Salicylic acid binding protein 2 (SABP2) that converts methyl salicylate to salicylic acid (SA) plays an obligatory role in the SA-mediated disease resistance pathway in plants. SABP2 interacts with SBIP24 in a yeast two-hybrid screening. SBIP24 belongs to the stearoyl-acyl carrier protein-desaturase protein family. To biochemically characterize the SBIP24, it was cloned from tobacco leaves using RT-PCR and expressed in E. coli. Recombinant SBIP24 was affinity purified using Ni-NTA chromatography. RT-PCR was performed to determine the role of SABP2 in modulating the expression SBIP24. TMV infected transgenic C3 (control tobacco plant containing empty silencing vector) and 1-2 (SABP2-silenced) transgenic tobacco plants were used. Preliminary results indicate that SABP2 may regulate the expression of SBIP24 in tobacco plants. Further studies are needed to confirm these preliminary results.

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.

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.

No description available.

There is a growing interest in documenting the effect of diet on the ability to convert saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA) by modulating expression of the SCD gene. We propose that if cattle were raised to a constant body weight, their MUFA:SFA ratio will be the same regardless of being calf-fed (CF) or yearling-fed (YF). Twenty-four Angus cattle were acquired for this study. Cattle were slaughtered at weaning at 8 mo of age (SFCF, n=4), eight steers were assigned to the CF group and slaughtered at 12 mo of age (MFCF, n=4) and 16 mo of age (LFCF, n=4). Twelve cattle were assigned to the YF group and slaughtered at 12 mo of age (SFYF, n=4) 16 mo of age (MFYF, n=4) and market weight of 525 kg (LFYF, n=4). Cattle were then statistically analyzed based on time on high energy diet. Fatty acids from digesta, plasma, liver, L. dorsi, and s.c. and i.m. adipose tissue were all analyzed by FAME. In s.c. 18:1 and 16:1 were greatest in LFCF (41.27% and 5.58%, respectively, P = 0.05), and 18:0 and 16:0 did not differ between groups (P > 0.10). MUFA:SFA ratios of s.c. tended to be higher in LFCF animals (1.26) vs. LFYF (1.06, P = 0.10). However, there was no difference seen when comparing CF to YF animals (P = 0.26). MUFA:SFA ratio was higher in i.m. (P = 0.03) and also increased with age (P < .01). A trained sensory panel saw no significant differences between palatability of flavor characteristics of cooked steaks from LFCF, MFYF, or LFYF (P > 0.05). We showed increased SCD gene expression in the LFYF (248.41 to 1528.69 SCD/GAPDH, P = 0.01). Expression was higher in YF (P = 0.04), but their initial deposits of SFA, combined with the lack of SCD expression while on pastures, prevented the MUFA:SFA ratio from increasing at a rate fast enough to change the final ratios in the animal.

There is a growing interest in documenting the effect of diet on the ability to convert saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA) by modulating expression of the SCD gene. We propose that if cattle were raised to a constant body weight, their MUFA:SFA ratio will be the same regardless of being calf-fed (CF) or yearling-fed (YF). Twenty-four Angus cattle were acquired for this study. Cattle were slaughtered at weaning at 8 mo of age (SFCF, n=4), eight steers were assigned to the CF group and slaughtered at 12 mo of age (MFCF, n=4) and 16 mo of age (LFCF, n=4). Twelve cattle were assigned to the YF group and slaughtered at 12 mo of age (SFYF, n=4) 16 mo of age (MFYF, n=4) and market weight of 525 kg (LFYF, n=4). Cattle were then statistically analyzed based on time on high energy diet. Fatty acids from digesta, plasma, liver, L. dorsi, and s.c. and i.m. adipose tissue were all analyzed by FAME. In s.c. 18:1 and 16:1 were greatest in LFCF (41.27% and 5.58%, respectively, P = 0.05), and 18:0 and 16:0 did not differ between groups (P > 0.10). MUFA:SFA ratios of s.c. tended to be higher in LFCF animals (1.26) vs. LFYF (1.06, P = 0.10). However, there was no difference seen when comparing CF to YF animals (P = 0.26). MUFA:SFA ratio was higher in i.m. (P = 0.03) and also increased with age (P < .01). A trained sensory panel saw no significant differences between palatability of flavor characteristics of cooked steaks from LFCF, MFYF, or LFYF (P > 0.05). We showed increased SCD gene expression in the LFYF (248.41 to 1528.69 SCD/GAPDH, P = 0.01). Expression was higher in YF (P = 0.04), but their initial deposits of SFA, combined with the lack of SCD expression while on pastures, prevented the MUFA:SFA ratio from increasing at a rate fast enough to change the final ratios in the animal.

Stearoyl-coA desaturase-1 (SCD1) activity has been linked to the development of obesity and the metabolic syndrome (MetS) through its central role in lipid metabolism. Understanding how changes in SCD1 activity affect obesity and MetS risk biomarkers and investigating how these changes in activity affect nutrient handling in non-hepatic tissues is also important. This study investigated how acute SCD1 inhibition effected plasma lipids, skeletal muscle nutrient handling and insulin signaling in mice fed a high-carbohydrate very-low fat diet for 10 weeks. This study demonstrated that SCD1 inhibition created acute dyslipidemia, altered nutrient handling protein activity and increased the percentage of saturated fatty acids (SFA) in hepatic and muscle tissue, independent of dietary oleic acid content. However, the molecular controls of protein synthesis in the mTOR pathway were not affected by the loss of SCD1 activity. In conclusion, we observed that inhibiting hepatic SCD1 activity and subsequently changing the monounsaturated fatty acid (MUFA) to SFA ratios in tissues alters normal nutrient handling in skeletal muscle.

Oleic acid (18:1) is one of the important monounsaturated fatty acids, which is synthesized upon desaturation of stearic acid and this reaction is catalyzed by the SSI2 encoded stearoyl-acyl-carrier-protein-desaturase. A mutation in SSI2 leads to constitutive activation of salicylic acid (SA)-mediated defense responses. Consequently, these plants accumulate high levels of SA and show enhanced resistance to bacterial and oomycete pathogens. Replenishing 18:1 levels in ssi2 plants, via a second site mutation in GLY1 encoded glycerol-3-phosphate (G3P) dehydrogenase, suppresses all the ssi2-triggered phenotypes. Study of mechanism(s) underlying gly1-mediated suppression of ssi2 phenotypes showed that 18:1 levels are regulated via acylation with G3P and a balance between G3P and 18:1 is critical for the regulation of defense signaling pathways. To establish a role for 18:1 and G3P during host defense, interaction between Colletotrichum higginsianum and Arabidopsis was studied. Resistance to C. higginsianum correlated with host G3P levels. The gly1 plants showed increased susceptibility while act1 plants, defective in utilization of G3P, showed enhanced resistance. Plant overexpessing GLY1 showed enhanced resistance in both wild type as well as camalexin deficient backgrounds. Together, these results suggested that G3P conferred resistance acted downstream or independent of camalexin. Exogenous application of glycerol lowered 18:1 levels and produced ssi2-like phenotypes in wild-type plants. Furthermore, glycerol application or the ssi2 mutation produced similar phenotypes in fatty acid desaturation mutants and mutants defective in SA/resistance gene signaling. Expression studies showed that ssi2 phenotypes were likely due to increased expression of resistance genes. Epistatic analysis suggested that certain components of SA pathway had redundant function and were required for 18:1-regulated signaling.

Stearoyl-coA desaturase-1 (SCD1) activity has been linked to the development of obesity and the metabolic syndrome (MetS) through its central role in lipid metabolism. Understanding how changes in SCD1 activity affect obesity and MetS risk biomarkers and investigating how these changes in activity affect nutrient handling in non-hepatic tissues is also important. This study investigated how acute SCD1 inhibition effected plasma lipids, skeletal muscle nutrient handling and insulin signaling in mice fed a high-carbohydrate very-low fat diet for 10 weeks. This study demonstrated that SCD1 inhibition created acute dyslipidemia, altered nutrient handling protein activity and increased the percentage of saturated fatty acids (SFA) in hepatic and muscle tissue, independent of dietary oleic acid content. However, the molecular controls of protein synthesis in the mTOR pathway were not affected by the loss of SCD1 activity. In conclusion, we observed that inhibiting hepatic SCD1 activity and subsequently changing the monounsaturated fatty acid (MUFA) to SFA ratios in tissues alters normal nutrient handling in skeletal muscle.

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.