SCD Activity Research Articles

516-P: High-Fat Feeding Alters Circulating Triglyceride Profile by Decreasing SCD Activity and Depleting n-3 Fatty Acids

516-P: High-Fat Feeding Alters Circulating Triglyceride Profile by Decreasing SCD Activity and Depleting n-3 Fatty Acids

Hepatic Stearoyl‐CoA desaturase deficiency‐mediated induction of the insulin‐like growth factor‐binding protein 1 requires FGF21

Stearoyl‐CoA desaturase‐1 (SCD1) catalyzes the rate‐limiting step in the biosynthesis of monounsaturated acids and is a key regulator of systemic glucose metabolism. Hepatic deletion of Scd1protects mice from high‐carbohydrate diet (HCD)‐induced hepatic steatosis and adiposity in part by suppressing hepatic lipogenesis, increasing adipose tissue lipogenesis, and increasing systemic glucose uptake. However, the mechanism leading to these phenotypes is unclear. Given that high carbohydrate diets can lead to chronic metabolic diseases such as obesity, diabetes, and hepatic steatosis, it is critical to understand how SCD1 deficiency alters metabolic pathways to produce metabolically beneficial phenotypes. Here we show that hepatic SCD1 deficiency enhances tissue glucose uptake and is correlated with dramatic increases in the expression and plasma levels of the fibroblast growth factor 21 (FGF21), and Insulin growth factor binding protein‐1 (IGFBP1), both liver derived insulin‐sensitizing hormones that are known to regulate whole body lipid and glucose homeostasis. Feeding both male and female LKO mice with triolein, but not tristearin, supplemented HCD reduced Fgf21 and Igfbp1 expression and restored plasma glucose levels. Inhibition of SCD activity in primary hepatocytes induced the expression of Fgf21 and Igfbp1 which was repressed by treatment with oleate but not stearate. Moreover, we show that the hepatic stearoyl‐CoA desaturase deficiency‐mediated induction of the Igfbp1 is through the oleate‐FGF21 axis.

DDRE-11. TARGETING FATTY ACID BIOSYNTHESIS IN GLIOBLASTOMA

We recently provided evidence that endoplasmic reticulum (ER) stress promotes fatty acid (FA) biosynthesis in glioblastoma (GBM) cancer stem cells (GSCs). We determined that Stearoyl CoA Desaturase 1 (SCD), a key FA desaturase, is essential for regulating ER homeostasis in GSCs, and showed that these cells are highly susceptible to pharmacological perturbation of SCD activity. An impaired SCD activity leads to the toxic accumulation of saturated FA and activates cell death signaling mediated by the ER sensor Inositol-requiring enzyme 1 (IRE1). This in turn promotes an IRE1-mediated mRNA decay of key DNA damage repair genes and impairs the ability of GSCs to repair DNA damage caused by radiation or chemotherapy. Consequently, combining SCD inhibition with temozolomide (TMZ) leads to major cytotoxicity both in TMZ-sensitive, and TMZ-resistant patient-derived GBM cells. Pharmacological inhibition of SCD delivered through the nasal route in mice, had a remarkable therapeutic benefit in patient-derived orthotopic GSCs mouse models, yet the modest brain permeability of the currently available SCD inhibitors precludes their clinical translation. To overcome this challenge, we have recently acquired a first-in-class, clinically relevant SCD inhibitor. This compound has undergone extensive pharmacokinetic and pharmacodynamic studies which confirmed brain permeability, efficacy, and safety in small animals and non-human primates. We show that the combination of this SCD inhibitor with TMZ is effective both in cultured GSCs, and in preclinical GSCs orthotopic mouse models. Our results support the clinical investigation of this new class of SCD inhibitors, in combination with TMZ, in patients diagnosed with GBM.

Iron, Oxidative Stress, and Δ9 Stearoyl-CoenzymeA Desaturase Index (C16:1/C16:0): An Analysis Applying the National Health and Nutrition Examination Survey 2003–04

BackgroundStearoyl-coenzyme A desaturase (SCD) is a key enzyme in fatty acid metabolism, and elevated SCD activity is associated with multiple adverse health outcomes. Diet, hormone levels, and environmental exposures are potential factors affecting SCD activity. Less is known about the relationship between micronutrients, including iron, and SCD activity. ObjectiveThe aim of this study was to investigate the association between serum ferritin level, a biomarker of circulating iron levels, and the Δ9 desaturase index (C16:1/C16:0), a biomarker of estimated SCD activity, among women in the United States. MethodsThe association between serum ferritin and the Δ9 desaturase index was assessed in a cross-sectional study of 447 female participants, aged 20–49 y, from NHANES 2003–2004. The multivariate analyses were performed utilizing generalized linear modeling, adjusting for potential confounders. Mediation of the relationship between serum ferritin and Δ9 desaturase index by γ-glutamyltranspeptidase (GGT), a biomarker of oxidative stress, was also assessed. ResultsIncreased ferritin was significantly associated with a higher Δ9 desaturase index. Adjusting for waist circumference, age, race, and cotinine levels, an interquartile range increase in serum ferritin corresponded to 3.92% (95% CI: 0.88%, 7.05%) higher Δ9 desaturase index. GGT, the biomarker used to measure oxidative stress level, did not appear to mediate the association between ferritin and Δ9 desaturase index. After stratifying by pregnancy status, these associations were limited to nonpregnant individuals. ConclusionsElevated SCD activity may be associated with increased iron storage inside the human body; the association did not appear to be mediated via oxidative stress, as estimated by GGT levels.

The reduction of starch in finishing diets supplemented with oil does not prevent the accumulation of trans-10 18:1 in lamb meat.

The experiment was conducted to test the hypothesis that the replacement of cereal with low-starch feed ingredients in lambs' finishing diets supplemented with oils could prevent the accumulation of -10-18:1 in meat. Forty lambs were fed 1 of 4 diets supplemented with soybean oil (5.9%) and fish oil (1%) for 6 wk before slaughter. The control (CON) diet contained 43% barley, and in the other diets, barley was completely replaced by dehydrated citrus pulp (DCP), dehydrated sugar beet pulp (DBP), or soybean hulls (SH). Growth performance, feed intake, and carcass and meat quality traits were analyzed. At slaughter, LM samples were collected for gene expression evaluation, and 3 d after slaughter, LM and subcutaneous (s.c.) fat samples were collected for fatty acid analysis. None of the diets affected meat quality, but the DCP diet reduced ADG ( < 0.05) and the DCP and SH diets decreased the feed-to-gain ratio ( < 0.01). The DCP diet increased ( < 0.05) the risk of parakeratosis and the severity of the lesions. Moreover, the DBP treatment led to increased a* (redness) and b* (yellowness) in s.c. fat compared with the CON treatment ( < 0.05). The lipid content of LM did not differ ( > 0.05) with treatment and averaged 34.4 g/kg of meat. Diets had no effect ( > 0.05) on SFA, PUFA, and -MUFA sums and on the -6:-3 ratio in both LM and s.c. fat. A lower expression of fatty acid synthase (FASN) was found with the DCP treatment than with the other treatments ( < 0.001). All treatments showed a high accumulation of -10-18:1, averaging 91 mg/g fatty acid in LM and 147 mg/g fatty acid in s.c. fat. The concentration of -11-18:1 in the tissues was considerably lower than that of -10-18:1, and thus the -10-18:1:-11-18:1 ratio was above 3 with all treatments. Despite this, the SH diet clearly promoted a larger deposition of -11-18:1 and -9,-11-18:2 in tissues compared with the other treatments. () gene expression and SCD activity index in LM were reduced with the SH diet compared with the CON and DCP diets. Overall, these results clearly showed, for the first time, that low-starch/high-NDF diets are not able to prevent the establishment of -10 shifted rumen biohydrogenation pathways, evaluated by the deposition of biohydrogenation intermediates in lamb meat and fat.

One-night sleep deprivation induces changes in the DNA methylation and serum activity indices of stearoyl-CoA desaturase in young healthy men.

BackgroundSleep deprivation has been associated with obesity among adults, and accumulating data suggests that stearoyl-CoA desaturase 1 (SCD1) expression has a relevant impact on fatty acid (FA) composition of lipid pools and obesity. The aim of this study was to investigate the effect of one-night total sleep deprivation (TSD) on DNA methylation in the 5’-prime region of SCD1, and whether detected changes in DNA methylation are associated with SCD activity indices (product to precursor FA ratios; 16:1n-7/16:0 and 18:1n-9/18:0) derived from serum phospholipids (PL).MethodsSixteen young, normal-weight, healthy men completed two study sessions, one with one-night TSD and one with one-night normal sleep (NS). Sleep quality and length was assessed by polysomnography, and consisted of electroencephalography, electrooculography, and electromyography. Fasting whole blood samples were collected on the subsequent morning for analysis of DNA methylation and FAs in serum PL. Linear regression analyses were performed to assess the association between changes in DNA methylation and SCD activity indices.ResultsThree CpG sites close to the transcription start site (TSS) of SCD1 (cg00954566, cg24503796, cg14089512) were significantly differentially methylated in dependency of sleep duration (−log10P-value > 1.3). Both SCD-16 and SCD-18 activity indices were significantly elevated (P < 0.05) following one-night TSD, and significantly associated with DNA methylation changes of the three mentioned probes in the 5’ region of SCD1.ConclusionOur results suggest a relevant link between TSD, hepatic SCD1 expression and de-novo fatty acid synthesis via epigenetically driven regulatory mechanisms.

Investigating mutual relationship among milk fatty acids by multivariate factor analysis in dairy cows

The interpretation of mutual relationship among milk fatty acids (FA) is not easy due to the high number of FA contained in milk fat and to the high degree of correlation among them. Multivariate analysis includes different statistical approaches that could help explaining complex pattern of variables. In this study, Multivariate Factor Analysis (MFA) was used to decompose the correlation matrix of 47 FA and milk production traits (milk yield and protein and fat content) measured in 300 Italian Holstein Friesian cows reared in the North of Italy in 23 commercial dairy farms, representative of the intensive dairy system. MFA was able to extract seven latent factors with specific biologic meaning: secretion of Long Chain FA (KLCFA), mammary FA de novo synthesis (Km), rumen biohydrogenation (Kbh), synthesis of odd chain FA (Ko), synthesis of branched chain FA (Kb), mammary desaturation activity (Kd), milk yield (Kmy). According to the pattern of communalities of the factor analysis, C18:3c9c12c15 was the only FA, along with C18:2t11c15, to be uncorrelated with the other variables and it seemed to be excluded by the metabolic pattern described by the seven factors. The desaturation products of the SCD enzyme were independently associated to three latent factors, suggesting new insights in the regulation of SCD activity. Factors were considered as new quantitative phenotypes related to prominent features of milk FA profile. With the aim of evaluating the feeding regimen and animal effects, latent factors were analysed with a mixed model, which considered the fixed effect of lactation stage, parity, some feeding regimen characteristics and the random effect of bull. Lactation stage significantly affected Km and Kmy factors. In perspective, the seven factors extracted by applying MFA analysis to milk FA composition could be considered as new and more informative traits to test the effect of endogenous and exogenous variation factors.

Hepatic Stearoyl‐CoA Desaturase deficiency suppresses gluconeogenesis by regulating lipid profile of mitochondria

Obesity plays a central role in the metabolic syndrome, which includes hyperinsulinemia, hypertension, hyperlipidemia and an increased risk of atherosclerotic cardiovascular disease. A common health complication observed in obese patients is insulin resistance, a condition in which increased insulin levels are required to maintain normal blood glucose levels. Without proper intervention, the condition often deteriorates into type 2 diabetes mellitus, characterized by increased hepatic glucose production, and decreased glucose clearance by insulin‐responsive tissues. It has been suggested that high glucose is associated with increased oxidative stress, enhanced leukocyte‐endothelial interaction, and glycosylation of lipoproteins, apolipoproteins, and clotting factors. Identification of new therapeutic approaches to improve glycemic management via the inhibition of hepatic glucose production will be beneficial for diabetes, cardiovascular diseases and related metabolic disorders.Stearoyl‐CoA desaturase (SCD) expressed at high levels in several human tissues is required for the biosynthesis of oleate (18:1) and palmitoleate (16:1), which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides and cholesterol esters. Given the multiple roles of monounsaturated fatty acids, variation in stearoyl‐CoA desaturase activity in mammals would be expected to have a major effect on lipid metabolism. We and others have reported that global deletion of SCD1 in mice dramatically increases whole body energy expenditure and the loss of SCD1 activity in the liver suppresses hepatic glucose production when mice were fed a lipogenic diet. We hypothesize that the deficiency of SCD alters energy metabolism at the cellular level via the regulation of mitochondria function. Our preliminary data suggest that hepatic SCD1 deficiency alters the lipid profile of mitochondria membrane, therefore alters the membrane potential and metabolic rate of the cell. In addition, we show that the pharmaceutical inhibition of SCD activity reduces hepatic glucose production in the cell culture conditions. These studies provide essential information on the role of SCD on energy metabolism and, at the same time validate hepatic SCD enzymes as therapeutic targets against hyperglycemia in disease conditions, such as diabetes and metabolic syndrome.Support or Funding InformationAmerican Diabetes Association 7‐13‐BS‐118

Effects of abomasal infusion of conjugated linoleic acids, Sterculia foetida oil, and fish oil on production performance and the extent of fatty acid Δ9-desaturation in dairy cows

The purpose of this study was to determine the effects of conjugated linoleic acid (CLA), Sterculia foetida oil (STO), and fish oil (FO) on milk yield and composition, milk FA profile, Δ9-desaturation activity, and mammary expression of 2 isoforms of stearoyl-coenzyme A desaturase (SCD-1 and SCD-5) in lactating dairy cows. Eight multiparous Holstein cows (69±13 d postpartum) were used in a double 4×4 Latin square design with 28-d periods. For the first 14 d of each period, cows received an abomasal infusion of (1) 406g of a saturated fatty acid (SFA) supplement (112g of 16:0 + 230g of 18:0) used as a control (CTL), (2) 36g of a CLA supplement (13.9g of trans-10,cis-12 18:2) + 370g of SFA, (3) 7g of STO (3.1g of 19:1 cyclo) + 399g of SFA, or (4) 406g of FO (55.2g of cis-5,-8,-11,-14,-17 20:5 + 59.3g of cis-4,-7,-10,-13,-16,-19 22:6). Infusions were followed by a 14-d washout interval. Compared with CTL, STO decreased milk yield from 38.0 to 33.0kg/d, and increased milk fat concentration from 3.79 to 4.45%. Milk fat concentration was also decreased by CLA (2.23%) and FO (3.34%). Milk fat yield was not affected by STO (1,475g/d) compared with CTL (1,431g/d), but was decreased by CLA (774g/d) and FO (1,186g/d). Desaturase indices for 10:0, 12:0, and 20:0 were decreased, whereas the extent of desaturation of 14:0, 16:0, 17:0, and 18:0 was not affected by CLA treatment compared with CTL. Infusion of STO significantly decreased all calculated desaturase indices compared with CTL; the 14:0 index was reduced by 80.7%. Infusion of FO decreased the desaturase indices for 10:0, 14:0, 20:0, trans-11 18:1, and 18:0. The effect of FO on the 14:0 index indicates a decrease in apparent Δ9-desaturase activity of 30.2%. Compared with CTL, mammary mRNA abundance of SCD-1 was increased by STO (+30%) and decreased by CLA (−24%), whereas FO had no effect. No effect was observed on mRNA abundance of SCD-5. In conclusion, abomasal infusion of CLA, STO, and FO were shown to exhibit varying and distinct effects on desaturase indices, an indicator of apparent SCD activity, and mammary mRNA abundance of SCD-1.

Dietary walnut oil modulates liver steatosis in the obese Zucker rat

PurposeNon-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. We aimed to clarify the impact of dietary walnut oil versus animal fat on hepatic steatosis, representing the initial step of multistage pathogenesis of NAFLD, in Zucker obese rats.MethodsZucker lean ad libitum (a.l.), Zucker obese a.l. or Zucker obese pair fed (p.f.) to the lean received isocaloric diets containing 8 % walnut oil (W8), W14 or 14 % lard (L14) (n = 10/group). Body weight, clinical serology, liver weight, lipid content and fatty acid composition and hepatic lipid metabolism-related transcripts were evaluated.ResultsCompared to lean, Zucker obese a.l. and p.f. showed hepatic triacylglyceride (TAG) accumulation. In Zucker obese p.f., W14 compared to W8 and L14 reduced liver lipids, TAG as well as hepatic omega-6 (n-6)/n-3 ratio and SCD activity index [(C18:0 + C18:1)/C18:0 ratio] paralleled by decreased lipoprotein lipase mRNA in obese p.f. and elevated microsomal triglyceride transfer protein mRNA in lean and obese. Further, W14 elevated the fasting blood TAG and reduced cholesterol levels in obese.ConclusionsIn our model, consumption of W14 inhibited hepatic lipid accumulation along with modulated hepatic gene expression implicated in hepatic fatty acid influx or lipoprotein assembly. These results provide first indication that dietary lipids from walnut oil are modulators of hepatic steatosis as the initial step of progressive NAFLD pathogenesis.

Plasma stearoyl‐CoA desaturase indices: Association with lifestyle, diet, and body composition

Stearoyl-coenzyme A desaturase-1 (SCD1) is a key enzyme in fatty acid and energy metabolism. Increased hepatic SCD1 activity is associated with obesity and obesity-related diseases. We examined the relations of two plasma SCD activity indices (16:1n-7/16:0, 18:1n-9/18:0) with body composition, and the association of lifestyle and dietary variables with the plasma SCD indices. This population-based, cross-sectional study of 2021 elderly (71-74 y) men and women from the Hordaland Health Study in Western Norway was conducted using a validated food frequency questionnaire, body composition measurements by dual-energy X-ray absorptiometry and determination of the plasma fatty acid profile. In multivariate regression analyses, plasma SCD indices were positively associated with BMI and body fat (P < 0.001 for both). From the 2.5th to 97.5th percentiles of plasma SCD-16 and SCD-18 indices, fat mass differed by about 8 kg and 5 kg, respectively. Intake of polyunsaturated fatty acids were negatively associated with SCD-16 (partial r = -0.30) and SCD-18 (partial r = -0.24) (P < 0.001 for both). Alcohol intake was positively associated with SCD-16 (partial r = 0.26) and SCD-18 (partial r = 0.16) (P < 0.001 for both), whereas coffee consumption and physical activity were inversely associated with SCD-16 (P = 0.026 and P = 0.006, respectively) and SCD-18 (P = 0.001 and P = 0.022, respectively). In this elderly population, plasma markers of SCD1 activity are associated with increased adiposity. Furthermore, modifiable dietary habits and lifestyle are associated with plasma SCD indices. These results suggest that SCD1 activity may be a promising target for weight control.

Stearoyl-CoA desaturase activity modulates the activation of epidermal growth factor receptor in human lung cancer cells

Stearoyl-CoA desaturase-1 (SCD1), the main enzyme that converts saturated fatty acids into monounsaturated fatty acids, is a key factor in the mechanisms of cancer cell proliferation, survival and tumorigenesis. Evidence indicates that SCD1 activity regulates these events in part by targeting the phosphatidylinositol-3 phosphate kinase/Akt and Ras/extracellular signal-regulated kinase (ERK) pathways, but the molecular mechanisms remain unknown. We now show that in H460 lung cancer cells, the suppression of SCD activity with CVT-11127, a specific small molecule SCD inhibitor, impairs the ligand-induced phosphorylation of epidermal growth factor (EGF) receptor, causing the inactivation of its downstream targets Akt, ERK and mammalian target of rapamycin. Importantly, the mitogenic response to EGF was markedly defective in SCD-depleted cancer cells. The inactivation of EGF receptor (EGFR) promoted by SCD inhibition may be caused by perturbations in the lipid microenvironment surrounding the receptor, since we detected significant alterations in the lateral mobility of plasma lipid microdomains. Finally, incubation of lung cancer cells with SCD blockers potentiated the antigrowth effect of gefitinib, an EGFR inhibitor employed in cancer treatment. Altogether, our data indicate that SCD activity may control cancer cell metabolism, proliferation and survival by modulating the EGFR→Akt/ERK signaling platforms. Our studies also suggest a value for SCD inhibitors as novel pharmacological agents in lung cancer, one of the most common and lethal forms of cancer for which therapeutic options remain very limited.

2-Aryl benzimidazoles: Human SCD1-specific stearoyl coenzyme-A desaturase inhibitors

A series of potent, benzimidazole-based SCD inhibitors which demonstrate selectivity for the hSCD1 enzyme over the hSCD5 isoform are described. The compounds possess suitable cellular activity and pharmacokinetic properties which render them capable of inhibiting liver SCD activity in a mouse pharmacodynamic assay. These 2-aryl benzimidazoles may serve as valuable tools for studying selective hSCD1-inhibition.

Inhibition of StearoylCoA Desaturase Activity Blocks Cell Cycle Progression and Induces Programmed Cell Death in Lung Cancer Cells

Lung cancer is the most frequent form of cancer. The survival rate for patients with metastatic lung cancer is ∼5%, hence alternative therapeutic strategies to treat this disease are critically needed. Recent studies suggest that lipid biosynthetic pathways, particularly fatty acid synthesis and desaturation, are promising molecular targets for cancer therapy. We have previously reported that inhibition of stearoylCoA desaturase-1 (SCD1), the enzyme that produces monounsaturated fatty acids (MUFA), impairs lung cancer cell proliferation, survival and invasiveness, and dramatically reduces tumor formation in mice. In this report, we show that inhibition of SCD activity in human lung cancer cells with the small molecule SCD inhibitor CVT-11127 reduced lipid synthesis and impaired proliferation by blocking the progression of cell cycle through the G1/S boundary and by triggering programmed cell death. These alterations resulting from SCD blockade were fully reversed by either oleic (18:1n-9), palmitoleic acid (16:1n-7) or cis-vaccenic acid (18:1n-7) demonstrating that cis-MUFA are key molecules for cancer cell proliferation. Additionally, co-treatment of cells with CVT-11127 and CP-640186, a specific acetylCoA carboxylase (ACC) inhibitor, did not potentiate the growth inhibitory effect of these compounds, suggesting that inhibition of ACC or SCD1 affects a similar target critical for cell proliferation, likely MUFA, the common fatty acid product in the pathway. This hypothesis was further reinforced by the observation that exogenous oleic acid reverses the anti-growth effect of SCD and ACC inhibitors. Finally, exogenous oleic acid restored the globally decreased levels of cell lipids in cells undergoing a blockade of SCD activity, indicating that active lipid synthesis is required for the fatty acid-mediated restoration of proliferation in SCD1-inhibited cells. Altogether, these observations suggest that SCD1 controls cell cycle progression and apoptosis and, consequently, the overall rate of proliferation in cancer cells through MUFA-mediated activation of lipid synthesis.

Inhibition of SCD activity enhances inflammation in RAW264.7 macrophages but improves cholesterol trafficking

Obesity is a major risk factor for atherosclerosis. Loss of stearoyl CoA desaturase (SCD) 1 in mice has been shown to protect mice from obesity. However, the role of SCD in atherosclerosis has not yet been fully understood. Macrophage inflammation is a key characteristic of atherosclerosis. Here, we tested the effects of SCD activity on the inflammation and cholesterol transport in RAW264.7 macrophages. Inhibition of the overall SCD activity by A939572, a pharmacological inhibitor targeting the catalytic domain of the enzyme, enhances the induction of inflammatory genes upon LPS treatment in RAW cells, which is prevented by addition of oleate (18:1n9) or palmitoleate (16:1n7), two products of SCD activity. While the total free cholesterol level is elevated in RAW cells treated with A939572, the uptake of oxLDL is, however, reduced. In addition, cholesterol efflux is increased with treatment of A939572 in these macrophages. The above results indicate that although inhibition of SCD activity promotes macrophage inflammation, it improves cholesterol trafficking in RAW macrophages. Thus, SCD might regulate inflammation and cholesterol trafficking through distinct pathways in macrophages.

De Novo Lipogenesis and Stearoyl-CoA Desaturase Are Coordinately Regulated in the Human Adipocyte and Protect against Palmitate-induced Cell Injury

De novo lipogenesis (DNL) is paradoxically up-regulated by its end product, saturated fatty acids (SAFAs). We tested the hypothesis that SAFA-induced up-regulation of DNL reflects coordinate up-regulation of elongation and desaturation pathways for disposal of SAFAs and production of monounsaturated fatty acids to protect cells from SAFA toxicity. Human preadipocytes were differentiated in vitro for 14 days with [U-(13)C]palmitate (0-200 microM) to distinguish exogenous fatty acids from those synthesized by DNL. Exogenous palmitate up-regulated DNL (p < 0.001) concomitantly with SCD and elongation (each p < 0.001). Adipocytes from some donors were intolerant to high palmitate concentrations (400 microM). Palmitate-intolerant cells showed lower TG accumulation. They had lower expression of SCD mRNA and less monounsaturated fatty acids in TG, emphasizing the importance of desaturation for dealing with exogenous SAFAs. There was greater [U-(13)C]palmitate incorporation in phospholipids. SCD knockdown with small interfering RNA caused down-regulation of DNL and of expression of DNL-related genes, with reduced membrane fluidity (p < 0.02) and insulin sensitivity (p < 0.01), compared with scrambled small interfering RNA controls. There was preferential channeling of DNL-derived versus exogenous palmitate into elongation and of DNL-derived versus exogenous stearate into desaturation. DNL may not act primarily to increase fat stores but may serve as a key regulator, in tandem with elongation and desaturation, to maintain cell membrane fluidity and insulin sensitivity within the human adipocyte.

Potent, orally bioavailable, liver-selective stearoyl-CoA desaturase (SCD) inhibitors

Two structurally distinct series of SCD (Δ9 desaturase) inhibitors (1 and 2) have been previously reported by our group. In the present work, we merged the structural features of the two series. This led to the discovery of compound 5b (CVT-12,012) which is highly potent in a human cell-based (HEPG2) SCD assay (IC50=6nM). This compound has 78% oral bioavailability in rats and is preferentially distributed into liver (76 times vs plasma) with relatively low brain penetration. In a five-day study (sucrose fed rats) compound 5b significantly reduced SCD activity in a dose-dependent manner as determined by GC analysis of fatty acid composition in plasma and liver, and significantly reduced liver triglycerides versus the control group (∼50%).

Orally bioavailable, liver-selective stearoyl-CoA desaturase (SCD) inhibitors

We discovered a structurally novel SCD (Δ9 desaturase) inhibitor 4a (CVT-11,563) that has 119nM potency in a human cell-based (HEPG2) SCD assay and selectivity against Δ5 and Δ6 desaturases. This compound has 90% oral bioavailability (rat) and excellent plasma exposure (dAUC 935ngh/mL). Additionally, 4a shows moderately selective liver distribution (three times vs plasma and adipose tissue) and relatively low brain penetration. In a five-day study (high sucrose diet, rat) compound 4a significantly reduced SCD activity as determined by GC analysis of fatty acid composition in plasma and liver. We describe the discovery of 4a from HTS hit 1 followed by scaffold replacement and SAR studies focused on DMPK properties.

Transcriptomic analysis of two sheep breeds during lactation, using a new custom microarray platform

Individual milk samples were weekly collected from 48 ewes during eight weeks. Ewes were at the second month of lactation and they were fed the same dietary regimen. The animals were grouped according to the amount of daily milk fat yield: A) 38-57g/d, B) 58-63g/d, C) 64-73g/d, D) 75-110g/d. Milk from group D showed a higher content of FA from C4 to C10 and a lower content of monounsaturated FA (MUFA) and conjugated linoleic acids (CLA) than Group A (+12%, -11%, and -18%, for C4 to C10, MUFA and CLA, respectively), while Group B and C showed intermediate values. CLA content increased with days of lactation in a similar way for all groups. The mean correlation among all records within each individual lactation was 0.45 for CLA content. Six milk FA pair ratios representing a proxy for SCD activity were also evaluated: group A showed higher values of FA ratios than Group D (+26% on average for all FA pairs). In conclusion, increasing ability of daily milk fat yield was associated with lower milk content of MUFA and CLA and higher content of FA from C4 to C10.

High content secondary screening assay to model NRTI-induced mitochondrial toxicity

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (Km = 10.5 μM). The assay was highly reproducible with an average Z′ value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC50 values of 0.88 and 0.12 μM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 μM – 14% conformation rate). Of the confirmed hits 172 had IC50 values of <10 μM, including 111 <1 μM and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.