Rozprawy doktorskie na temat „Stearoyl CoA desaturase”

Combination of the risk factors obesity, insulin resistance, dyslipidemia, and hypertension, often described as the "metabolic syndrome," increases the risk of developing diabetes and cardiovascular disease. Stearoyl-coenzyme A desaturase (SCD) activity has been implicated in the metabolic syndrome; however, earlier studies on the beneficial metabolic effects of SCD1 deficiency have been confined to normolipidemic mice, and the role of SCD in the context of atherosclerosis has not been examined. The primary purpose of this thesis was to investigate the effect of decreased SCD activity on susceptibility to atherosclerosis in mice. Thus, the overarching hypothesis driving the work was that SCD activity is atherogenic. As well, we examined the effect of absence of SCD1 on features of the metabolic syndrome and chronic inflammation in a mouse model of familial hyperlipidemia. An additional objective was to determine the effect of SCD1 deficiency on dextran sulfate sodium (DSS)-induced acute colitis with DSS dosing adjusted to account for genotypic differences in fluid consumption. These studies help us understand SCD functions in lipid metabolism and evaluate its attractiveness as a therapeutic target for the metabolic syndrome or atherosclerosis. After the Scd1ab-J mutation was characterized, mice carrying this allele were crossed with the low density lipoprotein receptor (LDLR)-deficient mouse strain. Features of the metabolic syndrome and atherosclerotic lesion area were evaluated in mice challenged with a Western diet for twelve weeks. In studies of colonic inflammation, wild-type controls were treated with 3.5% DSS for 5 days to induce moderately severe colitis, while the concentration of DSS given to SCD1-deficient mice was lowered to 2.5% to control for increased fluid consumption. Despite an antiatherogenic metabolic profile, SCD1 deficiency increased atherosclerosis in hyperlipidemic LDLR-deficient mice. Absence of SCD1 also led to chronic inflammation of the skin and increased plasma inflammatory markers, but did not accelerate inflammation in DSS-induced acute colitis when DSS intake was controlled. These findings reinforce the crucial role of chronic inflammation in promoting atherosclerosis, even in the presence of antiatherogenic metabolic characteristics, and suggest that inflammation must be closely monitored in studies of SCD inhibitors for treatment of the metabolic syndrome in humans.

L'importance de la stearoyl coa desaturase hepatique (scd1) a ete mise en evidence dans les mecanismes conduisant a la formation du tissu adipeux abdominal du poulet et plusieurs resultats suggerent l'implication du gene lui-meme dans la variabilite du caractere. Dans le but de verifier si l'hypothese d'une regulation essentiellement transcriptionnelle du gene scd1 hepatique etait verifiee ou non chez le poulet, l'etude de la regulation de l'expression de scd1 par des effecteurs hormonaux a ete entreprise. Ensuite, nous avons cherche a identifier des zones genomiques qui pourraient rendre compte d'une difference d'activite de scd1 correlee avec le taux de tissu adipeux des animaux. Pour cela, des regions cis de reponse aux principaux effecteurs de la regulation de l'expression de scd1 ont ete recherchees sur la partie 5 amont du gene. Nous avons etabli que le modele d'hepatocytes en culture primaire issu de poulet en croissance, d'environ 6 semaines, est un modele adapte a l'etude des genes de la lipogenese. Il permet d'eviter l'inconvenient majeur de la culture d'hepatocytes embryonnaires dans lesquels certains genes impliques dans la transduction de signaux hormonaux ne s'expriment pas. Ainsi, les interrogations concernant la difference de role joue par l'insuline sur la regulation des genes de la lipogenese entre les rongeurs et l'oiseau, ont commence a etre levees. En effet, nous avons montre que la regulation positive de l'expression de scd1 par l'insuline est essentiellement transcriptionnelle, ce qui est comparable aux observations faites chez les rongeurs. L'effet du glucagon sur l'expression de scd1 hepatique est egalement transcriptionnel. De plus, nous suggerons un effet du glucagon distinct de celui de l'insuline dans les hepatocytes de poulet. Par des experiences de transfection transitoire, nous avons montre que l'acide arachidonique, l'etya et le clofibrate avaient un effet inhibiteur sur la transcription du gene scd1 de poulet. Il apparait que l'effet du clofibrate sur la transcription de scd1 est oppose entre les especes souris et poulet. L'effet de l'etya sur la transcription de scd1 est plus rapide et plus court que celui de l'acide arachidonique, ce qui suggere que ces deux acides gras de meme structure (20 : 4) agissent par deux voies de regulation differentes. Le fragment de la partie 5 flanquante du gene scd1 compris entre 375 et +125 nucleotides contient les sequences necessaires a l'activite basale du promoteur de ce gene. De plus, les elements cis de reponse a l'insuline, a l'acide arachidonique, a l'etya et au clofibrate sont localises dans cette region. Les 83 nucleotides en 5 de ce fragment sont indispensables a l'activite de ce promoteur. Ils contiennent deux boites caat putatives et sont hautement conserves entre le rat, la souris et le poulet. Ce travail rassemble les premiers resultats concernant la regulation genetique de scd1 hepatique de poulet. Il permet de proposer des orientations pour des investigations futures vers la caracterisation d'elements regulateurs du gene scd1.

Recent investigations suggest that reducing stearoyl CoA desaturase (SCD) 1 expression confers protection against obesity and insulin resistance, whereas others show that increasing SCD1 expression protects cells from lipotoxicity. The overall aim of this thesis was to establish the role of SCD1 expression in fatty acid metabolism and insulin stimulated glucose disposal in skeletal muscle. In vitro and in vivo studies were conducted to investigate the relationship between fatty acid subtype, SCD1 expression and fuel metabolism. The role of fatty acid subtype on fatty acid metabolite accumulation and insulin resistance was initially examined in rats. Rats were provided with a low fat diet or a high fat diet consisting of predominantly saturated (SAT) or polyunsaturated fatty acids (PUFA). Rats fed a SAT diet were insulin resistant and had increased skeletal muscle diacylglycerol content whereas rats fed a PUFA diet retained insulin sensitivity and accumulated triacylglycerol rather than diacylglycerol. Interestingly, SCD1 mRNA and protein content were elevated in SAT rats compared with PUFA fed and control fed rats, indicating a possible involvement of SCD1 in the aetiology of insulin resistance. Subsequently, SCD1 expression was examined in the skeletal muscle of various rodent models of genetic and diet-induced obesity. SCD1 content was consistently upregulated in the skeletal muscle of obese rodents. To determine whether SCD1 contributes to or protects from fatty-acid induced insulin resistance, SCD1 levels were transiently altered in L6 skeletal muscle myotubes. Short interfering (si) RNA was used to decrease SCD1 content and a pcDNA3.1/HygromSCD1 vector was introduced to increase SCD1 content. Reducing SCD1 protein resulted in marked esterification of exogenous fatty acids into diacylglycerol and ceramide. Insulin-stimulated Akt (acute transforming retrovirus thymoma) phosphorylation and 2-deoxyglucose uptake were reduced with SCD1 siRNA. Exposure of L6 myotubes to palmitate abolished insulin-stimulated glucose uptake in both control and SCD1 siRNA myotubes. Transient overexpression of SCD1 resulted in triacylglycerol esterification but attenuated ceramide and diacylglycerol accumulation and protected myotubes from fatty acid-induced insulin resistance. Further, these changes were associated with reduced phosphorylation of c-Jun Amino-Terminal Kinase (JNK) and the inhibitor of IêB kinase (IKK), both of which impair insulin signalling. These studies indicated that SCD1 protects from cellular toxicity in L6 myotubes by preventing excessive accumulation of bioactive lipid metabolites. Collectively, these experiments indicate that increasing SCD1 expression may be a protective mechanism designed to prevent insulin resistance in obese phenotypes.

Malaria is an infectious disease caused by Plasmodium parasites that are transmitted by the bite of female Anopheles mosquitoes. Successful acquisition and transmission of malaria parasites requires a female mosquito obtaining a blood meal from human hosts. The blood meal, which is rich in protein, is required for egg development. Most of the ingested protein is converted to lipid and stored in the fat body where vitellogenesis takes place. In this process, saturated fatty acids are converted to unsaturated fatty acids by the stearoyl-CoA desaturase (SCD1). Unsaturated fatty acids are also essential for maintaining cell membrane fluidity and other housekeeping functions. The main aim of this thesis was to functionally and phenotypically characterize the function of SCD1 during blood meal metabolism in the African mosquito vector Anopheles coluzzii. RNA interference (RNAi) silencing of the SCD1 gene and administration of a small molecule inhibitor of SCD1 had a significant impact on the survival of female mosquitoes following a blood meal. SCD1 knockdown (KD) caused a 100% mortality within 48 h after a human blood meal, while addition of the SCD1 small molecule inhibitor sterculic acid (SA) in the blood meal caused a 50% mortality within 72 h of blood meal. Microscopic analysis showed that SCD1 KD mosquitoes failed to develop eggs in response to the blood meal, while their thorax was filled with blood at 24 h post blood meal. These findings were highly consistent with electron microscopy data that showed increased plasma membrane rigidity and depletion of lipid droplets in the midgut epithelial cells. Transcriptional profiling using A. coluzzii oligonucleotide DNA microarrays showed that genes involved in protein, lipid and carbohydrate metabolism, as well as a large number of immunity genes were the most affected in blood-fed SCD1 KD versus control mosquitoes. Metabolomics profiling highlighted the biochemical framework by which the SCD1 KD phenotype is manifested after a blood meal, revealing increased amounts of saturated fatty acids and TCA cycle (and other interlinked pathway) intermediates in SCD1 KD and SA-treated mosquitoes. The data reported in this thesis reveal that silencing of SCD1 in female A. coluzzii mosquitoes leads to a metabolic syndrome primarily associated with the increase of saturated fatty acids and TCA cycle intermediates, which affects important biological functions leading to premature mosquito death. The accumulation of saturated fatty acids is also the likely cause of a potent immune response observed in the absence of infection, which resembles an auto-inflammatory reaction. These data provide important leads for the development of novel interventions aiming to block transmission of mosquito-borne diseases.

Stearoyl-CoA desaturase (SCD) catalyses the rate-limiting step in the biosynthesis of monounsaturated fatty acids by the insertion of a a ^9-cis double bond into acyl-CoA substrates in the presence of NADH and molecular oxygen. Recent work with animals in which expression of the enzyme has been eliminated, suggests a critical role for this enzyme in regulating lipid metabolism and obesity.

Studies were conducted to investigate the tissue distribution of stearoyl-CoA desaturase-1 (SCD) and the regulation of SCD1 protein expression by dietary fat, insulin, polyunsaturated fatty acids (PUFA), and linoleic acid (cis-9, cis-12 18:2). The first study examined tissue distribution of SCD1 protein in Holstein calves (n=6/diet) fed one of four milk replacer diets for a nine wk period after which they were sacrificed. Milk replacer diets varied in fat content and were formulated and administered as follows: 0.4 kg/d 20% protein, 20% fat (20:20; CON), 0.97 kg/d (28:20; HPLF), 0.97 kg/d (28:28; HPHF), or 1.46 kg/d (28:28; HPHF+). Samples of subcutaneous adipose tissue (AT), perirenal AT, omental AT, duodenum, proximal jejunum, distal jejunum, ileum, and liver were collected from calves fed the HPHF+ diet to determine SCD1 tissue distribution. Tissue homogenates were prepared and used for Western blotting. Additionally, dietary effects were analyzed on tissues expressing SCD1 protein for all 24 calves. The second study investigated the regulation of SCD1 protein expression by insulin, fatty acids increasing in degree of unsaturation, and increasing concentrations of linoleic (18:2) acid. Subcutaneous AT was collected from Smith Valley Meats in Rich Creek, VA and used to prepare explants cultured in treatment media for 24 h. Treatments consisted of insulin at 0, 7, 14, and 21 nM; stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids at 100 μM; and linoleic (18:2) acid at concentrations of 0, 25, 50, 75, and 100 μM. Tissue explant homogenates were used for Western blotting to detect SCD1. In the first study, we found that SCD1 protein was detectable in subcutaneous AT, perirenal AT, and omental AT; however, it was not detectable in liver or small intestine samples. Also, the HPHF+ diet increased SCD1 protein expression in subcutaneous AT and perireanl AT. In the second study, SCD1 protein expression increased linearly with insulin concentration. There was no fatty acid treatment effect, but there was a negative linear effect with increase in degree of unsaturation. Finally, there was no effect on SCD1 protein expression with linoleic acid increasing in concentration. In conclusion, results indicate that SCD1 protein expression was detected in bovine AT depots, regulated by dietary fat, insulin, and by PUFA .
Master of Science

The development of the metabolic syndrome (MetS) and cardiovascular diseases have been suggested to be influenced more by the quality than the amount of dietary fat. The FA composition of serum lipids may be used as biomarkers of dietary fat quality. FAs can, however, also be endogenously synthesized by lipogenic enzymes such as elongases and desaturases. Three desaturases are important in humans: Stearoyl-CoA-desaturase (SCD), ∆6-desaturase (D6D) and ∆5-desaturase (D5D) and surrogate measures of desaturase activities can be estimated as product-to-precursor FA ratios.

In this thesis, we demonstrated that high SCD, D6D and low D5D estimated activities predicted MetS 20 years later, as well as cardiovascular and total mortality during a maximum of 33.7 years. The relation between D5D and MetS was independent of lifestyle and BMI, while the relation between SCD, D6D and MetS was confounded by BMI. Serum proportions of palmitic (16:0), palmitoleic (16:1) and dihomo-γ-linoleic acids were higher and the serum proportion of linoleic acid (LA) lower at baseline in those individuals who developed MetS. Further, LA was inversely related to mortality, while palmitic, palmitoleic and dihomo-γ-linoleic acids were directly associated with mortality. We also demonstrated that a diet rich in saturated fat “induced” a similar serum FA pattern (including estimated desaturase activities) that was associated with MetS, cardiovascular disease and mortality. We also propose that the SCD ratio [16:1/16:0] might be a novel and useful marker of dietary saturated fat, at least in Western high-fat diets. Finally, genetic variations in the human SCD1 gene were linked to obesity and insulin sensitivity, results that agree with data in SCD1 deficient mice.

This thesis suggests that dietary fat quality and endogenous desaturation may play a role in the development of metabolic and cardiovascular diseases and the results support current dietary guidelines.

This PhD dissertation is framed on a line of research aimed at improving pork quality and, particularly, intramuscular fat (IMF) and monounsaturated fatty acid (MUFA) content as two of the main traits affecting nutritional and sensorial pork attributes. Several strategies have been investigated to enhance IMF and MUFA without increasing the rest of fat depots, but one of the most promising approaches is to find out genetic markers specifically associated to them. There is a variant in the promoter of the stearoyl-CoA desaturase (SCD) gene (AY487830:g.2228T>C used as tag single nucleotide polymorphism) that specifically enhances MUFA. This polymorphism localizes in the core sequence of a putative retinoic acid response element. The primary objective of the thesis was to assess the impact of this SCD polymorphism in different production and commercial scenarios. The thesis comprises four studies. The first one was intended to show whether the effect of this SCD polymorphism is maintained at different market carcass weights. The second and third study examined whether the effect of the SCD polymorphism is still evident in dry-cured products from purebred Duroc and Duroc-sired Iberian crossbreds, respectively. The fourth study investigated the impact of carotenoid intake as a source of dietary retinoic acid on IMF and MUFA in pigs from opposite genotypes at the SCD gene polymorphism. Four experiments, one per objective, were designed. The first consisted of a series of 1-4 repeated samples of m. longissimus thoracis and subcutaneous fat of 214 Duroc barrows collected at 160, 180, 210 and at 220 days of age. The second and third experiments were based, respectively, on 125 dry-cured hams from purebred Duroc pigs (53 traced throughout curation and 72 randomly sampled) and on 74 dry-cured hams from Duroc × Iberian pigs (taken from sliced trays randomly purchased from the same supplier). Dry-cured hams were from barrows and gilts. The fourth experiment consisted of 32 Duroc barrows which were allocated in a 2 x 2 split-plot design consisting of two finishing diets (from 165 to 195 days of age) differing in pro-vitamin A carotenoid content and the two SCD homozygotes. The diets were identical except the corn line used in the feed. The carotenoid-rich diet was formulated with 20% of a carotenoid-fortified corn while the carotenoid-restricted diet used instead 20% of its near isogenic line, which did not contain pro-vitamin A carotenoids. The positive effect of the T allele at the SCD gene on fat desaturation and MUFA content was confirmed throughout the growing-finishing period and after the curing process on both purebred Duroc and Duroc × Iberian dry-cured hams. A strong relationship between MUFA in green and dry-cured samples was found, with TT pigs being more effective in retaining increased MUFA in green hams until the end of the curing period. Moreover, the SCD polymorphism had a greater impact on MUFA than using hams from barrows instead of gilts. The results of the last experiment indicated that pigs fed with the carotenoid-rich diet had 2.8-fold more retinoic acid and 4.5-fold more SCD gene expression in liver, around one fifth less fat and MUFA in liver and one third less IMF in m. gluteus medius. The TT genotype at the SCD gene increased MUFA in all tissues. Liver fat and MUFA content declined non-linearly with liver all-trans retinoic acid, suggesting a saturation point at relatively low all-trans retinoic acid content. The results obtained support that a pro-vitamin A carotenoid restricted diet at finishing and the TT genotype at the SCD gene complement well each other to simultaneously increase IMF and MUFA without increasing total fat content. The leptin receptor (LEPR) NM_001024587:g.1987C>T polymorphism was also segregating in Duroc, with the T allele positively affecting IMF and the saturated fatty acid (SFA) content. Selection for the SCD T allele, particularly in combination with selection for the LEPR C allele, is confirmed as a good strategy to enhance the MUFA/SFA ratio and therefore to produce healthier meat.
Aquesta tesi doctoral s'emmarca en una línia d'investigació dirigida a millorar la qualitat de la carn de porc i, principalment, el greix intramuscular (IMF) i els àcids grassos monoinsaturats (MUFA), per ser dos dels principals trets que afecten els atributs nutricionals i sensorials del porc. S'han investigat diverses estratègies per millorar IMF i MUFA sense augmentar la resta dels dipòsits de greix, i una de les més prometedores és la de trobar marcadors genètics associats específicament a ells. Hi ha una variant en el promotor del gen de la estearoil-CoA desaturasa (SCD) que millora específicament MUFA (AY487830: g.2228T> C, polimorfisme d'un sol nucleòtid utilitzat com a marcador de referència). Aquest polimorfisme es localitza en la seqüència central d'un putatiu element de resposta a l’àcid retinoic. L'objectiu principal de la tesi va ser avaluar l'impacte del polimorfisme SCD en diferents escenaris de producció i comercials. La tesi consta de quatre estudis. El primer tenia per objectiu mostrar si l'efecte del polimorfisme SCD es manté a diferents pesos comercials de la canal. El segon i tercer estudi es dedicaren a examinar si l'efecte del polimorfisme SCD és fa encara evident en productes curats de raça pura Duroc i en els seus creuaments amb Ibèric, respectivament. En el quart estudi es va investigar l'impacte de la ingesta de carotenoides, com a font d'àcid retinoic en la dieta, sobre IMF i MUFA en porcs de genotips oposats en el polimorfisme del gen SCD. Es van dissenyar quatre experiments, un per cada objectiu. El primer experiment consistí en un a sèrie de fins a 4 mostres repetides de m. longissimus thoracis i de greix subcutani presses a 160, 180, 210 i en 220 dies d'edat en 214 mascles castrats Duroc. El segon i tercer experiments es van basar, respectivament, en 125 pernils curats de porcs Duroc de pura raça (53 traçats des de fres fins el final de la curació i 72 escollits a l’atzar) i en 74 pernils curats procedents d’un encreuament Duroc × Ibèric (presos de safates de pernil llescat comprades a l'atzar del mateix proveïdor). Els pernils curats provenien de mascles castrats i femelles. El quart experiment va consistir en 32 mascles castrats Duroc disposats segons un disseny split-plot 2 x 2, amb dues dietes de finalització (des de 165 a 195 dies d'edat) que diferien en el contingut de provitamina A carotenoide i els dos homozigots SCD. Les dietes van ser idèntiques, excepte en la línia genètica de blat de moro utilitzat en el pinso. La dieta rica en carotenoides es va formular amb un 20% de blat de moro fortificat amb carotenoides, mentre que la dieta control va incloure en el seu lloc un 20% de blat de moro d’una línia quasi isogènica sense carotenoides precursors de provitamina A. Els resultats obtinguts confirmen que l'efecte positiu de l'al·lel T del gen SCD sobre la dessaturació del greix i el contingut de MUFA es manté durant tot el període de creixement i d'acabat i al final del procés de curació, tant en els pernils Duroc com en els Duroc × Ibèric. S’ha posat en evidencia una forta relació entre el contingut de MUFA en el pernil fresc i curat, de tal manera que els porcs TT són més eficaços retenint els MUFA fins al final del període de curació. D'altra banda, el polimorfisme SCD ha tingut un major impacte sobre MUFA que la utilització de porcs castrats en lloc de femelles per la producció de pernil. Els porcs alimentats amb una dieta enriquida en carotenoides mostraren 2.8 vegades més àcid retinoic i 4.5 vegades més expressió del gen SCD en fetge, aproximadament un cinquè menys de greix i MUFA en el fetge i un terç menys de IMF en m. gluteus medius. El genotip TT del gen SCD ocasionà un augment de MUFA en tots els teixits. El greix del fetge i el contingut de MUFA disminuïren de forma no lineal amb l'àcid holo-trans retinoic del fetge, el que suggereix l’existència d’un punt de saturació a un nivell relativament baix d'àcid holo-trans retinoic. Els resultats obtinguts han posat de manifest que els efectes d’una dieta restringida en carotenoides precursors de la vitamina A i el genotip TT del gen SCD es complementen bé per augmentar simultàniament IMF i MUFA sense variar el contingut de greix total. S’ha observat que el polimorfisme NM_001024587: g.1987C> T del gen del receptor de la leptina (LEPR) també segrega en Duroc, amb l'al·lel T afectant positivament IMF i el contingut d'àcids grassos saturats (SFA). La selecció a favor de l'al·lel T del gen SCD, particularment en combinació amb la selecció a favor de l'al·lel C de LEPR, es confirma com una bona estratègia per millorar la relació MUFA / SFA i per tant la producció de carn més saludable.
La presente tesis doctoral se enmarca en una línea de investigación dirigida a mejorar la calidad de la carne cerdo y, en particular, su contenido de grasa intramuscular (IMF) y ácidos grasos monoinsaturados (MUFA) al ser dos de los principales caracteres que afectan atributos tanto nutricionales como organolépticos de la carne. A la fecha, diversas estrategias han sido investigadas para mejorar IMF y MUFA sin aumentar el resto de depósitos grasos de la canal, siendo uno de los enfoques más prometedores el encontrar marcadores genéticos asociados específicamente a dichos caracteres. Existe una variante en el promotor del gen de la estearoil-CoA desaturasa (SCD) (AY487830: g.2228T>C ha sido utilizado como polimorfismo de un solo nucleótido de referencia) que mejora MUFA. Este polimorfismo se localiza en el núcleo de la secuencia de un elemento de respuesta putativo al ácido retinoico. El objetivo principal de la tesis fue evaluar el impacto del polimorfismo SCD en diferentes escenarios productivos y comerciales. La tesis está compuesta por cuatro estudios. El primero tuvo como objetivo evaluar si el efecto del polimorfismo SCD se mantiene a diferentes pesos comerciales. El segundo y tercer estudio examinaron si el efecto del polimorfismo SCD es evidente en jamones curados de raza pura Duroc y de Ibéricos cruzados con Duroc. El cuarto estudio investigó el impacto de la ingesta de carotenoides, como fuente de ácido retinoico en la dieta, sobre IMF y MUFA en cerdos de genotipos opuestos para el polimorfismo SCD. Para cada objetivo se diseñó un experimento. El primero consistió en un muestreo repetido (de 1 a 4) de m. longissimus thoracis y grasa subcutánea a las edades de 160, 180, 210 y 220 días en 214 machos castrados Duroc. El segundo y tercer experimento se basaron en 125 jamones curados de cerdos Duroc de raza pura (53 rastreados a lo largo de la curación y 72 muestreados al azar) y en 74 jamones curados Duroc × Ibérico (tomados de bandejas loncheadas compradas aleatoriamente del mismo proveedor), respectivamente. Los jamones curados provenían de machos castrados y hembras. El cuarto experimento consistió en 32 machos castrados Duroc que fueron asignados a un diseño split-plot 2 x 2 consistente en dos dietas de engorde (de 165 a 195 días de edad) que diferían en el contenido de carotenoides precursores de vitamina A y los dos genotipos homocigotos del gen SCD. Las dietas fueron idénticas excepto en la línea de maíz usada en la formulación. La dieta rica en carotenoides se formuló con 20% de maíz fortificado con carotenoides, mientras que la dieta restringida en carotenoides contuvo en su lugar un 20% de una línea casi-isogénica que no contenía carotenoides precursores de vitamina A. El efecto positivo del alelo T del gen SCD sobre la desaturación de la grasa y el contenido de MUFA se confirmó a lo largo del período de crecimiento-engorde así como al final del proceso de curado, tanto en los jamones de raza pura Duroc como en los Duroc × Ibérico. Se encontró una fuerte correlación en el contenido de MUFA entre muestras frescas y curadas, mientras que el genotipo TT fue más efectivo reteniendo el aumento de MUFA observado en los jamones frescos hasta el final del período de curación. Por otra parte, el polimorfismo SCD tuvo un mayor impacto sobre MUFA que el uso de jamones de machos castrados en lugar de hembras. Los resultados del último experimento mostraron que los cerdos alimentados con la dieta rica en carotenoides tuvieron 2.8 veces más ácido retinoico y 4.5 veces más expresión del gen SCD en hígado, alrededor de un quinto menos grasa y MUFA en el hígado, y un tercio menos de IMF en el m. gluteus medius. El genotipo TT del gen SCD aumentó MUFA en todos los tejidos. La grasa hepática y el contenido de MUFA disminuyeron de forma no lineal con el ácido retinoico hepático, sugiriendo un punto de saturación a un nivel relativamente bajo de ácido retinoico. Los resultados obtenidos evidencian que los efectos de una dieta restringida en carotenoides precursores de vitamina A durante el engorde y del genotipo TT del gen SCD se complementan bien para aumentar simultáneamente IMF y MUFA sin alterar el contenido total de grasa. El polimorfismo del gen del receptor de la leptina (LEPR) NM_001024587:g.1987C>T también segrega en esta población Duroc, con el alelo T afectando positivamente IMF y el contenido de ácidos grasos saturados (SFA). La selección del alelo T del gen SCD en combinación con la selección del alelo C de LEPR se confirma como una buena estrategia para aumentar el ratio MUFA / SFA y en consecuencia producir carne más saludable.

Studies were conducted to investigate: 1) desaturation of dietary trans-vaccenic acid (TVA, trans11-18:1) to the cis9,trans11-18:2 isomer of conjugated linoleic acid (9/11CLA), 2) effects of two conjugated linoleic acid isomers [9/11CLA or trans10,cis12-18:2 (10/12CLA)] and TVA on enzyme activities and mRNA abundance for lipogenic enzymes, and 3) regulation of stearoyl-CoA desaturase (SCD) gene transcription. In the first study, lactating mice were fed 3% linoleic acid (LA), or 2% LA plus 1% stearic acid (SA), 1% TVA, or 1% CLA mixture. Dietary TVA enriched the 9/11CLA content of carcass, liver, and mammary tissue of lactating mice. A similar enhancement of 9/11CLA also was observed in liver, but not carcass, of suckling pups nursing TVA-fed dams. The CLA mixture decreased mammary acetyl-CoA carboxylase (ACC) activity compared with other treatments. However, total fatty acid content of mammary tissue was reduced only when compared with TVA. In the second experiment, lactating mice were fed 3% canola oil (OA), or 2% OA plus 1% SA, 1% TVA, 1% 9/11CLA, or 1% 10/12CLA. Dietary TVA, 9/11CLA, and 10/12CLA decreased mRNA abundance for ACC and fatty acid synthase (FAS) in mammary tissue, suggesting each had the potential to reduce de novo fatty acid synthesis. However, only the CLA isomers decreased ACC activity in mammary tissue and concentration of medium-chain fatty acids (MCFA = 12:0+14:0+16:0) in milk fat. The 10/12CLA isomer caused greater reductions in MCFA and milk fat percentage than the 9/11CLA, indicating that 10/12CLA is the primary CLA isomer affecting lipid metabolism in the mammary gland. Dietary TVA, 9/11CLA, or 10/12CLA decreased SCD enzyme activity and mRNA abundance in mammary tissue. In study 3, mouse (COMMA-D/MME) and bovine (Mac-T) mammary epithelial cells were transfected with the putative promoter (600 bp) of SCD gene. The 9/11CLA reduced SCD gene transcription in mouse cells, but not bovine cells. Transcription, however, was reduced in both cell lines by 10/12CLA, linoleic acid, and linolenic acid. Thus, reduced SCD transcription in response to the CLA isomers in mouse mammary cells in vitro may provide an explanation for reduced SCD enzyme activity and mRNA abundance in mammary tissue when lactating mice were fed either of the CLA isomers. In contrast, stearic acid, oleic acid, and TVA did not affect SCD transcription. Although TVA did not reduce SCD transcription in mouse mammary cells in vitro, it did reduce SCD enzyme activity and mRNA abundance in mammary tissue when fed to lactating mice. The results suggested TVA may influence SCD mRNA processing or stability in the nucleus after transcription. Despite the reduction in SCD mRNA and enzyme activity, however, substantial quantities of TVA were desaturated to the 9/11CLA isomer when TVA was fed to lactating mice in the first two studies. Thus, dietary TVA provides an alternate supply of the anticarcinogenic 9/11CLA isomer in tissues.
Ph. D.

The effect of exogenous unsaturated fatty acids on cellular fatty acid biosynthesis in mammary cells was examined. Under normal situations, even though the diet of a dairy cow contains considerable amounts of unsaturated fatty acids, viz. oleic acid (18:1) and linoleic acid (18:2), the major 18-carbon fatty acid that enters the circulation post-ruminally for delivery to the mammary gland is saturated fatty acid, viz. stearic acid (18:0). This is due to extensive ruminal biohydrogenation of unsaturated fatty acids. Studies have indicated that saturated fatty acids such as 18:0 are enhancers and that certain unsaturated fatty acids are inhibitors of de novo fatty acid synthesis in tissues such as the liver and adipose tissue. The present study investigated the effect of cis and trans isomers of 18:1 and 18:2 on de novo fatty acid synthesis and desaturation in mouse and bovine mammary epithelial cell cultures, and compared it with the effect caused by 18:0. In the first experiment 12.5, 25, 50 or 100 micromoles stearic acid (SA), oleic acid (OA), elaidic acid (EA), trans-vaccenic acid (TVA), linoleic acid (LA) or conjugated linoleic acid (CLA) were supplemented in the media of mouse mammary epithelial (MME) cells that were grown to confluence in Dulbecco's modified Eagle's medium (DMEM). As indicated by cellular palmitic acid (16:0) content and fatty acid synthetase (FAS) activity, when compared with SA all unsaturated fatty acid treatments inhibited de novo fatty acid synthesis in MME cells. In addition, OA at all concentrations and LA and CLA at 50 and 100 micromoles inhibited cellular stearoyl-CoA desaturase (SCD) activity and mRNA abundance. However, EA and TVA, when compared with SA, enhanced SCD activity and mRNA abundance at 12.5 and 25 micromoles. In the second experiment 25, 50 or 100 micromoles SA, OA, TVA, LA or CLA were supplemented in the media of bovine mammary epithelial cells that were grown to confluence in DMEM. As indicated by cellular 16:0 content, acetyl-CoA carboxylase (ACC) activity and FAS activity, treatment with the unsaturated fatty acids inhibited de novo fatty acid synthesis at all concentrations, when compared with SA. Unsaturated fatty acid treatments also reduced the abundance of ACC and FAS mRNA in the cells. When compared with SA at all treatment-concentrations, OA and LA inhibited whereas TVA and CLA enhanced cellular SCD activity and mRNA abundance in the bovine cells. In both cell types, CLA and TVA appeared to be the most potent inhibitors of saturated fatty acid biosynthesis.
Ph. D.

Obesity is today a problem that has reached epidemic proportions. One of the causes of obesity is the over-consumption of energy. Fat is the most energy-dense nutrient, where the quality seems to be more important for the development of the metabolic diseases than the quantity. The fatty acid composition in serum lipid fractions can be used to mirror the dietary fat quality.

Stearoyl-CoA-desaturase (SCD) is an enzyme that converts saturated to monounsaturated fatty acids. A surrogate measure of SCD activity can be estimated as a fatty acid ratio; 16:1/16:0 (palmitoleic acid/palmitic acid) and 18:1/18:0 (oleic acid/stearic acid). The aim of this project was to investigate the intra-day variation in the SCD-ratio in humans eating a standardized diet. The results showed that triacylglycerol and nonesterified fatty acid fractions in serum lipids had a significant variance in the 16:1/16:0 ratio during the day, whereas 18:1/18:0 ratio in the same fractions did not exhibit the same pattern. In this study 16:1/16:0 ratio also seems to be a better marker than 18:1/18:0 ratio for estimating SCD activity. For further evaluation of the intra-day variation there need to be a more long-term study of the SCD-activity for a larger group of subjects.

El ácido 2-hidroxioleico (2OHOA) es un fármaco antitumoral diseñado para regular la estructura y composición de los lípidos de membrana y la función de importantes proteínas de membrana. El objetivo principal de este trabajo fue estudiar cómo el 2OHOA modula la composición lipídica y la estructura de membrana en las células tumorales. Se observó que el 2OHOA indujo profundas alteraciones en el contenido de fosfolípidos, aumentando el contenido de esfingomielina y disminuyendo el contenido de fosfatidiletanolamina y fosfatidilcolina. Este efecto fue específico contra las células cancerosas, ya que el tratamiento no afectó la composición lipídica de las células no tumorales MRC-5 de fibroblastos humanos. El aumento de SM se debió a una activación rápida y específica de las SM sintasas. Como consecuencia de la activación sostenida de la SMS, todo el metabolismo de los esfingolípidos se vio afectado. Finalmente, se evaluó el impacto de todos estos cambios sobre las propiedades biofísicas de membrana mediante espectroscopia de fluorescencia
2-Hydroxyoleic acid (2OHOA) is a potent antitumor drug that was designed to regulate membrane lipid composition and structure and the function of important membrane proteins. The main goal of this work was to study how 2OHOA modulates the membrane lipid composition and structure of tumor cells. 2OHOA induced dramatic alterations in phospholipid content, increasing sphingomyelin mass, and decreasing phosphatidyl-ethanolamine and phosphatidylcholine. This effect was specific against cancer cells as it did not affect non-tumor MRC-5 cells. The increased SM mass was due to a rapid and highly specific activation of SM synthases. As a consequence of the sustained activation of SMS, the whole sphingolipid metabolism was affected. Then, the impact of all these changes on membrane biophysical properties was evaluated by fluorescence spectroscopy

Stearoyl-CoA desaturase1 (SCD1) catalyzes the synthesis of conjugated linoleic acid (CLA) and mono-unsaturated fatty acids (MUFA) in the mammary gland of ruminant animals. We hypothesized that single nucleotide polymorphisms (SNPs) in the coding region, 5' and 3' untranslted regions (UTRs) of the SCD1 gene would influence the activity of SCD1 enzyme and consequently account for some within-breed variations in milk CLA and MUFA. Sequence analysis of the coding region of the SCD1 gene of Jerseys and Holsteins revealed c.702A→G, c.762T→C and c.878C→T SNPs in exon 5 in both breeds and c.435G→A in exon 3 in Holsteins. The SNPs resulted in: A (G435A702T 762C878), A1 (A435A702T 762C878), B (G435G702C 762T878) and B1 (A435G702C 762T878) coding variants in Holsteins and only variants A and B in Jerseys. Only SNP 878C→T resulted in a non-synonymous codon change resulting in p.293Ala and p.293Val protein variants or alleles at the SCD1 locus. Subsequent association studies found significantly higher C10 index, C12 index and C14 index and consequently higher concentrations of C10:1 and C12:1 in p.293AA cows compared to the p.293VV cows in both breeds. The SCD1 genotype had no influence on concentrations of C141, C16:1, C18:1 and CLA in both breeds.
Sequence analysis of the 5' and 3' UTRs revealed no SNPs in the 5'UTR and a total of 14 SNPs in the 3'UTR of both breeds. The SNPs were in complete linkage disequilibrium resulting in 3 haplotypes or regulatory variants: H1 (G1571G1644C1763C2053A2584 A3007C3107G3208 T3290G 3497G3682A4399C4533G4881), H2 (G1571G1644A1763C2053A 2584G3007 C3107G3208T3290G3497G 3682A4399C4533G4881) and H3 (T 1571C1644A1763 T2053G2584G3007T 3107A3208C3290A3497A3682T 4399T4533A4881) in Holsteins and only H1 and H3 variants in Jerseys. A subsequent association study involving 862 Holstein cows, found the H1 regulatory variant to be associated with higher C10 and C12 desaturase indices and consequently with higher concentrations of C10:1 and C12:1 compared with the H3 variant. The effects of the H2 variant were intermediate to those of H1 and H3. 3'UTR genotype had no influence on the concentrations of C14:1, C16:1, C18:1 and CLA. The concentrations of C10:1 and C12:1 in milk fat could therefore be due to effects of SNPs in the open reading frame and the 3'UTR regions of the SCD1 gene. These results indicate that SNPs in the coding and 3'UTR regions of the SCD1 gene could be used as markers for genetic selection for increased C10:1 and C12:1 contents of milk.

La sclérose latérale amyotrophique est une maladie neurodégénérative associée à un dysfonctionnement métabolique. Des altérations du métabolisme des lipides, décrites chez les patients SLA et les animaux modèles, pourraient participer à la mise en place des premières étapes de la maladie. L’objectif de cette thèse était d’étudier le rôle de la stéaroyl-coenzyme A désaturase 1 (SCD1), une enzyme clé du métabolisme des lipides, dans la SLA. En étudiant le profil d’acides gras périphériques dans un modèle de souris SLA, les souris SOD1m, nous avons vu une diminution de l’activité de la SCD1 dès les stades précoces (subcliniques) de la maladie. Cette diminution pourrait expliquer, à elle seule, les altérations du métabolisme des lipides caractéristiques de la SLA. La répercussion de la perte de l’activité de la SCD1 sur l’axe moteur a été étudiée. Une délétion du gène ou une inhibition pharmacologique de la SCD1 améliore la récupération fonctionnelle après lésion du nerf sciatique chez la souris sauvage. Nous avons cherché à voir si la perte d’activité de la SCD1 trouvée chez les souris SOD1m est un mécanisme de protection mis en place pour lutter contre l’évolution de la SLA. Nous avons traité des souris SOD1m avec un inhibiteur de l’activité de la SCD1. Le traitement a conduit à une augmentation du métabolisme oxydatif, une préservation de l’intégrité neuromusculaire ainsi qu’une amélioration de la survie des motoneurones. Nousconcluons que l’inhibition de la SCD1 représente une cible thérapeutique prometteuse dans la SLA
Amyotrophic lateral sclerosis is a neurodegenerative disease, associated with metabolic dysfunction. Alteration of lipid metabolism has been documented in ALS patients and animal models, and could participate to the first pathological steps of the disease. The objective of this thesis was to study the role of stearoyl-CoA desaturase 1 (SCD1), a key enzyme of lipid metabolism, in ALS. By studying the profile of peripheral fatty acids in an animal model of ALS, the SOD1 mice, we found that SCD1 activity was strongly reduced at early (sub-clinical) disease stage, and that this reduction could explain in itself the alteration of lipid metabolism characteristic of ALS. The impact of loss of SCD1 activity for the motor axis was then studied. Genetic deletion or pharmacological inhibition of SCD1 enhanced functional recovery after sciatic nerve injury in mice. Wefurther explored if the loss of SCD1 activity found in SOD1 mice is a protective mechanism elicited in response to ALS. We treated SOD1 mice with an inhibitor of SCD1 activity. The treatment resulted in exacerbated muscular oxidative metabolism,preservation of neuromuscular integrity and enhanced motor neuron survival. We conclude that inhibition of SCD1 represents a promising therapeutic target for ALS

Plasmodium falciparum et Mycobacterium tuberculosis sont deux pathogènes humains, causant respectivement le paludisme et la tuberculose. Les efforts pour combattre ces maladies et leurs ravages se heurtent à une adaptation des pathogènes vis-à-vis de leurs inhibiteurs respectifs. La recherche constante de nouveaux moyens de lutte et de nouvelles cibles d'intérêt pharmacologique sont une priorité dans l'éradication de ces deux organismes. Bien que phylogénétiquement éloignés, l'un étant un eucaryote hématozoaire et l'autre un procaryote, ils partagent des voies métaboliques communes, comme par exemple la biosynthèse des acides gras. La première partie de ce travail a consisté en l'étude de la synthèse et de la désaturation des acides gras chez P. Falciparum. Nous avons d'abord caractérisé l'activité d'une stéaroyl-CoA Δ9-désaturase chez P. Falciparum et déterminé ses caractéristiques biochimiques. Nous avons également démontré que l'enzyme responsable de la conversion d'acide stéarique en acide oléique est essentielle au développement intra-érythrocytaire du parasite, grâce à l'utilisation du méthyl-sterculate, un inhibiteur spécifique des Δ9-désaturases. Par ailleurs, nous avons participé à une étude centrée sur l'activité de l'énoyl-ACP réductase PfFabI, une enzyme clé du système de biosynthèse des acides gras de type II (FAS-II), lors de la schizogonie hépatique. Cette enzyme est essentielle au développement des schizontes hépatiques tardifs, la délétion du gène pffabI empêchant une maturation normale des parasites. Nous avons également mis en évidence une activité de synthèse des acides gras chez ce mutant au cours de la phase érythrocytaire, soulignant la présence d'une voie de synthèse des acides gras indépendante de FAS-II. La seconde partie de cette thèse traite de la biosynthèse de l'acide oléique et de son inhibition chez les mycobactéries. Nous avons d'abord démontré l'efficacité antituberculeuse de NAS-91 et NAS-21, originellement décrits comme inhibiteurs de la β-hydroxyacyl-ACP déhydratase PfFabZ chez P. Falciparum. Ces molécules inhibent la synthèse des acides mycoliques, acides gras particuliers impliqués dans la virulence des mycobactéries, vraisemblablement par une inhibition du système FAS-II. Elles inhibent également de manière prononcée la synthèse d'acide oléique chez M. Bovis BCG. Enfin, nous avons montré la participation de la monooxygénase mycobactérienne EthA dans l'activation d'agents antituberculeux ciblant la synthèse de l'acide oléique et/ou des acides mycoliques. Une fois activés, certains de ces composés, tels que l'isoxyl, inhibent fortement l'activité Δ9-désaturase chez M. Bovis BCG, réduisant considérablement de la production d'acide oléique
P. Falciparum and M. Tuberculosis are two major human pathogens, and the causative agents of malaria and tuberculosis, respectively. Efforts in fighting these diseases often are limited by the capacity of adaptation of these pathogens to the drugs. New means to combat these diseases are urgent for eradication of these organisms. Although being phylogenetically different, P. Falciparum being an eukaryotic hematozoan and M. Tuberculosis a prokaryot, they share common metabolic pathways, such as the fatty acid biosynthesis system. The first part of this thesis was devoted to the biosynthesis and desaturation of fatty acids in P. Falciparum. We first characterized the stearoyl-CoA Δ9-desaturase activity in P. Falciparum and determined its biochemical properties. We also demonstrated that the enzyme responsible for the conversion of stearic to oleic acid is essential for intra-erythrocytic growth by using methyl-sterculate, a specific inhibitor of Δ9-desaturases. In addition, we have participated in a study focusing on the P. Falciparum enoyl-ACP reductase PfFabI, a key enzyme of the type II fatty acid synthase (FAS-II), found to be only active during hepatic schizogony. This enzyme is essential for the development of late hepatic schizontes, and deletion of pffabI is deleterious for the normal maturation of parasites. Moreover, we provided evidence that a fatty acid biosynthesis activity occurred during the intra-erythrocytic cycle of this mutant, highlighting the presence of a FAS-II independent mechanism. The second part of this thesis deals with the oleic acid biosynthesis and its inhibition in mycobacteria. We first demonstrated the antitubercular activity of NAS-91 and NAS-21, known as inhibitors of the P. Falciparum β-hydroxyacyl-ACP dehydratase PfFabZ. These drugs inhibit the biosynthesis of mycolic acids, which are unique fatty acids involved in mycobacterial virulence, presumably by targeting the FAS-II system. These drugs also inhibit oleic acid synthesis in M. Bovis BCG. Finally, we demonstrated the participation of the mycobacterial monooxygenase EthA in the activation step of antitubercular drugs targeting oleic acid and/or mycolic acid biosynthesis. Once activated, some of these drugs, such as isoxyl, exhibit a strong inhibitory effect against the Δ9-desaturase in M. Bovis BCG, considerably reducing oleic acid production

The gastrointestinal tract (GIT) regulates nutrient uptake, secretes hormones and has a crucial gut flora and enteric nervous system. Of relevance for these functions are the G protein-coupled receptors (GPCRs) and the solute carriers (SLCs). The Adhesion GPCR subfamily is known to mediate neural development and immune system functioning, whereas SLCs transport e.g. amino acids, fatty acids (FAs) and drugs over membranes. We aimed to comprehensively characterize Adhesion GPCR and SLC gene expression along the rat GIT. Using qPCR we measured expression of 78 SLCs as well as all 30 Adhesion GPCRs in a twelve-segment GIT model. 21 of the Adhesion GPCRs had a widespread (≥5 segments) or ubiquitous (≥11 segments) expression. Restricted expression patterns were characteristic for most group VII members. Of the SLCs, we found the majority (56 %) of these transcripts to be expressed in all GIT segments. SLCs were predominantly found in the absorption-responsible gut regions. Both Adhesion GPCRs and SLCs were widely expressed in the rat GIT, suggesting important roles. The distribution of Adhesion GPCRs defines them as a potential pharmacological target. FAs constitute an important energy source and have been implicated in the worldwide obesity increase. FAs and their ratios – indices for activities of e.g. the desaturase enzymes SCD-1 (SCD-16, 16:1n-7/16:0), D6D (18:3n-6/18:2n-6) and D5D (20:4n-6/20:3n-6) – have been associated with e.g. overall mortality and BMI. We examined whether differences in FAs and their indices in five lipid fractions contributed to obesity susceptibility in rats fed a high fat diet (HFD), and the associations of desaturase indices between lipid fractions in animals on different diets. We found that on a HFD, obesity-prone (OP) rats had a higher SCD-16 index and a lower linoleic acid (LA) proportions in subcutaneous adipose tissue (SAT) than obesity-resistant rats. Desaturase indices were significantly correlated between many of the lipid fractions. The higher SCD-16 may indicate higher SCD-1 activity in SAT in OP rats, and combined with lower LA proportions may provide novel insights into HFD-induced obesity. The associations between desaturase indices show that plasma measurements can serve as proxies for some lipid fractions, but the correlations seem to be affected by diet and weight gain.

Les patients SLA et les souris modèles présentent un dysfonctionnement métabolique qui coïncide avec le changement de concentration de différentes espèces lipidiques. Notre hypothèse est qu'un tel dysfonctionnement métabolique au niveau musculaire conduirait aux premiers changements observés dans la SLA. Nous avons montré que l'expression de la stéaroyl-coenzyme A désaturase 1 (SCD1), une enzyme clé de la synthèse des acides gras mono-insaturés à partir des acides gras saturés, est diminuée dans le muscle avant les premiers symptômes moteurs observés chez les souris modèles de SLA. Dans ce modèle murin, les altérations en acides gras au niveau circulant et hépatique, traduisant les changements de SCD1,apparaissent lors des premiers symptômes de la pathologie. De plus, l'inhibition pharmacologique de l'activité de SCD1 mime le phénotype métabolique des souris modèles de SLA. Notre étude a ainsi montré que la diminution de la SCD1 joue un rôle important pour l'activité neuromusculaire. Elle module les besoins énergétiques, maintien l'activité musculaire par augmentation du métabolisme oxydatif et agit sur l'expression de gènes impliqués dans le développement et le fonctionnement de la jonction neuromusculaire. De plus, l'ablation du gène SCD1 stimule la récupération fonctionnelle musculaire après lésion du nerf. L'inhibition pharmacologique de SCD1 apporte également une protection au muscle. Nous avons pu conclure de cette étude qu'une modification de l'expression de SCD1 ainsi que du profil d'acides gras peut apporter une protection au muscle pour lutter contre la pathologie. En outre, des inhibiteurs de l'activité enzymatique de la SCD1 pourraient être développés comme traitement thérapeutique dans la SLA.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the Philadelphia chromosome (Ph) that arises from a reciprocal translocation between chromosomes 9 and 22, thereby resulting in the formation of the chimeric BCR-ABL oncogene encoding a constitutively activated tyrosine kinase. BCR-ABL tyrosine kinase inhibitors (TKIs) induce a complete hematologic and cytogenetic response in the majority of chronic phrase CML patients. However, TKIs cannot efficiently eradicate leukemia stem cells (LSCs) because of the insensitivity of LSCs to TKIs. Therefore, developing new strategies to target LSCs is necessary and critical for curing CML, and success of this approach depends on further understanding the molecular mechanisms by which LSCs survive and are maintained. In Chapter I, I briefly introduce CML disease, BCR-ABL oncoprotein, and TKIs. I also describe the identification and features of LSCs. Several key pathways in LSCs including Wnt/ß-catenin, hedgehog, FoxO, Bcl6 and HIF1, are discussed. I also propose our strategy to identify unique molecular pathways that are important for LSCs but not their normal stem cell counterparts. In Chapter II, I describe our finding about the function of the positive regulator, HIF1α, in CML development and LSC survival. I show that loss of HIF1α impairs the maintenance of CML through impairing cell cycle progression and inducing apoptosis of LSCs, and I also report that p16Ink4a and p19Arf mediate the effect of HIF1α on LSCs, as knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α-/- LSCs. As detailed in Chapter III and IV, through comparing the global gene expression profiles of LSCs and HSCs, I find two novel regulators, Blk and Scd1, which act as tumor suppressors in CML development. In Chapter III, I show that Blk is markedly down-regulated by BCR-ABL in LSCs, and that c-Myc and Pax5 mediate this down-regulation. Deletion of Blk accelerates CML development; conversely, Blk overexpression significantly delays the development of CML and impairs the function of LSCs. I also demonstrate that p27, as a downstream effector, is involved in the function of Blk in LSCs. Blk also functions as a tumor suppressor in human CML stem cells, and inhibits the colony-forming ability of human CML cells. In Chapter IV, I investigate the function of another negative regulator, Scd1, in CML LSCs, and find that expression of Scd1 is down-regulated in mouse LSCs and human CML cells. We report that Scd1 acts as a tumor suppressor in CML, as loss of Scd1 causes acceleration of CML development and overexpression of Scd1 delays CML development. Using a colony-forming assay, I demonstrate that Scd1 impairs the maintenance of LSCs due to the change of expression of Pten, p53 and Bcl2. Importantly, I find that both Blk and Scd1 do not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Taken together, our findings demonstrate that HIF1α is required for the maintenance of CML LSCs, and conversely that Blk and Scd1 suppress the function of LSCs, suggesting that combining TKI treatment with specific targeting of LSCs will be necessary for curing CML.

Absence of stearoyl-CoA desaturase-1 (SCD1) in mice leads to chronic inflammation of the skin and increased susceptibility to atherosclerosis, while also increasing plasma inflammatory markers. A recent report suggested that SCD1 deficiency also increases disease severity in a mouse model of inflammatory bowel disease, induced by dextran sulfate sodium (DSS). However, SCD1-deficient mice are known to consume increased amounts of water, which would also be expected to increase the intake of DSS-treated water. The aim of this study was to determine the effect of SCD1 deficiency on DSS-induced acute colitis with DSS dosing adjusted to account for genotype differences in fluid consumption. Wild-type controls were treated with 3.5% DSS for 5 days to induce moderately severe colitis, while the concentration of DSS given to SCD1-deficient mice was lowered to 2.5% to control for increased fluid consumption. Colonic inflammation was assessed by clinical and histological scoring. Although SCD1-deficient mice consumed a total intake of DSS that was greater than that of wild-type controls, colonic inflammation, colon length and fecal blood were not altered by SCD1-deficiency in DSS-induced colitis, while diarrhea and total weight loss were modestly improved. Despite SCD1 deficiency leading to chronic inflammation of the skin and increased susceptibility to atherosclerosis, it does not accelerate inflammation in the DSS-induced model of acute colitis when DSS intake is controlled. These observations suggest that SCD1 deficiency does not play a significant role in colonic inflammation in this model. [The original version of this article, along with updated information and services is located on the World Wide Web at: http://dx.doi.org/10.1016/j.bbalip.2009.08.001]

This thesis investigates the contributions of fatty acids (FA) to adipokine dysregulation and inflammation. Differentiated 3T3-L1 adipocytes were treated with palmitic, stearic, palmitoleic, and oleic acids and changes in adipokine gene expression were measured. Here it was determined that saturated FA (SFA) increased the expression of RANTES and monounsaturated FA (MUFA) decreased the expression of RANTES and IL-6; demonstrating that FA differentially regulate adipokine expression. Relationships between plasma levels of SFA, MUFA and C-reactive protein (CRP) were also identified in a human observational study, further demonstrating the link between FA and inflammation Moreover, an association was also found between stearoyl-CoA desaturase 1 (SCD1) activity and CRP, demonstrating that SCD1 activity contributes to the inflammatory state. Genetic variation in SCD1 was also found to alter plasma FA and CRP levels, thus contributing to systemic inflammation. Taken together, these results demonstrate that SFA and MUFA influence adipokine dysregulation and systemic inflammation.

La Stearoyl-CoA Désaturase-l (SCD1) est l'enzyme catalysant la synthèse des acides gras monoinsaturés, synthétisant le palmitoléyl-CoA (C16:1) et l'oléoyl-CoA (C18:1) à partir respectivement du palmitoyl-CoA (C16:0) et du stéaroyl-CoA (C18:0). Ces acides gras entrent ensuite dans la composition des triglycérides et des phospholipides membranaires. L'altération de la composition des phospholipides a été impliquée dans de nombreuses maladies incluant l'obésité et le syndrome métabolique associé. SCD1 est fortement exprimée au niveau du foie et du tissu adipeux. Elle est étroitement régulée par de nombreux facteurs nutritionnels et hormonaux principalement au niveau transcriptionnel mais aussi via une dégradation protéique rapide. Ceci permet à la cellule d'adapter l'activité enzymatique de SCD1 à la demande physiologique. Au niveau hépatique, l'insuline et la leptine sont impliquées respectivement dans la stimulation et l'inhibition de l'expression de SCD1. Leur fixation sur leur récepteur respectif précède l'activation de nombreuses voies de signalisation, qu'elles partagent pour la plupart, comme la voie 3-phosphoinositide-dependent protein kinase-1 (PI3K) et la voie des Mitogen Activated Protein Kinase Extracellular Regulated Kinase 1/2 (MAPK ERK1/2). Les travaux présentés dans cette thèse ont permis de caractériser les mécanismes impliqués dans l'action de l'insuline et de la leptine sur l'expression de SCD1. Dans un premier temps, nous avons mis en évidence l'implication de la voie PI3K Mammalian Target Of Rapamycin (mTor) et des facteurs de transcription Sterol Response Element Binding Protein-1 (SREBP-1) et Nuclear Factor Y (NF-Y) dans la stimulation de l'expression de SCD1 par l'insuline. Dans un deuxième temps, nous avons démontré l'implication de la voie des MAPK ERK1/2 dans l'inhibition de l'expression de SCD1 par la leptine. Nos résultats suggèrent aussi un rôle important du facteur de transcription Stimulating Protein 1 (Sp1) dans ce phénomène. Au niveau transcriptionnel, l'action de l'insuline et de la leptine semble indépendante l'une de l'autre. Cependant, il apparaît globalement que l'effet inhibiteur de la leptine supplante l'effet activateur de l'insuline au niveau de l'expression de SCD1. Le tissu adipeux est le lieu de stockage principal des triglycérides. L'analyse de la composition de ces triglycérides révèle que le C18:1 constitue l'acide gras le plus abondant. Des travaux réalisés sur des modèles animaux suggèrent fortement qu'une activité élevée de SCDl au niveau du tissu adipeux est fortement corrélée avec l'adiposité et l'obésité. Dans une troisième partie, nous avons donc testé l'hypothèse que la désaturation induite par SCD1 est positivement reliée à l'expansion du tissu adipeux. Nous avons montré que, indépendamment de leur apport en acides gras, le taux de C18:0 était diminuée dans le tissu adipeux omental de femmes atteintes d'obésité viscérale. De plus, chez ces patientes, ne présentant pas de syndrome métabolique, l'indice de désaturation (C18:1/C18:0) était augmenté. Nous avons aussi montré une association entre le niveau d'adiposité de ces patientes et la diminution du niveau de C18:0. L'adiposité de ces femmes a aussi été associée à l'augmentation de l'indice de. désaturation (C18:1/C18:0) et à l'augmentation du niveau d'expression de SCD1. En conclusion, nos travaux attestent la complexité de la régulation de l'expression de SCD1. Nos études confirment aussi le rôle clé de SCD1 au niveau du métabolisme lipidique, du développement de l'obésité et du syndrome métabolique associé. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Désaturase, SCD1, Insuline, Leptine, Protéines kinases, PI3K, mTor, MAPK, ERK1/2, SREBP-1, NF-Y, Sp1, foie, tissu adipeux.

碩士
國立臺灣大學
生化科學研究所
81
SCD2(Stearoyl CoA desaturase 2)的負調節序列PRE(Preadipocyte repressor element)的序列和一普遍存在之核內因子YY-1的結合序列甚為 相似,YY-1對轉錄的調控有時扮演促進子的角色,有時則扮演抑制子的角 色 ,端視所涉及的基因以及結合序列而定。目前已經四個不同實驗室分別 在研究不同基因的啟動區而找到相同的蛋白質,也就是YY-1,首先是Shenk 等人發現YY-1可以結合到adeno-associa ted P5這個病毒的啟動區以及起 動區上面,而且當在一般狀況之下,對於轉錄扮演是抑制的角色,但是這時 若是有adenoviruse E1A這個蛋白質出現時 ,則是扮演促進轉錄的角色,接 著下來Hariharan等人也發現YY-1具有促進一些核糖體蛋白質基因(rpL30, rpL32)進行轉錄的功能,同時Park以及Atch ison等人發現YY-1具有調節免 疫球蛋白基因上加強區之功能,因而他們也叫YY-1為NF-E1,最後,Flanagan 等人,則稱YY-1是UCRBP,因為他們發現它具有抑制老鼠白血球病毒的LTR進 行轉錄的功能。在本論文中,我經由比較UCR(upstream conserved region)結合序列 CGCCATTTT與SCD2啟動子PRE結合序列AGCCATTTC。發現 一個共同因子可以結合到這二者的結合序列上,從競爭性的原性電泳膠遲 緩試驗中,我已經証實UCR和SCD2的序列專一性的確很相似。更進一步研究 顯示UCR和SCD2上的結合因子具有相同抗原性,於是,我們可以下一個結論: 普遍存在之核內因子,YY-1,可以認知SCD2啟動子上的PRE。至於,YY-1在 SCD2上扮演何種角色,尚需進一步實驗才得以知曉。

碩士
臺北醫學大學
保健營養學研究所
101
Stearoyl-CoA desaturase (SCD), one of the lipid synthesizing enzymes, plays an important role in hyperglycemia. Previous studies found that SCD expression increased in the brains of Alzheimer’s disease patients, and the expression of SCD was negatively correlated with cognitive score. The purpose of the present study was to investigate the effect of high fat high fructose diet (HFHF) on blood insulin resistance, brain cognitive impairment, SCD expression in rats. Wistar rats were 32 male, 6 weeks old and divided into 3 groups: control group (n=8), soybean oil group (n=12) and coconut oil group (n=12). Soybean oil group and coconut oil group were fed high fat (39% kcal) high fructose (48% kcal) diets with soybean oil or coconut oil separately for 20 weeks to induce blood insulin resistance. After 20 weeks, rats were given cognitive tests and sacrificed. Serum analysis at week 20 showed that compared with control group coconut oil group had higher blood glucose, insulin and fructosamine, which indicated hyperglycemia and insulin resistance. Day-4 Morris water maze result showed coconut oil group had lowest cognitive function compared with control groups. Western results showed that coconut oil group and soybean oil group had higher β-amyloid accumulation in brain cortex. In fatty acid composition, both cortex and liver had higher oleic acid contents in coconut group which indicated that the rats had higher SCD activity. In RT-PCR results, coconut oil groups had higher SCD mRNA expression in brain cortex and hippocampus compare with control group.In conclusion, SCD activity could be activated by a 20-week high saturated fatty acid high fructose diet, SCD metabolism related fatty acid content could also be changed. We also found cognitive impairment in rat brain, which may relevant with brain SCD activity.

The effects of diet on milk fatty acid profile, including conjugated linoleic acid (CLA) have been well documented, however, there is limited information on the interaction of diet with genetics. The objective of this research was to determine the effect of single nucleotide polymorphisms (SNP) in the stearoyl CoA desaturase (SCD) gene on the ability to produce CLA in dairy cows fed low and high fat diets. The secondary objective was to determine the effect of a SNP in fatty acid synthase (FASN) on milk yield and composition. There was no effect of SCD genotype on CLA concentration, however there was an effect of SCD genotype on the ratio of C14:1/C14:0 which tended to be associated with CLA concentration when animals were fed a high fat diet. The FASN genotype affected milk yield, while composition remained constant.
Animal Science