Molecular Mechanisms Underlying the Elevated Expression of a Potentially Type 2 Diabetes Mellitus Associated SCD1 Variant

Abstract

Disturbances in lipid metabolism related to excessive food intake and sedentary lifestyle are among major risk of various metabolic disorders. Stearoyl-CoA desaturase-1 (SCD1) has an essential role in these diseases, as it catalyzes the synthesis of unsaturated fatty acids, both supplying for fat storage and contributing to cellular defense against saturated fatty acid toxicity. Recent studies show that increased activity or over-expression of SCD1 is one of the contributing factors for type 2 diabetes mellitus (T2DM). We aimed to investigate the impact of the common missense rs2234970 (M224L) polymorphism on SCD1 function in transfected cells. We found a higher expression of the minor Leu224 variant, which can be attributed to a combination of mRNA and protein stabilization. The latter was further enhanced by various fatty acids. The increased level of Leu224 variant resulted in an elevated unsaturated: saturated fatty acid ratio, due to higher oleate and palmitoleate contents. Accumulation of Leu224 variant was found in a T2DM patient group, however, the difference was statistically not significant. In conclusion, the minor variant of rs2234970 polymorphism might contribute to the development of obesity-related metabolic disorders, including T2DM, through an increased intracellular level of SCD1.

Keywords: fatty acids; genetic variation; lipotoxicity; metabolic disorders; obesity; oleate; single nucleotide polymorphism; stearoyl-CoA desaturase-1; type 2 diabetes mellitus.

Figure 1

Expression of M224L variants of stearoyl-CoA desaturase-1 in transiently transfected HEK293T cells. HEK293T cells were harvested and processed 24 h after transfection. Aliquots of cell lysates (5 µg) were loaded on 12% SDS-polyacrylamide gel, transferred to Immobilon-P membrane and SCD1 was detected with an anti-SCD1 (a) and an anti-Glu-Glu tag (b) antibodies, respectively. Actin was measured as loading control. Representative immunoblots of three independent experiments are shown. The band intensities were quantitated by densitometry and SCD1/Actin ratios are shown as bar graphs (c). Data are shown as mean values ± S.D. Statistical analysis was performed with the Tukey–Kramer Multiple Comparisons Test. ** p < 0.01; *** p < 0.001.

Figure 2

mRNA levels and structures of rs2234970 polymorphic SCD1 variants. (a) The mRNA level was measured in transiently transfected HEK293T cells. Samples were harvested and prepared as described in Section 4. qPCR was carried out using GAPDH, SCD1 and Glu-Glu tag sequence specific primers as indicated in Section 4. The diagram presented depicts the results of three independent measurements. Statistical analysis was performed with the Tukey–Kramer Multiple Comparisons Test. Data are shown as mean values ± S.D. *** p < 0.001. (b) The secondary MFE structures of +670A and +670C SCD1 were predicted by RNAfold server as indicated in Section 4. mRNA numbering begins at the first nucleotide of the start codon. The difference in the secondary structure of mRNAs is magnified in the left corner of the figure. (c) Mountain plot representation of MFE structure. The mountain plot represents a secondary structure in a plot of height vs. position, where the height is given by the number of base pairs enclosing the base at given position. Loops corresponding to plateaus, hairpin loops to peaks, and helices to slopes. The position of the polymorphic nucleotide change is indicated on horizontal axis. Red line: +670A; blue line: +670C. (d) HEK293T cells were transiently transfected with Glu-Glu tagged Met224 and Leu224 expressing constructs. 24 h after transfection cells were treated with actinomycin D (5 µg/mL) for 0, 1, 2, 4, 8, or 12 h. Samples were harvested and prepared as described in Section 4. qPCR was carried out using GAPDH and Glu-Glu tag sequence specific primers as indicated in Section 4. The diagram presents the average of three parallels. Statistical analysis was performed with the Tukey–Kramer Multiple Comparisons Test. Data are shown as mean values ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Intracellular degradation of M224L variants. (a) HEK293T cells were transiently transfected with Glu-Glu tagged Met224 and Leu224 expressing constructs. 24 h after transfection cells were treated with cycloheximide (50 mg/mL) for 0, 1, 2, 4, and 6 h. Western blot analysis of cell lysates (2 µg protein per lane) was carried out using anti-Glu-Glu tag and anti-Actin antibodies. Representative result of four independent experiments is shown. Data are shown as mean values ± S.D. The band intensities were quantitated by densitometry and SCD1-Glu-Glu/Actin ratios are shown as bar graphs. Statistical analysis was performed with the Tukey–Kramer Multiple Comparisons Test. Data are shown as mean values ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001. (b) Resolved crystal structure of Met224 human SCD1 was obtained from Protein Data Bank (file 4ZYO). 3D structure of Leu224 polymorphic variant was predicted by I-TASSER. Images were rendered using DeepView/Swiss-Pdb Viewer version 4.0.2. Met224 is shown in blue, Leu224 is shown in red. (c) Comparison of normalized B-factor values for Met224 and Leu224 SCD1 variants. The normalized-B factor (B-factor of each residue/B-factor of whole average) was plotted as the function of the amino acid residues. The blue line indicates the Met224 protein, and the red line shows Leu224. The position of the polymorphic residue change is indicated on the horizontal axis.

Figure 4

Effect of various fatty acids on the expression of M224L SCD1 variants in HEK293T cells. 24 h after transient transfection Met224 (a) and Leu224 (b) expressing cells were treated with BSA-conjugated oleate, palmitate, palmitoleate, linoleate, and stearate to a final concentration of 100 μM for 6 h. Immunoblot analysis of cell lysates (2 µg protein per lane) was carried out using anti-Glu-Glu tag and anti-Actin antibodies. Representative result of five independent experiments is shown. (c) The band intensities were determined by densitometry and the SCD1-Glu-Glu/Actin ratios were plotted. Statistical analysis was performed with the Tukey–Kramer Multiple Comparisons Test. Data are shown as mean values ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Changes in cellular fatty acid desaturation upon the over-expression of M224L variants. Transiently transfected HEK293T cells were harvested and the amount of major saturated (C18:0, C16:0) and mono-unsaturated ((a) C18:1 cisΔ9; (b) C16:1 cisΔ9) fatty acids was measured by GC-FID after saponification and methylation as described in Section 4, (c) and the ratio of unsaturated: saturated fatty acids was calculated. Data were normalized to the total protein content of the samples and are shown as mean values ± S.D.; n = 3. Statistical analysis was performed with the Tukey–Kramer Multiple Comparisons Test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Intracellular protein level of Ala224 artificial SCD1 variant. HEK293T cells were harvested and processed 24 h after transfection. Aliquotes of cell lysates (5 µg) were loaded on 12% SDS-polyacrylamide gel, transferred to Immobilon-P membrane and SCD1 was detected with an anti-SCD1 (a) and an anti-Glu-Glu tag (b) antibody, respectively. Actin was measured as loading control. Representative immunoblots of three independent experiments are shown. The band intensities were quantitated by densitometry and SCD1/Actin ratios are shown as bar graphs (c). Data are shown as mean values ± S.D. Statistical analysis was performed with the Tukey–Kramer Multiple Comparisons Test. n.s.: non-significant difference 4 h after the translation arrest, the intracellular amount of Met224 SCD1 was reduced to approximately 25%, while about 50% of the Leu224 and Ala224 proteins could still be detected at the same time (see Figure 3a and Figure 7a). Unlike the Leu224 version, however, the Ala224 protein level was not affected by oleate, palmitate, palmitoleate, linoleate, or stearate (Figure 7b). Also, to the contrary of Leu224 SCD1, the mRNA level of Ala224 SCD1 was significantly, by about 30%, lower than that of the Met224 variant (Figure 7c).

Figure 7

Expression, stability, and fatty acid sensitivity of Ala224 artificial SCD1 variant. (a) HEK293T cells were transiently transfected with Glu-Glu tagged Met224 and Ala224 constructs. 24 h after transfection cells were treated with cycloheximide (50 mg/mL) for 0, 1, 2, 4, and 6 h. Western blot analysis of cell lysates (2 µg protein per lane) was carried out using anti-Glu-Glu tag and anti-Actin antibodies. Representative result of four independent experiments is shown. The band intensities were determined by densitometry and the SCD1-Glu-Glu/Actin ratios were plotted. (b) 24 h after transient transfection, cells were treated with BSA-conjugated oleate, palmitate, palmitoleate, linoleate, and stearate to a final concentration of 100 μM for 6 h. Immunoblot analysis of cell lysates (2 µg protein per lane) was carried out using anti-Glu-Glu tag and anti-Actin antibodies. Representative result of three independent experiments is shown. The band intensities were quantitated by densitometry and SCD1-Glu-Glu/Actin ratios are shown as bar graphs. (c) qPCR was elaborated using GAPDH and SCD1-Glu-Glu tag sequence specific primers as indicated in Section 4. The diagram presented depicts the results of three independent measurements. Statistical analysis was performed with the Tukey–Kramer Multiple Comparisons Test. Data are shown as mean values ± S.D. * p < 0.05; *** p < 0.001.

Figure 8

Potential health impact of rs2234970 (M224L) SCD1 polymorphism and its proposed molecular background.