Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species.

Enhanced production of monounsaturated fatty acids (FA) derived from carbohydrate-enriched diets has been implicated in the development of obesity and insulin resistance. The FA elongases Elovl-5 and Elovl-6 are regulated by nutrient and hormone status, and have been shown using intact yeast and mammalian microsome fractions to be involved in the synthesis of monounsaturated FAs (MUFA). Herein, targeted knockdown and overexpression of Elovl-5 or Elovl-6 was used to determine their roles in de novo synthesis of specific MUFA species in mammalian cells. Treatment of rat insulinoma (INS)-1 cells with elevated glucose increased de novo FA synthesis and the ratio of MUFAs to saturated FAs. Elovl-5 knockdown decreased elongation of 16:1,n-7. Elovl-5 overexpression increased synthesis of 18:1,n-7; however, this increase was dependent on stearoyl-CoA desaturase–driven 16:1,n-7 availability. Knockdown of Elovl-6 decreased elongation of 16:0 and 16:1,n-7, resulting in accumulation of 16:1,n-7. Elovl-6 overexpression preferentially drove synthesis of 16:0 elongation products 18:0 and 18:1,n-9 but not 18:1,n-7. These findings demonstrate that coordinated induction of FA elongase and desaturase activity is required for balanced synthesis of specific n-7 versus n-9 MUFA species. Given the relative abundance of 16:0 to 16:1,n-7 and the specificity of Elovl-6 for 16:0, Elovl-6 is a major elongase for 18:1,n-9 production.


RNA extraction and quantitative real-time PCR
Total RNA was extracted with TRIZOL (Invitrogen, Carlsbad, CA) according to manufacturer's directions. First-stand cDNA was synthesized using the iScript cDNA Synthesis kit (BioRad, Hercules, CA). Quantitative real-time (qRT) PCR was conducted using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), synthesized cDNA and sets of gene-specifi c forward and reverse primers (supplemental Table I) (Integrated DNA Technologies, Coralville, IA). qRT-PCR reactions were carried out using a Mx3000P quantitative PCR system (Stratagene, La Jolla, CA). All samples were analyzed in triplicate. The relative amounts of mRNA were analyzed by the comparative cycle threshold method. Ribosomal protein L32 (RPL32) mRNA levels, which were unaffected by treatments, were used to normalize mRNA levels.

Overexpression of FA elongases and desaturase with adenoviruses
Elovl-5 and Elovl-6 cDNA were cloned and used for construction of recombinant adenoviruses as previously described ( 19 ). SCD2 cDNA was cloned in the same manner using cDNA synthesized from INS-1 cell mRNA and the following primers: sense 5 ′ -ATAGTCGACATGCCGGCTCACATACTGCAAGAG-3 ′ ; antisense 5 ′ -ATACTCGAGTCAGCCACTCTTGCAGCTCTCCTC-C C C-3 ′ (Acc. No. AB032243). Adenoviruses overexpressing SCD2 were constructed using the AdEasy Adenoviral Vector System (Stratagene) as previously described ( 17 ). An adenovirus overexpressing ␤ -galactosidase (B-gal) was obtained from Dr. Newgard, Duke University, NC. For gene transduction, 90% confl uent cell cultures were infected for 2 h with 5 or 10 pfu per cell and then cultured for an additional 24 h in 5.5 mM glucose medium to allow for gene expression. Cells were then cultured in INS-1 media containing 11.1 mM glucose with 1 Ci [2-14 C]acetic acid for 24 h prior to lipid extraction.

Lipid extraction and fatty acid analysis
Cells were harvested and lipids extracted as described by Wang et al. ( 19 ). Total lipid extracts from the [2-14 C]acetic acid-labeling studies were saponifi ed, extracted, and fatty acids were then fractionated by reverse phase-HPLC using a J'sphere ODS-H80 (YMC-Waters) column and quantifi ed by fl ow-through scintillation counting (IN/US).
Modulating the expression of FA elongase and desaturase genes has physiological signifi cance, as mice with knockdowns of either Elovl-6 or SCD1 are protected from diet-induced obesity (7)(8)(9). The role of FA elongases in regulating the determination of specifi c de novo-derived MUFA end products, however, remains to be defi ned. This study presents a comprehensive analysis of the effects of both decreased and increased expression of Elovl-5, Elovl-6, and SCDs on FAs synthesized de novo from glucose in a mammalian cell line. The results demonstrate that altering the expression of each enzyme causes signifi cant changes in de novo synthesis of specifi c MUFA species. levels were only modestly induced by glucose. Overall, the changes in lipogenic gene expression were associated with a 6-and 10-fold increase in de novo synthesized SFAs and MUFAs, respectively, as assessed by incorporation of 14 C-glucose into FAs ( Fig. 1C ). Elevated glucose thus led to a 1.6-fold increase in the total desaturation index ( Fig. 1D ). Of these newly synthesized MUFAs, there was a signifi cant increase in the n-7 relative to n-9 species ( Fig. 1E ). In subsequent experiments, INS-1 cells cultured in elevated glucose were used to examine the role of Elovl-5 and Elovl-6 on synthesis of specifi c MUFA species.

MATERIALS AND METHODS
Selective knockdown of Elovl-5 or Elovl-6 alters synthesis of specifi c MUFA species siRNAs were used to determine the relative contribution of Elovl-5, Elovl-6, or SCD1/2 on de novo synthesis of specifi c FAs species in INS-1 cells cultured in elevated glucose. siRNAs selective against Elovl-5 or Elovl-6 decreased expression of the target mRNA by 75% and 80%, respectively, with no signifi cant effect on nontarget mRNA levels ( Fig. 2A ). SCD siRNA reduced SCD1 and SCD2 by 90% and resulted in a 1.7-fold increase in

Statistical analysis
All experiments represent the average of three independent experiments done in duplicate. Data was analyzed by Student's t -test. P < 0.05 was considered signifi cant.

CONCLUSION
This study presents a comprehensive analysis of the effects of altered expression of Elovl-5 and Elovl-6 on de novo-synthesized MUFAs. Our results demonstrate that Elovl-5 preferentially converts 16:1,n-7 to 18:1,n-7, whereas Elovl-6 preferentially elongates 16:0 to 18:0, which can be further desaturated to 18:1,n-9. Loss of coordinate control of Elovl-5, Elovl-6, and SCD can disrupt production of specifi c MUFA species, which might infl uence cell function. As Elovl-5 and Elovl-6 can be differentially expressed under various physiologic and pharmacologic conditions and are also capable of elongating exogenous lipids ( 17,19 ), it is likely that FA elongases play a broader role in determining the balance of MUFA species.
Elovl-6 on the intracellular end products of de novoderived FAs in mammalian cells. The fi ndings reveal a signifi cant role for FA elongase activity in regulating the synthesis of de novo-derived MUFAs and establishing the balance among 16:1,n-7, 18:1,n-7, and 18:1,n-9.