduces lipid deposition in NASH.

Medium-chain acyl-CoA dehydrogenase (MCAD) is one of the significant enzymes involved in the β-oxidation of mitochondrial fatty acids. MCAD deficiency affects the β-oxidation of fatty acid and leads to lipid deposition in multiple organs, but little is known about its importance in nonalcoholic steatohepatitis (NASH). Empagliflozin is revealed to effectively improve NASH by increasing research, whereas the specific mechanism still has to be explored. Human liver tissues of patients with or without NASH were obtained for proteomic analysis to screen proteins of interest. db/db mice were given empagliflozin by gavage for 8 weeks. The expression of MCAD and signaling molecules involved in hepatic lipid metabolism was evaluated in human liver, mice and HL7702 cells. We found that the MCAD levels in the liver were significantly reduced in NASH patients compared to patients without NASH. Protein-protein interaction network analysis showed that MCAD was highly correlated with forkhead box A2 (FOXA2) and protein kinase AMP-activated catalytic subunit alpha (PRKAA). AMPK/FOXA2/MCAD signaling pathway was detected to be inhibited in the liver of NASH patients. Decreased expression of MCAD was also observed in the livers of db/db mice and hepatocyte treated with palmitic acid and glucose. Of note, empagliflozin could upregulate MCAD expression by activating AMPK/FOXA2 signaling pathway, reduce lipid deposition and improve NASH in vivo and in vitro. This research demonstrated that MCAD is a key player of hepatic lipid deposition and its targeting partially corrects NASH. MCAD thus may be a potential therapeutic target for the treatment of NASH.

The role of estrogen-related receptor α (ERRα) in metabolic adaptations by endurance training in skeletal muscle of streptozotocin-induced diabetic rats

The aim of present study was to investigate the effect of endurance training and ERRα inhibition (target for the discovery of new therapies for diseases such as diabetes) on the gene and protein expression of factors involved in lipid metabolism in diabetic rats. We designed the current study to evaluate the combination of ERRα inhibition and exercise training effects on the expression of ERRα, medium-chain acyl-CoA dehydrogenase (MCAD), carnitine palmitoyltransferase-1b (CPT-1b), pyruvate dehydrogenase kinase 4 (PDK4) and Citrate synthase (CS) in diabetic and non-diabetic rats. Sixty-four male Wistar rats were divided into 8 groups (n = 8) as follows: non-diabetic control, diabetic control (a single dose of 45 mg/kg of STZ), non-diabetic/ERRα inhibition group (received 0.48 mg/kg of XCT790), diabetic/ERRα inhibition group, non-diabetic/exercise training, diabetic/exercise training, non-diabetic rats which received XCT790 and performed exercise training, and diabetic rats which received XCT790 and also performed exercise training. The gene and protein expression were measured by real-time PCR (quantified by $$ 2^{{ - \Delta \Delta CT }}$$ method) and Western blotting method, respectively. Our results showed that the expression of ERRα (1.6-fold), MCAD (4.6-fold), CPT-1b (fivefold), CS (twofold), PDK4 (fourfold), and PGC-1α (fourfold) in the medial gastrocnemius muscle of the non-diabetic exercise training group was significantly higher than the non-diabetic control group. In sum, ERRα is a trainable factor, and its changes are parallel with higher expression of the enzymes which involved in lipid metabolism in healthy rats. ERRα inhibition and endurance training may have positive effects on the lipid metabolism in diabetic rats. This indirectly suggests a significant role of ERRα in the adaptation of lipid metabolism evoked by endurance training.

Reactivation of fatty acid oxidation by medium chain fatty acid prevents myocyte hypertrophy in H9c2 cell line.

Metabolic shift is an important contributory factor for progression of hypertension-induced left ventricular hypertrophy into cardiac failure. Under hypertrophic conditions, heart switches its substrate preference from fatty acid to glucose. Prolonged dependence on glucose for energy production has adverse cardiovascular consequences. It was reported earlier that reactivation of fatty acid metabolism with medium chain triglycerides ameliorated cardiac hypertrophy, oxidative stress and energy level in spontaneously hypertensive rat. However, the molecular mechanism mediating the beneficial effect of medium chain triglycerides remained elusive. It was hypothesized that reduction of cardiomyocyte hypertrophy by medium chain fatty acid (MCFA) is mediated by modulation of signaling pathways over expressed in cardiac hypertrophy. The protective effect of medium chain fatty acid (MCFA) was evaluated in cellular model of myocyte hypertrophy. H9c2 cells were stimulated with Arginine vasopressin (AVP) for the induction of hypertrophy. Cell volume and secretion of brain natriuretic peptide (BNP) were used for assessment of cardiomyocyte hypertrophy. Cells were pretreated with MCFA (Caprylic acid) and metabolic modulation was assessed from the expression of medium-chain acyl-CoA dehydrogenase (MCAD), cluster of differentiation-36 (CD36) and peroxisome proliferator-activated receptor (PPAR)-α mRNA. The signaling molecules modified by MCFA was evaluated from protein expression of mitogen activated protein kinases (MAPK: ERK1/2, p38 and JNK) and Calcineurin A. Pretreatment with MCFA stimulated fatty acid metabolism in hypertrophic H9c2, with concomitant reduction of cell volume and BNP secretion. MCFA reduced activated ERK1/2, JNK and calicineurin A expression mediated by AVP. In conclusion, the beneficial effect of MCFA is possibly mediated by stimulation of fatty acid metabolism and modulation of MAPK and Calcineurin A.

Effects of Sirt3‑autophagy and resveratrol activation on myocardial hypertrophy and energy metabolism.

The aim of the present study was to examine the role of sirtuin 3 (Sirt3)-autophagy in regulating myocardial energy metabolism and inhibiting myocardial hypertrophy in angiotensin (Ang) II-induced myocardial cell hypertrophy. The primary cultured myocardial cells of neonatal Sprague Dawley rats were used to construct a myocardial hypertrophy model induced with Ang II. Following the activation of Sirt3 by resveratrol (Res), Sirt3 was silenced using small interfering (si)RNA-Sirt3, and the morphology of the myocardial cells was observed under an optical microscope. Reverse transcription-polymerase chain reaction was used to detect the mRNA expression of the following myocardial hypertrophy markers; atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), Sirt3, medium-chain acyl-CoA dehydrogenase (MCAD) and pyruvate kinase (PK). Western blot analysis was used to detect the protein expression of Sirt3, light chain 3 (LC3) and Beclin1. Ang II may inhibit the protein expression of Sirt3, LC3 and Beclin1. Res, an agonist of Sirt3, may promote the protein expression of Sirt3, LC3 and Beclin1. Res inhibited the mRNA expression of ANP and BNP, and reversed the Ang II-induced myocardial cell hypertrophy. The addition of siRNA-Sirt3 decreased the protein expression of Sirt3, LC3 and Beclin1, increased the mRNA expression of ANP and BNP, and weakened the inhibitory effect of Res on myocardial cell hypertrophy. Res promoted the mRNA expression of MCAD, inhibited the mRNA expression of PK, and reversed the influence of Ang II on myocardial energy metabolism. siRNA-Sirt3 intervention significantly decreased the effect of Res in eliminating abnormal myocardial energy metabolism. In conclusion, Sirt3 may inhibit Ang II-induced myocardial hypertrophy and reverse the Ang II-caused abnormal myocardial energy metabolism through activation of autophagy.

Role of mitochondrial acyl-CoA dehydrogenases in the metabolism of dicarboxylic fatty acids

Dicarboxylic fatty acids, taken as a nutritional supplement or produced endogenously via omega oxidation of monocarboxylic fatty acids, may have therapeutic potential for rare inborn errors of metabolism as well as common metabolic diseases such as type 2 diabetes. Breakdown of dicarboxylic acids yields acetyl-CoA and succinyl-CoA as products, the latter of which is anaplerotic for the TCA cycle. However, little is known about the metabolic pathways responsible for degradation of dicarboxylic acids. Here, we demonstrated with whole-cell fatty acid oxidation assays that both mitochondria and peroxisomes contribute to dicarboxylic acid degradation. Several mitochondrial acyl-CoA dehydrogenases were tested for activity against dicarboxylyl-CoAs. Medium-chain acyl-CoA dehydrogenase (MCAD) exhibited activity with both six and 12 carbon dicarboxylyl-CoAs, and the capacity for dehydrogenation of these substrates was significantly reduced in MCAD knockout mouse liver. However, when dicarboxylic acids were fed to normal mice, the expression of MCAD did not change, while expression of peroxisomal fatty acid oxidation enzymes was greatly upregulated. In conclusion, mitochondrial fatty acid oxidation, and in particular MCAD, contributes to dicarboxylic acid degradation, but feeding dicarboxylic acids induces only the peroxisomal pathway.

Genistein, daidzein, and resveratrols stimulate PGC‐1β‐mediated gene expression

PGC‐1β is a transcriptional co‐activator of nuclear receptors such as the estrogen receptor‐related receptor (ERR). Transgenic overexpression of PGC‐1β in mice increases energy expenditure and suppresses high‐fat diet‐induced obesity. In this study, we screened various food‐derived and natural compounds using a reporter assay system to measure the transcriptional activity of PGC‐1β. Soy‐derived isoflavones, genistein and daidzein, and several resveratrols activated PGC‐1β. Genistein, daidzein, and trans‐oxyresveratrol activated ERR‐responsive element‐mediated reporter activity in the presence of PGC‐1β. Stable overexpression of PGC‐1β in C2C12 myoblasts increased the expression of medium‐chain acyl‐CoA dehydrogenase (MCAD), an important enzyme in fatty acid β‐oxidation. Genistein and daidzein increased MCAD mRNA levels and mitochondrial content in PGC‐1β‐expressing C2C12 cells. These compounds activated ERR/PGC‐1β complex‐mediated gene expression, and our findings may be a practical foundation for developing functional foods targeting obesity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genistein, daidzein, and resveratrols stimulate PGC-1β-mediated gene expression

PGC-1β is a transcriptional co-activator of nuclear receptors such as the estrogen receptor-related receptor (ERR). Transgenic overexpression of PGC-1β in mice increases energy expenditure and suppresses high-fat diet-induced obesity. In this study, we screened various food-derived and natural compounds using a reporter assay system to measure the transcriptional activity of PGC-1β. Soy-derived isoflavones, genistein and daidzein, and several resveratrols activated PGC-1β. Genistein, daidzein, and trans-oxyresveratrol activated ERR-responsive element-mediated reporter activity in the presence of PGC-1β. Stable overexpression of PGC-1β in C2C12 myoblasts increased the expression of medium-chain acyl-CoA dehydrogenase (MCAD), an important enzyme in fatty acid β-oxidation. Genistein and daidzein increased MCAD mRNA levels and mitochondrial content in PGC-1β-expressing C2C12 cells. These compounds activated ERR/PGC-1β complex-mediated gene expression, and our findings may be a practical foundation for developing functional foods targeting obesity.

Bezafibrate induces autophagy and improves hepatic lipid metabolism in glycogen storage disease type Ia.

Glucose-6-phosphatase α (G6Pase) deficiency, also known as von Gierke's Disease or Glycogen storage disease type Ia (GSD Ia), is characterized by decreased ability of the liver to convert glucose-6-phosphate to glucose leading to glycogen accumulation and hepatosteatosis. Long-term complications of GSD Ia include hepatic adenomas and carcinomas, in association with the suppression of autophagy in the liver. The G6pc-/- mouse and canine models for GSD Ia were treated with the pan-peroxisomal proliferator-activated receptor agonist, bezafibrate, to determine the drug's effect on liver metabolism and function. Hepatic glycogen and triglyceride concentrations were measured and western blotting was performed to investigate pathways affected by the treatment. Bezafibrate decreased liver triglyceride and glycogen concentrations and partially reversed the autophagy defect previously demonstrated in GSD Ia models. Changes in medium-chain acyl-CoA dehydrogenase expression and acylcarnintine flux suggested that fatty acid oxidation was increased and fatty acid synthase expression associated with lipogenesis was decreased in G6pc-/- mice treated with bezafibrate. In summary, bezafibrate induced autophagy in the liver while increasing fatty acid oxidation and decreasing lipogenesis in G6pc-/- mice. It represents a potential therapy for glycogen overload and hepatosteatosis associated with GSD Ia, with beneficial effects that have implications for non-alcoholic fatty liver disease.

Abstract P175: Chronic Exercise Attenuates Blood Pressure Elevation and Albuminuria With Improvement of Renal Lipid Metabolism in High Fructose-fed Rats

High fructose diet (HFr) can lead to metabolic disorder, hypertension, and renal disease. Although chronic exercise (Ex) provides various beneficial effects on hypertension and kidney disease, the precise mechanism is not fully clarified. Thus, present study examined the effects of Ex on the blood pressure, renal function and renal lipid metabolism in rats fed with HFr. Sprague-Dawley rats were allocated to 3 groups. The HFr and Ex groups were fed with HFr (60%, w/w), the control group was fed with the diet in which fructose was replaced by starch. The Ex group underwent treadmill exercise at aerobic intensity. After 12 weeks, renal triglyceride (TG) content were measured, and expression of enzymes and regulators of fatty acid metabolism were analyzed by Western blot. HFr increased systolic blood pressure (SBP) and albuminuria and Ex decreased the HFr-increased SBP and albuminuria (85±4 vs. 122±9 vs. 91±4 mmHg, P<0.01, 326±67 vs. 534±79 vs. 176±54 mg/day, P<0.01). HFr increased plasma TG and uric acid (UA) and Ex decreased the HFr-increased TG and UA (124±20 vs. 474±35 vs. 238±23 mg/dL, P<0.01, 1.15±0.10 vs. 2.14±0.10 vs. 1.50±0.13 mg/dL, P<0.01), whereas HFr or Ex did not affect plasma creatinine. HFr increased renal TG content and Ex decreased the HFr-increased TG content (12.2±0.5 vs. 14.1±0.5 vs. 10.3±1.1 mg/100mg tissue, P<0.01). Among enzymes of fatty acid synthesis, HFr increased the renal expression of fatty acid synthase (FAS), and Ex decrease the expression of FAS (P<0.01). Among enzymes and regulators of fatty acid oxidation, HFr decreased the renal expression of PPARα, carnitine palmitoyltransferase I (CPTI), medium-chain acyl-CoA dehydrogenase (MCAD) (P<0.05), and Ex increased the expression of PPARα, CPTI, MCAD, and acyl-coenzyme A oxidase (ACOX). These results indicated that Ex attenuates blood pressure elevation, albuminuria with an improvement of renal lipid metabolism in the HFr-fed rats. These effects of Ex may relate to an improvement of the renal lipid metabolism.

Metabolic heterogeneity of activated beige/brite adipocytes in inguinal adipose tissue

Sustained β3 adrenergic receptor (ADRB3) activation simultaneously upregulates fatty acid synthesis and oxidation in mouse brown, beige, and white adipose tissues; however, the cellular basis of this dual regulation is not known. Treatment of mice with the ADRB3 agonist CL316,243 (CL) increased expression of fatty acid synthase (FASN) and medium chain acyl-CoA dehydrogenase (MCAD) protein within the same cells in classic brown and white adipose tissues. Surprisingly, in inguinal adipose tissue, CL-upregulated FASN and MCAD in distinct cell populations: high MCAD expression occurred in multilocular adipocytes that co-expressed UCP1+, whereas high FASN expression occurred in paucilocular adipocytes lacking detectable UCP1. Genetic tracing with UCP1-cre, however, indicated nearly half of adipocytes with a history of UCP1 expression expressed high levels of FASN without current expression of UCP1. Global transcriptomic analysis of FACS-isolated adipocytes confirmed the presence of distinct anabolic and catabolic phenotypes, and identified differential expression of transcriptional pathways known to regulate lipid synthesis and oxidation. Surprisingly, paternally-expressed genes of the non-classical gene imprinted network were strikingly enriched in anabolic phenotypes, suggesting possible involvement in maintaining the balance of metabolic phenotypes. The results indicate that metabolic heterogeneity is a distinct property of activated beige/brite adipocytes that might be under epigenetic control.

Mitoprotective antioxidant EUK-134 stimulates fatty acid oxidation and prevents hypertrophy in H9C2 cells.

Oxidative stress is an important contributory factor for the development of cardiovascular diseases like hypertension-induced hypertrophy. Mitochondrion is the major source of reactive oxygen species. Hence, protecting mitochondria from oxidative damage can be an effective therapeutic strategy for the prevention of hypertensive heart disease. Conventional antioxidants are not likely to be cardioprotective, as they cannot protect mitochondria from oxidative damage. EUK-134 is a salen-manganese complex with superoxide dismutase and catalase activity. The possible role of EUK-134, a mitoprotective antioxidant, in the prevention of hypertrophy of H9C2 cells was examined. The cells were stimulated with phenylephrine (50μM), and hypertrophy was assessed based on cell volume and expression of brain natriuretic peptide and calcineurin. Enhanced myocardial lipid peroxidation and protein carbonyl content, accompanied by nuclear factor-kappa B gene expression,confirmed the presence of oxidative stress in hypertrophic cells. Metabolic shift was evident from reduction in the expression of medium-chain acyl-CoA dehydrogenase. Mitochondrial oxidative stress was confirmed by the reduced expression of mitochondria-specific antioxidant peroxiredoxin-3 and enhanced mitochondrial superoxide production. Compromised mitochondrial function was apparent from reduced mitochondrial membrane potential. Pretreatment with EUK-134 (10μM) was effective in the prevention of hypertrophic changes in H9C2 cells, reduction of oxidative stress, and prevention of metabolic shift. EUK-134 treatment improved the oxidative status of mitochondria and reversed hypertrophy-induced reduction of mitochondrial membrane potential. Supplementation with EUK-134 is therefore identified as a novel approach to attenuate cardiac hypertrophy and lends scope for the development of EUK-134 as a therapeutic agent in the management of human cardiovascular disease.

Abstract B51: Effects of dietary fatty acids on pancreatic cancer

Abstract Introduction: Fatty acids (FAs) are principal constituents of dietary fat. Epidemiological studies show that high fat diets (HFDs) regulate pancreatic cancer. However, the nature of the regulation may depend on the major types of dietary FAs namely saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and n6- and n3 polyunsaturated fatty acids (n6PUFAs and n3PUFAs). We undertook the present study to investigate effects of these FAs on pancreatic cancer. Methods: We made four HFDs that all had 15% fat and 4 kcal/g. Fat contents in these HFDs were derived from cocoa butter, olive oil, soybean oil and flaxseed oil, respectively. These fat origins were chosen because in each of them more than 50% of total FAs are from a single FA type. Consequently, four HFDs were enriched with SFAs, MUFAs, n6PUFAs and n3PUFAs, respectively. Two normal-fat (4%-5%) diets were used as controls. One of them was iso-caloric to HFDs (IsoC control) and the other was normal chow (NC control, 3 kcal/g). The 6 diets were used in 6 groups of nude mice whose pancreas carried human pancreatic cancer cells. Fourteen weeks later, all mice were sacrificed. FA profiling was determined in liver and skeletal muscle by chromatography. Tumor grafts were analyzed by histology and Western blots. Results: The intake of HFDs that were rich in certain types of FAs increased the same types of FAs in the liver and skeletal muscle. The tumors whose hosts were fed MUFA diet were significantly greater than those whose hosts were fed NC, IsoC, and n3PUFA diets. The tumors whose hosts were fed SFA and n6PUFA diets were marginally greater than those in NC, IsoC, and n3PUFA groups. Necrotic areas were seen in all tumors. Big necrotic areas (>25% of total tumor areas) were most frequent in the n3PUFA group, which suggests that n3PUFA diet promoted cancer-cell death. Medium-chain acyl-CoA dehydrogenase (MCAD) is known to regulate the beta-oxidation of FAs. When MCAD expression was determined by immunocytochemistry, the expression was more profound in tumors from SFA, MUFA, and n6PUFA groups than from NC, IsoC, and n3PUFA groups. When lipid droplets were stained in tumor sections, the droplets were scarce in control groups and were numerous in all HFD groups. When transcription factor hypoxia-inducible factor-1 (HIF-1) and proliferating cell nuclear antigen (PCNA) were stained by immunocytochemistry, a HIF-1/PCNA co-expression was seen in tumors from MUFA and n6PUFA groups. Previous studies have shown that cyclooxygenase-2 (COX-2) and superoxide dismutase-1 (SOD-1) may be involved in FA-induced regulation. Our Western blotting data suggest that COX-2 and SOD-1 were increased in tumors from four HFD groups, particularly SFA, MUFA, and n6PUFA groups. Conclusion: Pancreatic cancer cells are in a favorable condition when their hosts are fed diets rich in SFAs, MUFAs and n6PUFAs. In contrast, the diet rich in n3PUFAs has anticancer effects on pancreatic cancer cells. Citation Format: Hongyi Liu, Ming Yu, Yijie Duan, Dapeng Zhang, Shasha Li, Yue Chen, Feng Wang. Effects of dietary fatty acids on pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B51.

Effects of castration on the adiposity and expression of lipid metabolism genes in various fat depots of Korean cattle

Castration increases intramuscular (IM) fat deposition in the musculus longissimus dorsi, a highly desirable trait in Korean beef cattle. However, castration may also affect accumulation in other fat depots. We examined whether castration affects adipose cellularity and lipid metabolism gene expression in various fat depots, including subcutaneous (SC), abdominal (AB), perirenal (PR), and IM fat. First, frozen sections taken from fat depots were stained with hematoxylin and eosin and the mean fat cell size was determined. Steers showed larger cell sizes in AB (P=0.01), SC (P=0.05), and PR (P=0.01) fat compared to bulls. Next, lipid metabolism gene expression in AB fat was compared between bulls and steers. In AB fat, steers showed increased mRNA expression of CCAAT/enhancer binding protein alpha (C/EBPα), a gene related to adipogenesis, compared to bulls (P<0.01). In contrast, steers showed decreased protein expression of medium-chain acyl-CoA dehydrogenase (MCAD), an enzyme involved in fatty acid β-oxidation, compared to bulls (P<0.05). Our results demonstrate that castration induces hypertrophy in body fat cells, and that the up-regulation of adipogenesis and down-regulation of fatty acid β-oxidation may in part contribute to this effect. We compared fat cell sizes among the various fat depots in Korean cattle steers. The order of fat cell size was AB>PR>SC>IM. The expression levels of lipid metabolism genes were compared among various fat depots. The mRNA levels of C/EBPα and peroxisome proliferator-activated receptor-γ were highest (P<0.05) in AB fat and lowest in IM fat. The mRNA levels of fatty acid binding protein-4 and lipogenic acetyl-coenzyme A carboxylase and fatty acid synthase genes were highest (P<0.05) in AB fat, and lowest in IM fat. Expression levels of lipolytic hormone-sensitive lipase and fatty oxidation MCAD genes were also highest (P<0.05) in AB fat and lowest in IM fat. Our results suggest that combined effects of higher adipogenesis, lipogenesis, and cellular fatty acid transport are responsible for the largest AB and smallest IM fat cells among the fat depots examined.

Increased dietary fat contributes to dysregulation of the LKB1/AMPK pathway and increased damage in a mouse model of early-stage ethanol-mediated steatosis

ObjectiveThe objective of the study was to examine the interaction of moderate and high dietary fat and ethanol with respect to formation of steatosis and regulation of the AMP-activated protein kinase (AMPK) pathway in a mouse model of chronic ethanol consumption. MethodsMale C57BL/6J mice were pair-fed a modified Lieber–DeCarli diet composed of either moderate fat [30% fat-derived calories (MF)] or high fat [45% fat-derived calories (HF)] combined with increasing concentrations of ethanol (2%–6%) for 6 weeks. ResultsChronic ethanol consumption resulted in significant increases in plasma alanine aminotransferase in MF (1.84-fold) and HF mice (2.33-fold), yet liver triglycerides only increased significantly in the HF model (1.62-fold). Ethanol addition significantly increased plasma adiponectin under conditions of MF but not HF. In combination with MF, the addition of ethanol significantly decreased total and hepatic pThr172AMPKα and acetyl CoA Carboxylase (ACC). HF plus ethanol decreased pSer108AMPKβ, yet a marked 1.5-fold increase in pThr172AMPKα occurred. No change was evident in pSer79ACC under conditions of ethanol and HF ingestion. In both models, nuclear levels of sterol response element binding protein 1c and carbohydrate response element binding protein were decreased. Surprisingly, MF plus ethanol significantly elevated protein expression of medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and very long chain acyl-CoA dehydrogenase but did not significantly affect mRNA expression of other proteins involved in β-oxidation and fatty acid synthesis. HF plus ethanol significantly reduced mRNA expression of both stearoyl CoA desaturase 1 and fatty acid elongase 5, but did not have an effect on MCAD or LCAD. ConclusionThese data suggest that, when co-ingested with ethanol, dietary fat differentially contributes to dysregulation of adiponectin-dependent activation of the AMPK pathway in the liver of mice.

Carnitine palmitoyltransferase 2 and carnitine/acylcarnitine translocase are involved in the mitochondrial synthesis and export of acylcarnitines

Acylcarnitines are commonly used in the diagnosis of mitochondrial fatty acid β-oxidation disorders (mFAODs). It is generally assumed that this plasma acylcarnitine profile reflects the mitochondrial accumulation of acyl-CoAs. The identity of the enzymes and the mitochondrial and plasmalemmal transporters involved in the synthesis and export of these metabolites have remained undefined. We used lentiviral shRNA to knock down the expression of medium-chain acyl-CoA dehydrogenase (MCAD) in control and carnitine palmitoyltransferase 2 (CPT2)-, carnitine/acylcarnitine translocase (CACT)-, and plasmalemmal carnitine transporter (OCTN2)-deficient human fibroblasts. These cell lines, including mock-transduced controls, were loaded with decanoic acid and carnitine, followed by the measurement of the acylcarnitine profile in the extracellular medium. In control fibroblasts, MCAD knockdown markedly increased the production of octanoylcarnitine (3-fold, P<0.01). OCTN2-deficient cell lines also showed extracellular accumulation of octanoylcarnitine (2.8-fold, P<0.01), suggesting that the cellular export of acylcarnitines does not depend on OCTN2. In contrast, in CPT2- and CACT-deficient cells, the accumulation of octanoylcarnitine in the medium did not significantly increase in the MCAD knockdown. Similar results were obtained using pharmacological inhibition of CPT2 in fibroblasts from MCAD-deficient individuals. This shows that CPT2 and CACT are crucial for mitochondrial acylcarnitine formation and export to the extracellular fluids in mFAOD.

Selenium Significantly Inhibits Adipocyte Hypertrophy and Abdominal Fat Accumulation in OLETF Rats via Induction of Fatty Acid β-Oxidation

A combination of selenium (Se) with other trace element is associated with partially modulate fatty acid distribution as well as reduction of the body weight and feed efficiency. To investigate whether or not Se treatment has an impact on lipid metabolism, we examined the levels of lipid metabolism-related factors, including abdominal fat, adiponectin, cholesterol, very long chain dehydrogenase (VLCAD), and medium chain acyl-CoA dehydrogenase (MCAD) in 20-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats following sodium selenite treatment for 2weeks. Herein, we observed that (a) Se treatment induced insulin-like effects by lowering the serum glucose level in rats; (b) Se-treated rats showed significance values decreases in abdominal fat mass, adipocyte size, and adiponectin, which are associated with lipid metabolism; (c) Se treatment led to reduced levels of cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol; (d) fat tissue in Se-treated rats displayed significantly lower expression of adipocyte marker genes along with increased expression of VLCAD and MCAD; and (e) fatty liver formation and β-oxidation gene expression were both significantly reduced in liver tissue of Se-treated rats. Therefore, our results suggest that Se may induce inhibition of adipocyte hypertrophy and abdominal fat accumulation along with suppression of fatty liver formation by the differential regulation of the gene expression for fatty acid β-oxidation in the OLETF model.

Tissue-Specific Strategies of the Very-Long Chain Acyl-CoA Dehydrogenase-Deficient (VLCAD−/−) Mouse to Compensate a Defective Fatty Acid β-Oxidation

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD−/−) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD−/− mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD−/− mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD−/− mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD−/− mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.

Low Receptor Interacting Protein 140 (RIP140) Expression Adversely Impacts Proximal Insulin Signaling in L6 Skeletal Muscle Cells

We have shown that reduced expression of receptor interacting protein 140 (RIP140) alters the regulation of muscle fatty acid (FA) uptake and FA oxidation under basal and insulin‐stimulated conditions. To provide mechanistic insights for these changes, we measured the gene expression and protein content of proteins known to be involved in metabolic regulation. L6 myotubes were silenced using RNAi sequences for either RIP140 (RIP) or a negative control (NC) and incubated ± insulin (I: 1000 nM). As expected, RIP140 protein content (53%) and gene expression (37%) were decreased (P&lt;0.05) in RIP cells. Low RIP140 expression increased (P&lt;0.05) nerve growth factor IB (Nur77) gene expression (92%) but did not affect medium chain acyl‐CoA dehydrogenase (MCAD) expression. In NC cells, insulin did not affect MCAD or Nur77 expression while it decreased (P&lt;0.05) Nur77 expression in RIP cells. Insulin increased fibroblast growth factor 21(FGF21) protein content in NC cells, but it had no effect in RIP cells. Low RIP140 expression increased basal AKTSer473 phosphorylation and prevented the insulin‐induced increase in AKTSer473, AKTThr308 or PKC‐ξThr408/410 phosphorylation. We conclude that low RIP140 expression directly affects the expression of several proteins associated with metabolic regulation and adversely impacts the induction of proximal insulin signaling.USC Women in Science and Engineering, USC Integrative and Evolutionary Biology and USC Zumberge Research Innovation Fund