Fatty Acid Synthase Abundance Research Articles

Triazolo[4,5-d]pyrimidin-5-amines based ERK3 inhibitors fail to demonstrate selective effects on adipocyte function

Extracellular signal-regulated kinase 3 (ERK3 also designated MAPK6 — mitogen-activated protein kinase 6) is a ubiquitously expressed kinase participating in the regulation of a broad spectrum of physiological and pathological processes. Targeted inhibition of the kinase may allow the development of novel treatment strategies for a variety of types of cancer and somatic pathologies, as well as preserving metabolic health, combat obesity and diabetes. We chose and synthesized three triazolo [4,5-d]pyrimidin-5-amines proposed previously as putative ERK3 inhibitors to assess their selectivity and biological effects in terms of metabolic state impact in living cells. As it was previously shown that ERK3 is a major regulator of lipolysis in adipocytes, we focused on this process. Our new results indicate that in addition to the previously identified lipolytic enzyme ATGL, ERK3 also regulates hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL). Moreover, this kinase also promotes the abundance of fatty acid synthase (FASN) as well as protein kinase cAMP-activated catalytic subunit alpha (PKACα). To investigate various effects of putative ERK3 inhibitors on lipolysis, we utilized different adipocyte models. We demonstrated that molecules exhibit lipolysis-modulating effects; however, the effects of triazolo [4,5-d]pyrimidin-5-amines based inhibitors on lipolysis are not dependent on ERK3. Subsequently, we revealed a wide range of the compounds’ possible targets using a machine learning-based prediction.Therefore, the tested compounds inhibit ERK3 in vitro, but the biological effect of this inhibition is significantly overlapped and modified by some other molecular events related to the non-selective binding to other targets.

Exercise Down-Regulates Hepatic Lipogenic Enzymes in Food-Deprived and Refed Rats

The effects of an acute bout of prolonged exercise on the activities of several hepatic lipogenic enzymes and the abundance of fatty acid synthase (FAS) mRNA were evaluated using a food deprivation-refeeding protocol in which diets contained 50% of the energy from either fructose or cornstarch. Food was withheld from male rats for 48 h and refed for 0, 4, 8, 12, 24 or 48 h. At each time point, half of each dietary group was subjected to a single bout of treadmill running until exhaustion and killed immediately. The other half of each group rested without food for the same amount of time before being killed. Exercise significantly decreased FAS activity by 57, 46, 10, 26 and 70% at 4, 8, 12, 24 and 48 h of refeeding, respectively, in the fructose-fed rats; and by 70 and 63% at 24 and 48 h of refeeding, respectively, in the cornstarch-fed rats. Activities of L-type pyruvate kinase and glucose 6-phosphate dehydrogenase were significantly decreased after exercise in the fructose-fed, but not cornstarch-fed rats. In rested rats, FAS mRNA abundance increased ∼fourfold above the unfed levels after 8 and 12 h of refeeding. Exercise attenuated the diet-induced increases in FAS mRNA abundance. At 8 h of refeeding, both cornstarch- and fructose-fed exercised rats had 71% (P < 0.05) of the FAS mRNA levels of their rested counterparts; at 12 h, these exercised rats showed only 46 and 27% (P < 0.05) of FAS mRNA levels compared with rested rats fed the same diet. We conclude that dietary induction of FAS activity and mRNA abundance can be inhibited by prolonged exercise, suggesting that exercise may influence FAS transcription and/or mRNA stability.

Long-Term Dietary Supplementation with Betaine Improves Growth Performance, Meat Quality and Intramuscular Fat Deposition in Growing-Finishing Pigs.

This study was designed to investigate the effects of dietary betaine supplementation on growth performance, meat quality and muscle lipid metabolism of growing-finishing pigs. Thirty-six crossbred pigs weighing 24.68 ± 0.97 kg were randomly allotted into two treatments consisting of a basal diet supplemented with 0 or 1200 mg/kg betaine. Each treatment included six replications of three pigs per pen. Following 119 days of feeding trial, dietary betaine supplementation significantly enhanced average daily gain (ADG) (p < 0.05) and tended to improve average daily feed intake (ADFI) (p = 0.08) and decreased the feed intake to gain ratio (F/G) (p = 0.09) in pigs during 100~125 kg. Furthermore, a tendency to increase ADG (p = 0.09) and finial body weight (p = 0.09) of pigs over the whole period was observed in the betaine diet group. Betaine supplementation significantly increased a*45 min and marbling and decreased b*24 h and cooking loss in longissimus lumborum (p < 0.05), tended to increase intramuscular fat (IMF) content (p = 0.08), however had no significant influence on carcass characteristics (p > 0.05). Betaine supplementation influenced the lipid metabolism of pigs, evidenced by a lower serum concentration of low-density lipoprotein cholesterol (p < 0.05), an up-regulation of mRNA abundance of fatty acid synthase and acetyl-CoA carboxylase (p < 0.05), and a down-regulation of mRNA abundance of lipolysis-related genes, including the silent information regulators of transcription 1 (p = 0.08), peroxisome proliferator-activated receptorα (p < 0.05), peroxisome proliferator-activated receptor gamma coactivator-1α (p = 0.07) and carnitine palmitoyl transferase 1 (p < 0.05) in longissimus lumborum. Moreover, betaine markedly improved the expression of microRNA-181a (miR-181a) (p < 0.05) and tended to enhance miR-370 (p = 0.08). Overall, betaine supplementation at 1200 mg/kg could increase the growth performance of growing-finishing pigs. Furthermore, betaine had a trend to improve meat quality and IMF content via increasing lipogenesis and down-regulating the abundance of genes associated with lipolysis, respectively, which was associated with the regulation of miR-181a and miR-370 expression by betaine.

Oxidative stability of milk and expression of oxidative stress response, lipogenic-related, and IGF-1 genes in early-lactation cows supplemented with peanut and sunflower oils

Context Feeding dairy cows with plant oils rich in unsaturated fatty acids is a recommended strategy for increasing the proportion of polyunsaturated fatty acids in milk for human consumption. Aims We evaluated the effects of supplementation with peanut oil (high oleic acid content) and sunflower oil (high linoleic acid content) on the fatty acid profile and oxidative stability of milk and the expression of oxidative stress response, lipogenic-related and insulin-like growth factor 1 (IGF-1) genes in pasture-fed Jersey cows in early lactation. Methods Twenty-four cows were distributed in a randomised block design. Treatments were four dietary supplements of concentrate alone, or concentrate plus oil (59.6 g/kg dry matter): peanut oil, sunflower oil, or a 1:1 (v/v) mixture of peanut and sunflower oils. Analyses were performed at 30 and 60 days in milk. Key results Both peanut oil and mixed oil diets increased the hypocholesterolemic/hypercholesterolemic ratio (P &lt; 0.001) and health-promoting index (P &lt; 0.001) of milk. The sunflower oil diet increased the thrombogenicity index (P = 0.005) and conjugated diene production (P &lt; 0.001). mRNA abundance of fatty acid synthase (P = 0.003), acetyl-CoA carboxylase alpha (P = 0.001), and stearoyl-CoA desaturase (P &lt; 0.001) decreased with the sunflower oil diet. mRNA abundance of superoxide dismutase decreased (P = 0.01) with sunflower oil and mixed oil diets. The control diet tended (P = 0.08) to reduce IGF-1 mRNA abundance. Conclusions and implications Supplementation with peanut oil increases the health-promoting index of milk, whereas supplementation with sunflower oil increases susceptibility to lipid peroxidation. Supplementation with sunflower oil reduces the expression of lipogenic and antioxidant enzyme genes, and supplementation with unsaturated fats may reduce IGF-1 gene expression in early-lactation cows. The relatively small sample size means that more studies are needed.

Size, numberand phospholipid composition of milk fat globules are affected by dietary conjugated linoleic acid.

Milk fat globules (MFGs) surround the triacylglycerol core that composes milk fat. The aim of this study isto induce milk fat depression via dietary conjugated linoleic acid (CLA) supplementation to study MFG size parameters, numberand glycerophospholipid composition. Eighteen Holstein dairy cows (136 ± 28 days in milk, 571 ± 37.9 kg body weight, 27.6 ± 2.1 kg milk/day) were selected and randomly assigned to a control or CLA group for a 14-dayperiod. Cows were fed a basal diet (control, n = 8) or the control plus 400 g/day CLA (C18:2 cis-9, trans-11 38.1% and C18:2 trans-10, cis-12 36.8%) (n = 10) for 7 days after which the CLA group was switched to the basal diet for another 7 days along with the control group. Cow performance, milk composition, MFG size and numbers were measured daily. On the seventh day after the start of the experiment, milk samples were identified and the quantification of glycerophospholipid compounds, and RNA were isolated from milk fat samples for a real-time polymerase chain reaction. Compared with control, at Day 7 from the start of feeding, supplemental CLA did not affect milk production (28.09vs. 28.50 kg/day), dry matter intake (14.9vs. 15.4 kg/day), or milk protein (3.55/100vs. 3.70 g/100 ml) and lactose contents (5.11/100vs. 5.17 g/100 ml). However, although the specific surface area of MFG (2138vs. 1815 m²/kg) was greater, CLA reduced milk fat content (1.95/100vs 3.64 g/100 ml on Day 7) and particle size parameters of MFG. The number of MFG gradually decreased until Day 7 of feeding, and then increased by Day 14 (2.96 × 109 on Day 1, 1.63 × 109 on Day 7 and 2.28 × 109 on Day 14) in the CLA group. Compared with control, glycerophospholipid analysis revealed that concentrations of phosphatidylcholine (PC) (e.g., PC [16:0/18:1] 20322vs. 29793 nmol/L), lysophosphatidylethanolamine (LPE) (e.g., LPE [18:1] 956vs. 4610 nmol/L)and phosphatidylethanolamine (PE) (e.g., PE [16:0/18:1] 7000 vs. 9769 nmol/L) in milk lipids decreased during CLA feeding. In contrast, concentrations of phosphatidylinositol (PI) (e.g., PI [18:0/18:1] 4052vs. 1799 nmol/L) and phosphatidylserine (PS) (e.g., PS [18:1/18:2] 9500 vs. 6843 nmol/L) increased. The messenger RNA abundance of fatty acid synthase, diacylglycerol O-acyltransferase 1, glycerol-3-phosphate acyltransferase 4and phosphate cytidylyltransferase 1, choline, alpha (PCYT1A) were downregulated in the CLA group, confirming published data demonstrating a negative effect of CLA on lipogenesis in the mammary gland. Overall, these results provided evidence for the important role of lipogenic gene expression in the regulation of MFG size, number and glycerophospholipid composition.

Effect of maternal dietary starch-to-fat ratio and daily energy intake during late pregnancy on the performance and lipid metabolism of primiparous sows and newborn piglets.

The present study evaluated the effects of maternal dietary energy intake and starch-to-fat ratio during late gestation on the performance and lipid metabolism of sows and their offspring. On day 84 of gestation, 80 Landrace × Yorkshire primiparous sows were assigned to 2×2 factorial arrangements according to body weight following a randomized complete block design. The factors were daily energy intake (8,375 kcal ME/d [CE] vs. 9,600 kcal ME/d [HE]) and dietary starch-to-fat ratio (10:1 [CR] vs. 15:1 [HR]). All sows were fed one of four diets from day 85 of gestation until farrowing. Data were analyzed using the GLM procedure in SPSS. High energy intake increased the body weight of sows on day 110 of gestation (P = 0.031) as well as the weight of piglets at birth (P = 0.018). Increased energy intake elevated the plasma triglyceride concentrations in sows (P = 0.027) and piglets (P = 0.044). Maternal high energy intake altered the liver metabolome of newborn piglets in terms of metabolites related to carbohydrate and linoleic acid metabolism. Moreover, maternal high energy intake increased hepatic total cholesterol (P = 0.023) and triglyceride (P = 0.026) concentration in newborn piglets. Furthermore, maternal high energy intake significantly increased the transcript abundance of fatty acid synthase (FAS; P = 0.001) and protein abundance of phosphorylated protein kinase B (P =0.001) in the liver of newborn piglets. A high starch-to-fat ratio reduced low-density lipoprotein cholesterol (LDL-C) concentration in the plasma of sows (P = 0.044) and newborn piglets (P = 0.048) as well as in the liver of newborn piglets (P = 0.015). Furthermore, maternal high starch-to-fat ratio increased the transcript abundances of FAS (P = 0.004) in newborn piglets. In conclusion, high daily energy intake of sows increased the birth weight of newborn piglets. Moreover, maternal high daily energy intake and high dietary starch-to-fat ratio improved the lipid metabolism of newborn piglets.

Stearic acid does not overcome conjugated linoleic acid trans-10, cis-12-induced milk fat depression in lactating ewes.

The objective of this study was to test the hypothesis that stearic acid (SA) supplementation increases milk fat content and overcomes the antilipogenic effects of trans-10, cis-12 conjugated linoleic acid (CLA) in lactating ewes. Twenty-eight Lacaune ewes (36 (sd 2) days in lactation; 70·5 (sd) 9·6 kg of body weight), producing 1·8 (sd 0·4) kg of milk/d, were used in a completely randomised design (seven ewes/treatment) for 21 d. The treatments were: (1) Control; (2) CLA (6·4 g/d of trans-10, cis-12 CLA); (3) SA (28 g/d of SA) and (4) SA in association with trans-10, cis-12 CLA (CLASA; 6·4 g/d of trans-10, cis-12 CLA plus 28 g/d of SA). All data were analysed using a mixed model that included the fixed effect of treatment and the random effect of ewe. SA did not alter milk fat content and yield relative to Control (91·9 v. 91·2 (sd 4·1) g/d). CLASA was not able to overcome the reduction in fat content and fat yield induced by CLA (75 v. 82 (sd 0·14) g/d). SA increased the relative abundance of CD36, fatty acid-binding protein 4 (FABP4) and PPAR-γ mRNA by 140, 112 and 68 % compared with CLASA. SA also reduced the relative abundance of acetyl-CoA carboxylase α promoter II and stearoyl-CoA desaturase (SCD) when compared with Control (45 and 39 %). Compared with CLA, CLASA treatment had no effect on the mRNA abundance of fatty acid synthase, lipoprotein lipase, CD36, SCD, FABP4, acylglycerolphosphate acyltransferase 6, sterol regulatory element-binding protein 1 and PPAR-γ. In conclusion, SA supplementation did not increase milk fat synthesis and did not overcome the CLA-induced milk fat depression when associated with trans-10, cis-12 CLA.

Methionine supplementation during a hydrogen peroxide challenge alters components of insulin signaling and antioxidant proteins in subcutaneous adipose explants from dairy cows

Enhanced postruminal supply of methionine (Met) during the peripartal period alters protein abundance of insulin, AA, and antioxidant signaling pathways in subcutaneous adipose tissue (SAT). Whether SAT is directly responsive to supply of Met and can induce molecular alterations is unknown. Our objective was to examine whether enhanced Met supply during an oxidative stress challenge in vitro alters insulin, AA, inflammation, and antioxidant signaling-related protein networks. Four late-lactation Holstein cows (average 27.0 kg of milk per day) were used for SAT collection. Tissue was incubated in duplicate for 4 h in a humidified incubator with 5% CO2 at 37°C according to the following experimental design: control medium with an "ideal" profile of essential AA (CTR; Lys:Met 2.9:1), CTR plus 100 μM H2O2 (HP), or CTR with greater Met supply plus 100 μM H2O2 (HPMET; Lys:Met 2.5:1). Molecular targets associated with insulin signaling, lipolysis, antioxidant nuclear factor, erythroid 2 like 2 (NFE2L2), inflammation, and AA metabolism were determined through reverse-transcription quantitative PCR and western blotting. Data were analyzed using the MIXED procedure of SAS 9.4 (SAS Institute Inc.). Among proteins associated with insulin signaling, compared with CTR, HP led to lower abundance of phosphorylated AKT serine/threonine kinase (p-AKT) and solute carrier family 2 member 4 (SLC2A4; insulin-induced glucose transporter). Although incubation with HPMET restored abundance of SLC2A4 to levels in the CTR and upregulated abundance of fatty acid synthase (FASN) and phosphorylated 5'-prime-AMP-activated protein kinase (p-AMPK), it did not alter p-AKT, which remained similar to HP. Among proteins associated with AA signaling, compared with CTR, challenge with HP led to lower abundance of phosphorylated mechanistic target of rapamycin (p-MTOR), and HPMET did not restore abundance to CTR levels. Among inflammation-related targets studied, incubation with HPMET led to greater protein abundance of nuclear factor kappa B subunit p65 (NFKB-RELA). The response in NFKB observed with HPMET was associated with a marked upregulation of the antioxidant transcription regulator NFE2L2 and the antioxidant enzyme glutathione peroxidase 1 (GPX1). No effects of treatment were detected for mRNA abundance of proinflammatory cytokines or antioxidant enzymes, underscoring the importance of post-transcriptional regulation. Overall, data indicated that short-term challenge with H2O2 was particularly effective in reducing insulin and AA signaling. Although a greater supply of Met had little effect on those pathways, it seemed to restore the protein abundance of the insulin-induced glucose transporter. Overall, the concomitant upregulation of key inflammation and antioxidant signaling proteins when a greater level of Met was supplemented to oxidant-challenged SAT highlighted the potential role of this AA in regulating the inflammatory response and oxidant status. Further studies should be conducted to assess the role of postruminal supply of Met and other AA in the regulation of immune, antioxidant, and metabolic systems in peripartal cow adipose tissue.

Insulin signaling and glucose metabolism in different hepatoma cell lines deviate from hepatocyte physiology toward a convergent aberrant phenotype

Hepatoma cell lines are widely used to model the hepatocyte for insulin signaling and fatty liver disease. However, a direct comparison of insulin action in primary hepatocytes and in hepatoma cell lines is needed to validate this model and to better understand liver cancer. Here we have investigated insulin signaling, gluconeogenic gene expression, glucose production, and fatty acid synthase abundance in primary hepatocytes and in HepG2, Hepa 1–6, and McARH7777 hepatoma cell lines. Differences in the electrophoretic profiles of protein extracts from human and mouse primary hepatocytes and the hepatoma cells lines are shown. Compared to primary hepatocytes, hepatoma cells showed high basal phosphorylation of AKT at Thr 308 and constitutively activated RAS-MAPK signaling, which were resistant to the dominant negative Ras mutant H-Ras17N. Hepatoma cell lines also showed defective expression of gluconeogenic enzymes, insulin unresponsive GSK phosphorylation, and marginal glucose production. Hepatoma cells also showed lower protein levels of fatty acid synthase and a largely distinct protein electrophoresis profile from hepatocytes but similar between different hepatoma lines. We conclude that hepatoma cell lines do not accurately model the hepatocyte for insulin action but may be valuable tools to investigate the proteomic changes conferring to hepatocellular carcinoma its peculiar metabolisms.

Knockout of butyrophilin subfamily 1 member A1 (BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells

BackgroundMilk lipids originate from cytoplasmic lipid droplets (LD) that are synthesized and secreted from mammary epithelial cells by a unique membrane-envelopment process. Butyrophilin 1A1 (BTN1A1) is one of the membrane proteins that surrounds LD, but its role in bovine mammary lipid droplet synthesis and secretion is not well known.MethodsThe objective was to knockout BTN1A1 in bovine mammary epithelial cells (BMEC) via the CRISPR/Cas9 system and evaluate LD formation, abundance of lipogenic enzymes, and content of cell membrane phospholipid (PL) species. Average LD diameter was determined via Oil Red O staining, and profiling of cell membrane phospholipid species via liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsLentivirus-mediated infection of the Cas9/sgRNA expression vector into BMEC resulted in production of a homozygous clone BTN1A1(−/−). The LD size and content decreased following BTN1A1 gene knockout. The mRNA abundance of fatty acid synthase (FASN) and peroxisome proliferator-activated receptor-gamma (PPARG) was downregulated in the BTN1A1(−/−) clone. Subcellular analyses indicated that BTN1A1 and LD were co-localized in the cytoplasm. BTN1A1 gene knockout increased the percentage of phosphatidylethanolamine (PE) and decreased phosphatidylcholine (PC), which resulted in a lower PC/PE ratio.ConclusionsResults suggest that BTN1A1 plays an important role in regulating LD synthesis via a mechanism involving membrane phospholipid composition.

Changes in lipid metabolism and β-adrenergic response of adipose tissues of periparturient dairy cows affected by an energy-dense diet and nicotinic acid supplementation.

Dairy cattle will mobilize large amounts of body fat during early lactation as an effect of decreased lipogenesis and increased lipolysis. Regulation of lipid metabolism involves fatty acid synthesis from acetate and β-adrenergic-stimulated phosphorylation of hormone-sensitive lipase (HSL) and perilipin in adipocytes. Although basic mechanisms of mobilizing fat storage in transition cows are understood, we lack a sufficiently detailed understanding to declare the exact regulatory network of these in a broad range of dairy cattle. The objective of the present study was to quantify 1) protein abundance of fatty acid synthase (FAS), 2) extent of phosphorylation of HSL and perilipin in vivo, and 3) β-adrenergic stimulated lipolytic response of adipose tissues in vitro at different stages of the periparturient period. We fed 20 German Holstein cows an energy-dense or an energetically adequate diet prepartum and 0 or 24 g/d nicotinic acid (NA) supplementation. Biopsy samples of subcutaneous and retroperitoneal adipose tissue were obtained at d 42 prepartum (d -42) and at d 1, 21, and 100 postpartum (d +1, d +21, d +100, respectively). To assess β-adrenergic response, tissue samples were incubated with 1 μ isoproterenol for 90 min at 37°C. The NEFA and glycerol release, as well as HSL and perilipin phosphorylation, was measured as indicators of in vitro stimulated lipolysis. In addition, protein expression of FAS and extent of HSL and perilipin phosphorylation were measured in fresh, nonincubated samples. There was no effect of dietary energy density or NA on the observed variables. The extent of HSL and perilipin phosphorylation under isoproterenol stimulation was strongly correlated with the release of NEFA and glycerol, consistent with the functional link between β-adrenergic-stimulated protein phosphorylation and lipolysis. In the nonincubated samples, FAS protein expression was decreased at d +1 and d +21, whereas HSL and perilipin phosphorylation increased from d -42 to d +1 and remained at an increased level throughout the first 100 d of lactation. In vitro lipolytic response was significant in prepartum samples at times when in vivo lipolysis was only minimally activated by phosphorylation. These data extend our understanding of the complex nature of control of lipolysis and lipogenesis in dairy cows and could be useful to the ongoing development of systems biology models of metabolism to help improve our quantitative knowledge of the cow.

Dietary supplementation of chinese ginseng prevents obesity and metabolic syndrome in high-fat diet-fed mice.

Obesity and diabetes are growing health problems worldwide. In this study, dietary provision of Chinese ginseng (0.5 g/kg diet) prevented body weight gain in high-fat (HF) diet-fed mice. Dietary ginseng supplementation reduced body fat mass gain, improved glucose tolerance and whole body insulin sensitivity, and prevented hypertension in HF diet-induced obese mice. Ginseng consumption led to reduced concentrations of plasma insulin and leptin, but had no effect on plasma adiponectin levels in HF diet-fed mice. Body temperature was higher in mice fed the ginseng-supplemented diet but energy expenditure, respiration rate, and locomotive activity were not significantly altered. Dietary intake of ginseng increased fatty acid oxidation in the liver but not in skeletal muscle. Expression of several transcription factors associated with adipogenesis (C/EBPα and PPARγ) were decreased in the adipose tissue of HF diet-fed mice, effects that were mitigated in mice that consumed the HF diet supplemented with ginseng. Abundance of fatty acid synthase (FASN) mRNA was greater in the adipose tissue of mice that consumed the ginseng-supplemented HF diet as compared with control or un-supplemented HF diet-fed mice. Ginseng treatment had no effect on the expression of genes involved in the regulation of food intake in the hypothalamus. These data suggest that Chinese ginseng can potently prevent the development of obesity and insulin resistance in HF diet-fed mice.

Effect of Breed on Fatty Acid Composition and Lipogenic Enzyme Abundance in the Subcutaneous Adipose Tissue of Pigs

This study investigated the role of lipogenic enzyme expression in breed-specific fat deposition in pigs. (i) determine effect of breed on the relative abundance of the key lipogenic enzymes stearoyl-CoA desaturase (SCD), delta-6 desaturase (Δ6D), and fatty acid synthase (FAS) in pig subcutaneous adipose tissue. (ii) Investigate breed-specific relationships between lipogenic enzyme abundance and fatty acid composition. Large White × Piétrain, Piétrain, and Duroc × Piétrain pigs were used. Expression of SCD, Δ6D, and FAS was analyzed by Western blotting. Fatty acid composition was determined by gas chromatography. FAS protein in Large White × Piétrain pigs was similar to the Piétrain breed, but was significantly higher than Duroc × Piétrain. A positive relationship was found between FAS abundance and the saturated fatty acids (SFAs), for Large White × Piétrain pigs, but not for the other breeds. Δ6D was significantly higher in Large White × Piétrain compared with Duroc × Piétrain and Piétrain. This was accompanied by significantly higher total n-3 poly-unsaturated fatty acids (PUFAs) in the Large White × Piétrain when compared to the other breeds. (i) increased subcutaneous adipose tissue SFA content in Large White × Piétrain pigs (but not Piétrain and Duroc × Piétrain) is related to increased abundance of FAS protein; (ii) high n-3 PUFA content in Large White × Piétrain pigs is related to activation of Δ6D protein synthesis; (iii) SCD and Δ6D abundance does not contribute to between-breed differences in MUFA and n-6 PUFA content of pig subcutaneous adipose tissue.

Intravenous infusions of glucose stimulate key lipogenic enzymes in adipose tissue of dairy cows in a dose-dependent manner

The present study was investigated whether increasing amounts of glucose supply have a stimulatory effect on the mRNA abundance and activity of key lipogenic enzymes in adipose tissue of midlactation dairy cows. Twelve Holstein-Friesian dairy cows in midlactation were cannulated in the jugular vein and infused with either a 40% glucose solution (n=6) or saline (n=6). For glucose infusion cows, the infusion dose increased by 1.25%/d relative to the initial net energy for lactation (NEL) requirement until a maximum dose equating to a surplus of 30% NEL was reached on d 24. This maximum dose was maintained until d 28 and stopped thereafter (between d 29–32). Cows in the saline infusion group received an equivalent volume of 0.9% saline solution. Samples of subcutaneous adipose tissue were taken on d 0, 8, 16, 24, and 32 when surplus glucose reached 0, 10, 20, and 30% of the NEL requirement, respectively. The mRNA abundance of fatty acid synthase, cytoplasmic acetyl-coenzyme A synthetase, cytoplasmic glycerol 3-phosphate dehydrogenase-1, and glucose 6-phosphate dehydrogenase showed linear treatment × dose interactions with increasing mRNA abundance with increasing glucose dose. The increased mRNA abundance was paralleled by a linear treatment × dose interaction for fatty acid synthase and acetyl-coenzyme A synthetase enzymatic activities. The mRNA abundance of ATP-citrate lyase showed a tendency for linear treatment × dose interaction with increasing mRNA abundance with increasing glucose dose. The mRNA abundance of all tested enzymes, as well as the activities of fatty acid synthase and acetyl-coenzyme A synthetase, correlated with plasma glucose and serum insulin levels. In a multiple regression model, the predictive value of insulin was dominant over that of glucose. In conclusion, gradual increases in glucose supply upregulate key lipogenic enzymes in adipose tissue of midlactating dairy cows with linear dose dependency. Insulin appears to be critically involved in this regulation.

Modulation of Fatty Acid Synthase Enzyme Activity and Expression during Hepatitis C Virus Replication

The hepatitis C virus (HCV) induces alterations of host cells to facilitate its life cycle. Fatty acid synthase (FASN) is a multidomain enzyme that plays a key role in the biosynthesis of fatty acids and is upregulated during HCV infection. Herein, we applied activity-based protein profiling (ABPP) that allows for the identification of differentially active enzymes in complex proteomic samples, to study the changes in activity of FASN during HCV replication. For this purpose, we used an activity-based probe based on the FASN inhibitor Orlistat, and observed an increase in the activity of FASN in the presence of a subgenomic and a genomic HCV replicon as well as in chimeric SCID/Alb-uPA mice infected with HCV genotype 1a. To study the molecular basis for this increase in FASN activity, we overexpressed individual HCV proteins in Huh7 cells and observed increased expression and activity of FASN in the presence of core and NS4B, as measured by western blots and ABPP, respectively. Triglyceride levels were also elevated in accordance with FASN expression and activity. Lastly, immunofluorescence and ABPP imaging analyses demonstrated that while the abundance and activity of FASN increases significantly in the presence of HCV, its localization does not change. Together these data suggest that the HCV-induced production of fatty acids and neutral lipids is provided by an increase in FASN abundance and activity that is sufficient to allow HCV propagation without transporting FASN to the replication complexes.

Activation of liver X receptor (LXR) enhances de novo fatty acid synthesis in bovine mammary epithelial cells

Liver X receptor (LXR) is a nuclear receptor and a known regulator of lipid synthesis in rodents; however, the role of LXR in the regulation of fatty acid synthesis in bovine mammary epithelial cells has not yet been defined. The objective of the study was to evaluate the effect of LXR activation on the de novo synthesis of fatty acids in bovine mammary epithelial cells (BME-UV). Bovine mammary epithelial cells were treated with T0901317 (T09), an LXR agonist. Treatment of BME-UV with T09 increased the transcription of ATP-binding cassette transporter-G1, an LXR target gene, without modifying LXRα mRNA abundance. Acute and chronic treatment of BME-UV with T09 dramatically increased de novo fatty acid synthesis. Activation of LXR resulted in the upregulation of transcription, translation, and proteolytic cleavage of sterol regulatory element-binding protein-1 (SREBP1), a lipogenic transcription factor expressed in the bovine mammary gland. Additionally, the mRNA abundance of fatty acid synthase, an LXR and SREBP1 target gene, increased in response to LXR activation. Our data indicate that SREBP1 is regulated by LXR activation in BME-UV. Controlling LXR activation may prove useful in regulating milk fat production in lactating dairy cows.

Hyperglycaemia confers resistance to chemotherapy on breast cancer cells: the role of fatty acid synthase

The prognosis for women with breast cancer is adversely affected by the comorbidities of obesity and diabetes mellitus (DM), which are conditions associated with elevated levels of circulating fatty acids, hyperglycaemia and hyperinsulinaemia. We investigated the effects of exposure of non-malignant and malignant human breast epithelial cells to elevated levels of fatty acids and glucose on their growth, survival and response to chemotherapeutic agents. We found that palmitate induced cell death in the non-malignant cells but not in the malignant cells, which was abrogated through the inhibition of ceramide production and by oleate but not by IGF1. Fatty acid synthase (FAS) is responsible for the de novo synthesis of fatty acids from sugars, and is over-expressed in many epithelial cancers. Abundance of FAS was higher in malignant cells than in non-malignant cells, and was up-regulated by IGF1 in both cell types. IGF-induced growth of non-malignant cells was unaffected by suppression of FAS expression, whereas that of malignant cells was blocked as was their resistance to palmitate-induced cell death. Palmitate did not affect cell proliferation, whereas oleate promoted the growth of non-malignant cells but had the opposite effect, that is, inhibition of IGF1-induced growth of malignant cells. However, when the phosphatidylinositol 3-kinase pathway was inhibited, oleate enhanced IGF1-induced growth in both cell types. Hyperglycaemia conferred resistance on malignant cells, but not on non-malignant cells, to chemotherapy-induced cell death. This resistance was overcome by inhibiting FAS or ceramide production. Understanding the mechanisms involved in the associations between obesity, DM and breast cancer may lead to more effective treatment regimens and new therapeutic targets.

Trans-11 Vaccenic Acid Reduces Hepatic Lipogenesis and Chylomicron Secretion in JCR:LA-cp Rats1–3

Trans-11 vaccenic acid (VA) is the predominant trans isomer in ruminant fat and a major precursor to the endogenous synthesis of cis9,trans11-conjugated linoleic acid in humans and animals. We have previously shown that 3-wk VA supplementation has a triglyceride (TG)-lowering effect in a rat model of dyslipidemia, obesity, and metabolic syndrome (JCR:LA-cp rats). The objective of this study was to assess the chronic effect (16 wk) of VA on lipid homeostasis in both the liver and intestine in obese JCR:LA-cp rats. Plasma TG (P < 0.001), total cholesterol (P < 0.001), LDL cholesterol (P < 0.01), and nonesterified fatty acid concentrations, as well as the serum haptoglobin concentration, were all lower in obese rats fed the VA diet compared with obese controls (P < 0.05). In addition, there was a decrease in the postprandial plasma apolipoprotein (apo)B48 area under the curve (P < 0.05) for VA-treated obese rats compared with obese controls. The hepatic TG concentration and the relative abundance of fatty acid synthase and acetyl-CoA carboxylase proteins were all lower (P < 0.05) in the VA-treated group compared with obese controls. Following acute gastrointestinal infusion of a VA-triolein emulsion in obese rats that had been fed the control diet for 3 wk, the TG concentration was reduced by 40% (P < 0.05) and the number of chylomicron (CM) particles (apoB48) in nascent mesenteric lymph was reduced by 30% (P < 0.01) relative to rats infused with a triolein emulsion alone. In conclusion, chronic VA supplementation significantly improved dyslipidemia in both the food-deprived and postprandial state in JCR:LA-cp rats. The appreciable hypolipidemic benefits of VA may be attributed to a reduction in both intestinal CM and hepatic de novo lipogenesis pathways.

Feeding conjugated linoleic acid partially recovers carcass quality in pigs fed dried corn distillers grains with solubles

Dried corn distillers grains with solubles (DDGS) fed to swine may adversely affect carcass quality due to the high concentration of unsaturated fat. Feeding CLA enhances pork quality when unsaturated fat is contained in the diet. The effects of CLA on growth and pork quality were evaluated in pigs fed DDGS. Diets containing 0, 20, or 40% DDGS were fed to pigs beginning 30 d before slaughter. At 10 d before slaughter, one-half of each DDGS treatment group was fed 0.6% CLA or 1% choice white grease. Carcass data, liver- and backfat-samples were collected at slaughter. Longissimus muscle area, 10th-rib back-fat depth, last rib midline backfat depth, LM color, marbling, firmness and drip loss, and bacon collagen content were not altered by DDGS or CLA. Outer layer backfat iodine values were increased (P <or= 0.05) with DDGS feeding and were 65.07, 69.75, and 74.25 for 0, 20, and 40% DDGS, respectively. Addition of CLA decreased (P <or= 0.05) outer layer backfat iodine values from 71.11 to 68.31. Diets containing DDGS decreased (P <or= 0.05) percent lean tissue contained in bacon from 48% for controls to 38% for pigs fed 40%. Abundance of fatty acid synthase, carnitine palmitoyl transferase Ia, acetyl-CoA-carboxylase, stearoyl-CoA desaturase, and glycerol-3-phosphate dehydrogenase mRNA in adipose or liver were not different (P > 0.05) for pigs fed DDGS. Feeding CLA decreased (P <or= 0.05) the Delta(9) de-saturase index in adipose tissue. The data indicate that decreased carcass firmness with DDGS feeding is not reflected by changes in lipogenic gene expression. Feeding 20% or more DDGS to finishing swine decreases bacon leanness, but inclusion of 0.6% CLA in the finishing diet can partially reverse these effects.

Effects of trans-10, cis-12 conjugated linoleic acid on gene expression and lipid metabolism of adipose tissue of growing pigs

The objective of the present study was to determine the effects of trans-10, cis-12 conjugated linoleic acid (CLA) in adipose tissue explant cultures of growing pigs on the following responses: lipogenesis (measured as rate of (14)C-labeled glucose incorporation over a subsequent 2-h incubation in the presence or absence of insulin), lipolysis (release of non-esterified fatty acid over a 2-h incubation in the presence or absence of isoproterenol), activities of lipogenic enzymes, and mRNA abundance of fatty acid synthase (FAS). Adipose tissue explants from nine growing pigs (78 +/- 3 kg) were cultured in 199 medium with insulin, dexamethasone and antibiotics for 4, 12, 24, and 48 h. The treatments were 1) control: 100 microM polyvinyl alcohol (PVA); 2) pGH: 100 ng/mL porcine growth hormone (pGH) plus 100 microM PVA; 3) CLA200: 200 microM trans-10, cis-12 CLA; 4) CLA50: 50 microM trans-10, cis-12 CLA, and 5) LA: 200 microM linoleic acid. Fatty acids were added along with PVA (2:1), respectively, for 24 h. Explants were collected after each culture period and assayed for lipogenesis. Transcripts of FAS mRNA were quantified by real-time RT-PCR after 24 and 48 h. Lipolysis and activities of FAS, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-malate dehydrogenase were determined after 48 h. As expected, glucose incorporation was decreased (P < 0.05) in response to pGH treatment (positive control). LA had no effect on any parameter evaluated. Treatment with trans-10, cis-12 CLA decreased FAS activity (P < 0.05), but NADPH-generating enzymes were unaffected by treatments. Consistent with reduction in FAS activity, both lipid synthesis and FAS mRNA abundance were reduced with chronic CLA treatment, pGH increased baseline and stimulated lipolysis (P < 0.05) after 48 h of culture, while CLA treatment had no effect on non-esterified fatty acid release. Results of this study showed that trans-10, cis-12 CLA alters lipogenesis but has no effect on lipolysis in cultures of pig adipose tissue.