steroyl-CoA Desaturase Research Articles

THU631 Steroyl-CoA Desaturase Is A Progesterone Receptor Target Gene In Uterine Leiomyoma

Abstract Disclosure: A.S. Komorowski: None. A. Zuberi: None. J.S. Coon V: None. M.L. Anton: None. S.E. Bulun: None. P. Yin: None. Uterine leiomyomas (LM) are common benign neoplasms affecting approximately one in four reproductive-age women. LM can cause menorrhagia, dysmenorrhea, anemia, and infertility. Steroyl-CoA desaturase (SCD) is a delta-9 fatty acid desaturase which converts saturated fatty acids to monounsaturated fatty acids in a reaction that is critical to maintenance of fatty acid homeostasis. Prior work has demonstrated upregulation of SCD in multiple cancer types as part of lipid metabolic reprogramming, and SCD has been identified as a potential drug target for cancer and non-alcoholic steatohepatitis. Growth of LM tumors is progesterone-dependent, and we found that progesterone receptor (PR)-binding sites were enriched in the SCD gene locus. Normalized RNA-seq count demonstrated that knockdown of PR decreased SCD expression in LM cells. Further experiments with knockdown of PR in LM cells demonstrated decreased SCD expression on both quantitative real-time PCR and Western blot. Treatment of LM cells with progesterone for 6 hours lead to mild upregulation of SCD expression. In summary, steroyl-CoA desaturase, an enzyme involved in lipid homeostasis, is upregulated by progesterone and may represent a novel target for treatment of uterine LM. Presentation: Thursday, June 15, 2023

Development of real-time PCR assay for genotyping SNP rs41255693 in cattle <em>SCD</em> gene

Increased content of monounsaturated fatty acids with low melting points in cattle milk and meat known to strongly affect their taste and nutritional value. Steroyl-CoA-desaturase (SCD) is a key enzyme in the biosynthesis of monounsaturated fatty acids. The gene encoding SCD in cattle is located on chromosome 26 and consists of 6 exons and 5 introns and is expressed in the cells of cow udder, adipose, muscles and other tissues. To date, several SNPs in cattle SCD gene have been found. For SNP rs41255693 (g.10329T > C), a correlation was shown between the content of monounsaturated fatty acids in cattle milk/meat and milk productivity. This article describes the development of a real-time PCR assay for genotyping SNP rs41255693 in cattle SCD gene with allele-specific TaqMan probes. The use of this assay allows to significantly reduce the time needed for cattle genotyping compared to the commonly used PCR-RFLP method.

3158 - SCD Takes Leukemia to the CNS

Background: In acute lymphoblastic leukemia (ALL), central nervous system (CNS) involvement is a major clinical concern with no specific therapy. Prophylactic CNS-directed intrathecal chemotherapy or irradiation are required for relapse-free survival but imply long term toxicity. Therefore, mechanisms mediating leukemia-cell entry and survival into the CNS need to be understood to develop alternative CNS-directed treatment strategies. Previous studies showed an increased expression of Steroyl CoA desaturase (SCD) in BCP-ALL cells isolated from cerebrospinal fluid (CSF) of patients at CNS relapse. Aim: Investigate if SCD have a role in leukemia trafficking, homing and survival into the CNS in order to design new diagnostic and reduced toxicity- treatments to prevent relapse. Methods:Microarray profiling of cells isolated from CNS and BM of xeno-transplanted mice was performed.Human BCP-ALL cell line 018z was transduced to knock-down (KD) or overexpress human SCD. In vivo, SCD KD and overexpressing cells were transplanted in NSG mice who were sacrificed upon the first symptoms of CNS involvement. BM, spleen and meninges were collected and analyzed to check human engraftment by FACS. In Competition assays, SCD overexpressing and WT cells were transplanted in the same mouse in a 1:1 ratio. Results: Microarray profiling of cells collected from BM and meninges of leukemic mice revealed up-regulation of the genes belonging to the signaling pathway of sterol regulatory element binding proteins (SREBPs) in ALL cells that had entered the CNS, includingSCD. In vitro, SCD-KD 018z cells showed reduced proliferation than WT while SCD overexpression did not alter proliferation.Mice transplanted with SCD KD cells showed a general lower infiltration whereas SCD overexpression led to a faster onset of CNS disease.Competition assays showed that in the CNS the balance was skewed towards engraftment of SCD overexpressing cells over WT. Conclusion: SCD has a role in homing and survival of leukemic cells in the CNS.The research can lead to innovative therapies that will minimize the need for intensive chemotherapy in the CNS.

Zingiber officinale extract administration diminishes steroyl-CoA desaturase gene expression and activity in hyperlipidemic hamster liver by reducing the oxidative and endoplasmic reticulum stress

BackgroundStearoyl CoA desaturases (SCD) are enzymes that convert saturated to monounsaturated fatty acids and have increased activity in hepatic steatosis. PurposeWe aimed to investigate the potential of ginger extract (GIN) to modulate the liver SCD1 expression and activity in hyperlipidemic (HL) conditions, in order to lower lipid accumulation in the steatotic liver. Study design/MethodsMale Golden Syrian hamsters were divided in three groups: (i) fed with standard chow (N), (ii) fed with standard chow plus 3% cholesterol and 15% butter for 21 weeks (HL), (iii) HL treated with GIN (800 µg/kg body weight/day) in the last 5 weeks of fat diet (HL-GIN). Cholesterol (C), triglycerides (TG), non-esterified fatty acids (NEFA), SCD1 estimated activity (C16:1n7/C16:0; C18:1n9/C18:0) and gene expression, acetyl-CoA carboxylase (ACC), thiobarbituric acid reactive substances (TBARS), paraoxonase1 (PON1) and myeloperoxidase (MPO) were determined in the plasma and liver of all hamsters. We measured protein expression of endoplasmic reticulum stress (ERS) markers, gene and protein expression of liver X receptor α/β (LXRα/β), peroxisome proliferator-activated receptor γ (PPARγ), ATP-binding cassette sub-family G member 5/8 (ABCG5/G8) and 7α-hydroxylase1 (CYP7A1) in all hamsters’ livers. ResultsIn plasma, in HL-GIN versus HL hamsters, SCD1 estimated activity was lower (27%; 15%, p < 0.05), NEFA levels decreased by 91%, p < 0.001, while C and TG levels did not vary; the oxidative stress expressed as MPO and TBARS levels decreased (15%; 11%, p < 0.01), while PON1 protein increased (75%, p < 0.05). In the liver of HL-GIN versus HL, C, TG, NEFA, MPO and TBARS levels decreased (8–40%, p < 0.05) and PON1 protein levels increased (30%, p < 0.05), SCD1 estimated activity decreased (8%; 9%, p < 0.05), in parallel with the reduced gene expression of SCD1 and ACC (70–80%, p < 0.05). The protein expression of the ERS sensors decreased (30–65%, p < 0.05), while that of ABCG5/G8, CYP7A1, LXRα/β and PPARγ increased in HL-GIN (20–30%, p < 0.05) versus HL liver. ConclusionGIN reduces SCD1 estimated activity and expression, as well as the lipids accumulated in the livers of HL hamsters. This is achieved through a mechanism involving the decrease of the oxidative and ERS, and the enhancement of cholesterol efflux.

Hepatocyte‐specific depletion of augmenter of liver regeneration (ALR) protein alters miRNA signature linked to lipid homeostasis leading to excessive steatosis

Augmenter of liver regeneration (ALR) is a fundamental life protein expressed in all mammalian organs. ALR's presence in mitochondria is essential for their function and survival. Depletion of ALR from liver (hepatocyte)‐specific ALRflox/flox/Alb‐Cre (ALR‐L‐KO) mice causes mitochondrial injury, robust lipid accumulation and apoptosis between 1 and 2 weeks post‐birth. Steatosis is regressed upon reappearance of ALR by 4 weeks but continued death of Cre‐expressing hepatocytes induces regeneration, inflammation and fibrosis. To delineate mechanisms of this pathological progression, we investigated whether ALR depletion alters expression of micro‐RNAs (miRNAs), which play important roles in numerous biological processes including lipid homeostasis. We performed miRNA‐seq analysis and profiled the hepatic miRNA expression in 1‐, 2‐, and 4‐week old mice. Of the 765 micro‐RNAs identified in WT and ALR‐L‐KO livers, 203 showed differential expression: 125, 106 and 141 miRNAs were up‐regulated and 70, 97 and 61 miRNAs down‐regulated at 1, 2 and 4 weeks respectively in ALR‐L‐KO compared to the WT mice. miR‐708‐3p, miR‐540‐3p and miR‐541‐5p, which have 3′UTR‐binding sites for peroxisome proliferator‐activated receptor (PPAR)α, carnitine palmitoyl transferase a (CPT1a) and mitochondrial transcription factor A (TFAM) (all down‐regulated at 2 weeks in ALR‐L‐KO mice), were all found to be up‐regulated at 2 weeks in ALR‐L‐KO compared to the WT mice. Increase in these miRNAs upon ALR depletion was also concurrent with increased expression of elongation of very long fatty acids 6 (ELOVl6) and steroyl CoA desaturase (SCD1) that are involved in de novo lipogenesis, and decreased expression of acyl CoA oxidase 1 (ACOX1) and CPT1 that promote β‐oxidation of fatty acids. The effect of ALR depletion on these miRNAs was recapitulated in vitro in cultured hepatocytes. Likewise, hepatic expression of miRNAs implicated in inflammation and fibrosis (miR‐199a‐1‐5p, miR‐146b‐5p, miR‐194‐1‐5p, miR‐1843a, miR‐410‐3p and miR‐434‐3p) was also altered in a way to promote these pathologies. These findings suggest that ALR's regulation of miRNAs is critical to hepatic lipid homeostasis with potential implications in nonalcoholic fatty liver disease/steatohepatitis.Support or Funding InformationThis work was supported by DoD grant W81XWH‐14‐PRMRP‐IIRA and NIH R21 AA020846 to CRG.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Maternal Obesity on Placental Lipid Metabolism.

Obese women, on average, give birth to babies with high fat mass. Placental lipid metabolism alters fetal lipid delivery, potentially moderating neonatal adiposity, yet how it is affected by maternal obesity is poorly understood. We hypothesized that fatty acid (FA) accumulation (esterification) is higher and FA β-oxidation (FAO) is lower in placentas from obese, compared with lean women. We assessed acylcarnitine profiles (lipid oxidation intermediates) in mother-baby-placenta triads, in addition to lipid content, and messenger RNA (mRNA)/protein expression of key regulators of FA metabolism pathways in placentas of lean and obese women with normal glucose tolerance recruited at scheduled term Cesarean delivery. In isolated trophoblasts, we measured [3H]-palmitate metabolism. Placentas of obese women had 17.5% (95% confidence interval: 6.1, 28.7%) more lipid than placentas of lean women, and higher mRNA and protein expression of FA esterification regulators (e.g., peroxisome proliferator-activated receptor γ, acetyl-CoA carboxylase, steroyl-CoA desaturase 1, and diacylglycerol O-acyltransferase-1). [3H]-palmitate esterification rates were increased in trophoblasts from obese compared with lean women. Placentas of obese women had fewer mitochondria and a lower concentration of acylcarnitines, suggesting a decrease in mitochondrial FAO capacity. Conversely, peroxisomal FAO was greater in placentas of obese women. Altogether, these changes in placental lipid metabolism may serve to limit the amount of maternal lipid transferred to the fetus, restraining excess fetal adiposity in this population of glucose-tolerant women.

Differential expression of skeletal muscle genes following administration of clenbuterol to exercised horses.

BackgroundClenbuterol, a beta2-adrenergic receptor agonist, is used therapeutically to treat respiratory conditions in the horse. However, by virtue of its mechanism of action it has been suggested that clenbuterol may also have repartitioning affects in horses and as such the potential to affect performance. Clenbuterol decreases the percent fat and increases fat-free mass following high dose administration in combination with intense exercise in horses. In the current study, microarray analysis and real-time PCR were used to study the temporal effects of low and high dose chronic clenbuterol administration on differential gene expression of several skeletal muscle myosin heavy chains, genes involved in lipid metabolism and the β2-adrenergic receptor. The effect of clenbuterol administration on differential gene expression has not been previously reported in the horse, therefore the primary objective of the current study was to describe clenbuterol-induced temporal changes in gene expression following chronic oral administration of clenbuterol at both high and low doses.ResultsSteady state clenbuterol concentrations were achieved at approximately 50 h post administration of the first dose for the low dose regimen and at approximately 18–19 days (10 days post administration of 3.2 μg/kg) for the escalating dosing regimen. Following chronic administration of the low dose (0.8 μg/kg BID) of clenbuterol, a total of 114 genes were differentially expressed, however, none of these changes were found to be significant following FDR adjustment of the p-values. A total of 7,093 genes were differentially expressed with 3,623 genes up regulated and 3,470 genes down regulated following chronic high dose administration. Of the genes selected for further study by real-time PCR, down-regulation of genes encoding myosin heavy chains 2 and 7, steroyl CoA desaturase and the β2-adrenergic receptor were noted. For most genes, expression levels returned towards baseline levels following cessation of drug administration.ConclusionThis study showed no evidence of modified gene expression following chronic low dose administration of clenbuterol to horses. However, following chronic administration of high doses of clenbuterol alterations were noted in transcripts encoding various myosin heavy chains, lipid metabolizing enzymes and the β2-adrenergic receptor.

The Crystal Structure of an Integral Membrane Fatty Acid α-Hydroxylase

Neuronal electrical impulse propagation is facilitated by the myelin sheath, a compact membrane surrounding the axon. The myelin sheath is highly enriched in galactosylceramide (GalCer) and its sulfated derivative sulfatide. Over 50% of GalCer and sulfatide in myelin is hydroxylated by the integral membrane enzyme fatty acid 2-hydroxylase (FA2H). GalCer hydroxylation contributes to the compact nature of the myelin membrane, and mutations in FA2H result in debilitating leukodystrophies and spastic paraparesis. We report here the 2.6 Å crystal structure of sphingolipid α-hydroxylase (Scs7p), a yeast homolog of FA2H. The Scs7p core is composed of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum. The structure contains two zinc atoms coordinated by the side chains of 10 highly conserved histidines within a dimetal center located near the plane of the cytosolic membrane. We used a yeast genetic approach to confirm the important role of the dimetal-binding histidines in catalysis and identified Tyr-322 and Asp-323 as critical determinants involved in the hydroxylase reaction. Examination of the Scs7p structure, coupled with molecular dynamics simulations, allowed for the generation of a model of ceramide binding to Scs7p. Comparison of the Scs7p structure and substrate-binding model to the structure of steroyl-CoA desaturase revealed significant differences in the architecture of the catalytic cap domain and location of the dimetal centers with respect to the membrane. These observations provide insight into the different mechanisms of substrate binding and recognition of substrates by the hydroxylase and desaturase enzymes.

Effect of High Linoleic Acid Diet on Carbohydrate‐stimulated Expression of Lipogenic Genes

Linoleic acid, an ω‐6 PUFA, has been proposed to inhibit de novo lipogenesis (DNL) by decreasing expression of SREBP‐1c, a major regulator of DNL. To investigate whether previous intake of a high linoleic acid diet inhibits carbohydrate‐stimulated SREBP‐1c expression and DNL in vivo, we compared the expression of SREBP‐1c and its lipogenic targets in the fasted and post‐meal states in male Sprague‐Dawley rats fed: 1) a 55% fat (78% linoleic acid) diet (n=15, P); 2) a 55% fat (linoleic acid‐free) diet (n=15, S); or 3) a no‐fat diet (n=15, N) for 1 wk. All rats were fasted for 24 h; liver samples were obtained at fasting, 16h after refeeding on a 74% carbohydrate, fat‐free meal, or 45 min after injection with insulin + glucose.&#x0D; Body weights and liver lipid levels were greater in P and S than N rats (P=S&gt;N; p&lt; .01) but % body fat was higher in P than S rats (P&gt;S=N, p&lt; .01). Refeeding increased mRNA for SREBP‐1c and the lipogenic enzymes Acetyl CoA Carboxylase, Steroyl CoA Desaturase 1, and Pyruvate Kinase compared with fasting in all groups similarly, but expression of Fatty Acid Synthase was lower in the P vs. S group (p&lt; .02). In all groups, refeeding or insulin decreased Phosphoenolpyruvate Carboxykinase mRNA and increased Glucokinase mRNA, indicating normal insulin sensitivity. Refeeding decreased expression of INSIG2, a protein which inhibits maturation of SREBP‐1c, in the S and N groups (p&lt; .002), but not the P group.The minimal reduction in the stimulation of lipogenic gene expression seen in the high‐linoleic acid group was not associated with decreased expression SREBP‐1c, but was associated with lack of suppression of INSIG2 expression, suggesting that ω‐6 PUFA may impair processing rather than expression of SREBP‐1c.

Abstract 4339: Acute lymphoblastic leukemia cells stimulate adipocyte lipolysis and utilize adipocyte-derived free-fatty acids for proliferation

Abstract Obesity promotes the pathogenesis of many cancers, including acute lymphoblastic leukemia (ALL), the most common childhood malignancy. We have shown that ALL cells interact with adipocytes in vivo and in vitro, and that adipocytes protect ALL cells from chemotherapies. Since cancer cells require free fatty acids (FFA) for energy and as molecular building blocks, we hypothesize that ALL cells gain a survival advantage by stimulating adipocyte lipolysis and using adipocyte-derived FFAs. To test whether ALL cells stimulate adipocyte lipolysis, 3T3-L1 adipocytes were exposed to ALL-conditioned media for 72 hours, and glycerol release and adipocyte lipid content were measured. To determine whether ALL cells take up adipocyte-derived FFA, adipocytes were pre-labeled with fluorescent FFA (BODIPY), and then co-cultured with murine (8093) and human (BV173, RS4;11, Molt4, Nalm6, SEM, SupB15) ALL cells in a Transwell system. ALL uptake of BODIPY-FFAs was analyzed via flow cytometry. To determine the fate of FFA in ALL cells, ALL cells were incubated with BODIPY-FFA and evaluated by confocal and live cell microscopy, and lipid classes resolved through thin layer chromatography (TLC). Real-time PCR was employed to analyze adipocyte-induced alterations in the transcription of key enzymes in the ALL de novo lipogenesis pathway. Finally, TOFA, an acetyl-CoA carboxylase 1 (ACC1) and steroyl-CoA desaturase 1 (SCD1) inhibitor, was used to block de novo lipogenesis in ALL cells in vitro, and evaluate the ability of ALL cells to use adipocyte-derived FFAs for proliferation. ALL-conditioned media stimulated 3T3-L1 lipolysis (191.0 vs. 165.5 mg/L glycerol, n=1), reducing adipocyte lipid content as quantified with Oil Red O by 16.8 ± 7.1% (p = 0.047). When murine or human ALL cells were co-cultured with BODIPY-labeled adipocytes, they accumulated the fluorescent label within 48 hours (97-98% of cells labeled positive). TLC, live cell imaging and confocal microscopy demonstrated the majority of this label accumulated within ALL cell phospholipid membranes and triglyceride-laden lipid droplets. Adipocyte co-culture reduced expression of lipogenic enzymes such as fatty acid synthase (FAS), ACC1 and SCD1 in ALL cells. Finally, while TOFA inhibited ALL cell proliferation (6.1 ± 1.1x104 vs. 41.9 ± 4.7x104 cells, p=0.004, n=3), this proliferation was partially rescued by adipocyte conditioned media (ACM, 29.8 ± 9.2 x104, p=0.045 vs. TOFA alone, n=3). Therefore, we have demonstrated that ALL cells stimulate adipocyte lipolysis and use adipocyte-derived FFAs to supplement de novo lipogenesis and proliferation. This may contribute to the effect of obesity on ALL development and relapse. Citation Format: Jonathan Tucci, Xia Sheng, Steven D. Mittelman. Acute lymphoblastic leukemia cells stimulate adipocyte lipolysis and utilize adipocyte-derived free-fatty acids for proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4339. doi:10.1158/1538-7445.AM2014-4339

Characterization of Hair Follicle Development in Engineered Skin Substitutes

Generation of skin appendages in engineered skin substitutes has been limited by lack of trichogenic potency in cultured postnatal cells. To investigate the feasibility and the limitation of hair regeneration, engineered skin substitutes were prepared with chimeric populations of cultured human keratinocytes from neonatal foreskins and cultured murine dermal papilla cells from adult GFP transgenic mice and grafted orthotopically to full-thickness wounds on athymic mice. Non-cultured dissociated neonatal murine-only skin cells, or cultured human-only skin keratinocytes and fibroblasts without dermal papilla cells served as positive and negative controls respectively. In this study, neonatal murine-only skin substitutes formed external hairs and sebaceous glands, chimeric skin substitutes formed pigmented hairs without sebaceous glands, and human-only skin substitutes formed no follicles or glands. Although chimeric hair cannot erupt readily, removal of upper skin layer exposed keratinized hair shafts at the skin surface. Development of incomplete pilosebaceous units in chimeric hair corresponded with upregulation of hair-related genes, LEF1 and WNT10B, and downregulation of a marker of sebaceous glands, Steroyl-CoA desaturase. Transepidermal water loss was normal in all conditions. This study demonstrated that while sebaceous glands may be involved in hair eruption, they are not required for hair development in engineered skin substitutes.

Type 2 Diabetes Associated Changes in the Plasma Non-Esterified Fatty Acids, Oxylipins and Endocannabinoids

Type 2 diabetes has profound effects on metabolism that can be detected in plasma. While increases in circulating non-esterified fatty acids (NEFA) are well-described in diabetes, effects on signaling lipids have received little attention. Oxylipins and endocannabinoids are classes of bioactive fatty acid metabolites with many structural members that influence insulin signaling, adipose function and inflammation through autocrine, paracrine and endocrine mechanisms. To link diabetes-associated changes in plasma NEFA and signaling lipids, we quantitatively targeted >150 plasma lipidome components in age- and body mass index-matched, overweight to obese, non-diabetic (n = 12) and type 2 diabetic (n = 43) African-American women. Diabetes related NEFA patterns indicated ∼60% increase in steroyl-CoA desaturase activity and ∼40% decrease in very long chain polyunsaturated fatty acid chain shortening, patterns previously associated with the development of nonalcoholic fatty liver disease. Further, epoxides and ketones of eighteen carbon polyunsaturated fatty acids were elevated >80% in diabetes and strongly correlated with changes in NEFA, consistent with their liberation during adipose lipolysis. Endocannabinoid behavior differed by class with diabetes increasing an array of N-acylethanolamides which were positively correlated with pro-inflammatory 5-lipooxygenase-derived metabolites, while monoacylglycerols were negatively correlated with body mass. These results clearly show that diabetes not only results in an increase in plasma NEFA, but shifts the plasma lipidomic profiles in ways that reflect the biochemical and physiological changes of this pathological state which are independent of obesity associated changes.

Comparison of gene expression and fatty acid profiles in concentrate and forage finished beef1

Fatty acid profiles and intramuscular expression of genes involved in fatty acid metabolism were characterized in concentrate- (CO) and forage- (FO) based finishing systems. Intramuscular samples from the adductor were taken at slaughter from 99 heifers finished on a CO diet and 58 heifers finished on a FO diet. Strip loins were obtained at fabrication to evaluate fatty acid profiles of LM muscle for all 157 heifers by using gas chromatography fatty acid methyl ester analysis. Composition was analyzed for differences by using the General Linear Model (GLM) procedure in SAS. Differences in fatty acid profile included a greater atherogenic index, greater percentage total MUFA, decreased omega-3 to omega-6 ratio, decreased percentage total PUFA, and decreased percentage omega-3 fatty acids in CO- compared with FO-finished heifers (P<0.05). Fatty acid profiles from intramuscular samples were ranked by the atherogenic index, and 20 heifers with either a high (HAI; n=10) or low (LAI; n=10) atherogenic index were selected for gene expression analysis using real-time PCR (RT-PCR). Gene expression data for the 20 individuals were analyzed as a 2 by 2 factorial arrangement of treatments using the GLM procedure in SAS. There was no significant diet × atherogenic index interaction identified for any gene (P>0.05). Upregulation was observed for PPARγ, fatty acid synthase (FASN), and fatty acid binding protein 4 (FABP4) in FO-finished compared with CO-finished heifers in both atherogenic index categories (P<0.05). Upregulation of diglyceride acyl transferase 2 (DGAT2) was observed in FO-finished heifers with a HAI (P<0.05). Expression of steroyl Co-A desaturase (SCD) was upregulated in CO-finished heifers with a LAI, and downregulated in FO-finished heifers with a HAI (P<0.05). Expression of adiponectin (ADIPOQ) was significantly downregulated in CO-finished heifers with a HAI compared with all other categories (P<0.05). The genes identified in this study which exhibit differential regulation in response to diet or in animals with extreme fatty acid profiles may provide genetic markers for selecting desirable fatty acid profiles in future selection programs.

Akt2 Is Required for Hepatic Lipid Accumulation in Models of Insulin Resistance

Insulin drives the global anabolic response to nutrient ingestion, regulating both carbohydrate and lipid metabolism. Previous studies have demonstrated that Akt2/protein kinase B is critical to insulin's control of glucose metabolism, but its role in lipid metabolism has remained controversial. Here, we show that Akt2 is required for hepatic lipid accumulation in obese, insulin-resistant states induced by either leptin deficiency or high-fat diet feeding. Lep(ob/ob) mice lacking hepatic Akt2 failed to amass triglycerides in their livers, associated with and most likely due to a decrease in lipogenic gene expression and de novo lipogenesis. However, Akt2 is also required for steatotic pathways unrelated to fatty acid synthesis, as mice fed high-fat diet had reduced liver triglycerides in the absence of hepatic Akt2 but did not exhibit changes in lipogenesis. These data demonstrate that Akt2 is a requisite component of the insulin-dependent regulation of lipid metabolism during insulin resistance.

Suppression of VLDL secretion by cultured hepatocytes incubated with chylomicron remnants enriched in n−3 polyunsaturated fatty acids is regulated by hepatic nuclear factor-4α

Dietary n− 3 polyunsaturated fatty acids (PUFA) suppress the secretion of very low density lipoprotein (VLDL) directly when delivered to the liver in chylomicron remnants (CMR). The role of sterol regulatory element-binding proteins (SREBPs) and hepatic nuclear factor-4α (HNF-4α) in the regulation of this effect was investigated. Chylomicron remnant-like particles (CRLPs) containing triacylglycerol (TG) from palm (rich in saturated fatty acids (SFA)) or fish (rich in n− 3 PUFA) oil were incubated with cultured rat hepatocytes (24 h) and the expression of protein and mRNA for SREBP-1, SREBP-2 and HNF-4α, and levels of mRNA for their target genes were determined. SREBP-1 and -2 protein expression in the membrane and nuclear fractions was unaffected by either type of CRLPs. mRNA abundance for SREBP-1c and -2 was also unchanged by CRLP-treatment, as were levels of mRNA for target genes of SREBP-1, including steroyl CoA desaturase, acetyl CoA carboxylase, fatty acid synthase and ATP citrate lyase, and SREBP-2 (3-hydroxy-3-methylglutaryl CoA reductase). In contrast, HNF-4α protein and mRNA levels were significantly decreased by CRLPs enriched in n− 3 PUFA, but not SFA, and the expression of mRNA for HNF-4α target genes, including HNF-1α, apolipoprotein B and the microsomal TG transfer protein, was also lowered by n− 3 PUFA-, but not SFA-enriched CRLPs. These findings suggest that the direct suppression of VLDL secretion by dietary n− 3 PUFA delivered to the liver in CMR is mediated via decreased expression of HNF-4α.

LXRα activation perturbs hepatic insulin signaling and stimulates production of apolipoprotein B-containing lipoproteins

Liver X receptor-alpha (LXRalpha) is considered a master regulator of hepatic lipid metabolism; however, little is known about the link between LXR activation, hepatic insulin signaling, and very low-density lipoprotein (VLDL)-apolipoprotein B (apoB) assembly and secretion. Here, we examined the effect of LXRalpha activation on hepatic insulin signaling and apoB-lipoprotein production. In vivo activation of LXRalpha for 7 days using a synthetic LXR agonist, TO901317, in hamsters led to increased plasma triglyceride (TG; 3.6-fold compared with vehicle-treated controls, P = 0.006), apoB (54%, P < 0.0001), and VLDL-TG (eightfold increase compared with vehicle). As expected, LXR stimulation activated maturation of sterol response element binding protein-1c (SREBP-1c) as well as the SREBP-1c target genes steroyl CoA desaturase (SCD) and fatty acid synthase (FAS). Metabolic pulse-chase labeling experiments in primary hamster hepatocytes showed increased stability and secretion of newly synthesized apoB following LXR activation. Microsomal triglyceride transfer protein (MTP) mRNA and protein were unchanged, however, likely because of the relatively short period of treatment and long half-life of MTP mRNA. Examination of hepatic insulin-signaling molecules revealed LXR-mediated reductions in insulin receptor (IR)beta subunit mass (39%, P = 0.014) and insulin receptor substrate (IRS)-1 tyrosine phosphorylation (24%, P = 0.023), as well as increases in protein tyrosine phosphatase (PTP)1B (29%, P < 0.001) protein mass. In contrast to IRS-1, a twofold increase in IRS-2 mass (228%, P = 0.0037) and a threefold increase in IRS-2 tyrosine phosphorylation (321%, P = 0.012) were observed. In conclusion, LXR activation dysregulates hepatic insulin signaling and leads to a considerable increase in the number of circulating TG-rich VLDL-apoB particles, likely due to enhanced hepatic assembly and secretion of apoB-containing lipoproteins.

Activation of the lipogenic phenotype predicts poor prognosis in human hepatocellular carcinoma

Emerging evidence indicates that lipogenic enzymes are induced in hepatocarcinogenesis, but their role liver cancer development and progression remains poorly delineated. In the present investigation, we determined the levels and biologic role of the main lipogenic enzymes in a human hepatocellular carcinoma (HCC) collection. Levels of Fatty Acid Synthase (FASN), Acetyl-CoA Carboxylase (ACACA), ATP citrate lyase (ACLY), and Steroyl-CoA Desaturase (SCD) were all upregulated in HCC compared with corresponding noncancerous livers. Of note, the highest expression of lipogenic enzymes occurred in tumors with poor prognosis, implying their role in tumor aggressiveness. Suppression of FASN, ACACA, ACLY, and SCD by either siRNA or chemical inhibitors strongly reduced the growth of HCC cells in vitro, leading to massive apoptosis, whereas overexpression of FASN, ACACA, ACLY, and SCD induced a significant increase in proliferation and resistance to chemically-induced apoptosis in HCC cells. At the molecular level, inhibition of FASN was paralleled by downregulation of genes involved in cell proliferation and survival, including Akt, epidermal growth factor receptor (EGFR or ERBB1), S-phase kinase-associated protein 2 (SKP2), and NF- kB, and re-expression of putative oncosuppressor genes, including p21, p27, and death associated protein kinase 2 (DAPK2). Furthermore, FASN expression was regulated at post-transcriptional level by the isopeptidase ubiquitin-specific protease-2a (USP2a), whose suppression resulted in FASN downregulation in HCC cells. Taken together, the present data assign a pathogenetic and prognostic role to deregulation of the fatty acid biosynthetic pathway in liver cancer, and envisage the usefulness of FASN inhibitors as therapeutic modality for human HCC.

Comparing Subcutaneous Adipose Tissue in Beef and Muskox with Emphasis on trans 18:1 and Conjugated Linoleic Acids

Muskox (Ovibos moschatus) are ruminant animals native to the far north and little is known about their fatty acid composition. Subcutaneous adipose tissue (backfat) from 16 wild muskox was analyzed and compared to backfat from 16 barley fed beef cattle. Muskox backfat composition differed substantially from beef and the most striking difference was a high content of 18:0 (26.8 vs. 9.77%). This was accompanied by higher levels of most other saturated fatty acids except beef had more 16:0. Muskox backfat also had a lower level of cis-18:1 and this was related to a lower expression of steroyl-CoA desaturase mRNA. Beef backfat had a higher level of total trans-18:1 (4.25 vs. 2.67%). The most prominent trans-18:1 isomers in beef backfat were 10t-18:1 (2.13%) and 11t-18:1 (0.77%) whereas the most prominent isomers in muskox backfat were 11t-18:1 (1.41%), 13t/14t- (0.27%) and 16t-18:1 (0.23%). The total conjugated linoleic acid (CLA) content was higher in beef backfat than muskox (0.67 vs. 0.50%) with 9c,11t-18:2 as the most abundant CLA isomer. The second most abundant CLA isomer in beef backfat was 7t,9c-18:2 (0.10%) whereas in muskox it was 11t13c-18:2 (0.04%). Muskox backfat had a higher content of 18:3n-3 and its elongation and desaturation products 20:5n-3, 22:5n-3 and 22:6n-3 and a lower n-6/n-3 ratio. Overall, the high forage diet of muskox seemed to produce a healthier fatty acid profile and highlighted the need to develop feeding strategies for intensively raising beef that will not negatively impacting fatty acid composition.

Evidence that oestrogen receptor-α plays an important role in the regulation of glucose homeostasis in mice: insulin sensitivity in the liver

We used oestrogen receptor-alpha (ERalpha) knockout (ERKO) and receptor-beta (ERbeta) knockout (BERKO) mice to investigate the mechanism(s) behind the effects of oestrogens on glucose homeostasis. Endogenous glucose production (EGP) was measured in ERKO mice using a euglycaemic-hyperinsulinaemic clamp. Insulin secretion was determined from isolated islets. In isolated muscles, glucose uptake was assayed by using radiolabelled isotopes. Genome-wide expression profiles were analysed by high-density oligonucleotide microarray assay, and the expression of the genes encoding steroyl-CoA desaturase and the Leptin receptor (Scd1 and Lepr, respectively) was confirmed by RT-PCR. ERKO mice had higher fasting blood glucose, plasma insulin levels and IGT. The plasma leptin level was increased, while the adiponectin concentration was decreased in ERKO mice. Levels of both glucose- and arginine-induced insulin secretion from isolated islets were similar in ERKO and wild-type mice. The euglycaemic-hyperinsulinaemic clamp revealed that suppression of EGP by increased insulin levels was blunted in ERKO mice, which suggests a pronounced hepatic insulin resistance. Microarray analysis revealed that in ERKO mice, the genes involved in hepatic lipid biosynthesis were upregulated, while genes involved in lipid transport were downregulated. Notably, hepatic Lepr expression was decreased in ERKO mice. In vitro studies showed a modest decrease in insulin-mediated glucose uptake in soleus and extensor digitorum longus (EDL) muscles of ERKO mice. BERKO mice demonstrated normal glucose tolerance and insulin release. We conclude that oestrogens, acting via ERalpha, regulate glucose homeostasis mainly by modulating hepatic insulin sensitivity, which can be due to the upregulation of lipogenic genes via the suppression of Lepr expression.

Regulatory Role of Glycogen Synthase Kinase 3 for Transcriptional Activity of ADD1/SREBP1c

Adipocyte determination- and differentiation-dependent factor 1 (ADD1) plays important roles in lipid metabolism and insulin-dependent gene expression. Because insulin stimulates carbohydrate and lipid synthesis, it would be important to decipher how the transcriptional activity of ADD1/SREBP1c is regulated in the insulin signaling pathway. In this study, we demonstrated that glycogen synthase kinase (GSK)-3 negatively regulates the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitors enhanced a transcriptional activity of ADD1/SREBP1c and expression of ADD1/SREBP1c target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and steroyl-CoA desaturase 1 (SCD1) in adipocytes and hepatocytes. In contrast, overexpression of GSK3beta down-regulated the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitor-mediated ADD1/SREBP1c target gene activation did not require de novo protein synthesis, implying that GSK3 might affect transcriptional activity of ADD1/SREBP1c at the level of post-translational modification. Additionally, we demonstrated that GSK3 efficiently phosphorylated ADD1/SREBP1c in vitro and in vivo. Therefore, these data suggest that GSK3 inactivation is crucial to confer stimulated transcriptional activity of ADD1/SREBP1c for insulin-dependent gene expression, which would coordinate lipid and glucose metabolism.