Brown but not white adipose cells synthesize omega-3 docosahexaenoic acid in culture

Abstract

Adipose tissue is a complex endocrine organ which coordinates several crucial biological functions including fatty acid metabolism, glucose metabolism, energy homeostasis, and immune function. Brown adipose tissue (BAT) is most abundant in young infants during the brain growth spurt when demands for omega-3 docosahexaenoic acid (DHA, 22:6n-3) is greatest for brain structure. Our aim was to characterize relative biosynthesis of omega-3 long chain polyunsaturated fatty acids (LCPUFA) from precursors in cultured white (WAT) and brown (BAT) cells and study relevant gene expression. Mouse WAT and BAT cells were grown in regular DMEM media to confluence, and differentiation was induced. At days 0 and 8 cells were treated with albumin bound d5-18:3n-3 (d5-ALA) and analyzed 24h later. d5-ALA increased cellular eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) in undifferentiated BAT cells, whereas differentiated BAT cells accumulated 20:4n-3, EPA and DPA. DHA as a fraction of total omega-3 LCPUFA was greatest in differentiated BAT cells compared to undifferentiated cells. Undifferentiated WAT cells accumulated EPA, whereas differentiated cells accumulated DPA. WAT accumulated trace newly synthesized DHA. Zic1 a classical brown marker and Prdm16 a key driver of brown fat cell fate are expressed only in BAT cells. Ppargc1a is 15 fold higher in differentiated BAT cells. We conclude that in differentiated adipose cells accumulating fat, BAT cells but not WAT cells synthesize DHA, supporting the hypothesis that BAT is a net producer of DHA.

Keywords: Brown adipose tissue; Differentiated; Omega-3 long chain polyunsaturated fatty acids; Undifferentiated; White adipose tissue.

Figure 1. Oil Red O staining of undifferentiated and differentiated WAT and BAT cells

No lipid droplets were detected in undifferentiated BAT and WAT cells (day 0) but both differentiated BAT and WAT cells (day 8) accumulated lipid droplets. As expected, BAT cells had more lipid droplets than WAT.

Figure 2. mRNA expression levels of gene markers in WAT and BAT cells

Images of semi-quantitative real time PCR products run on 2% agarose gels. Gapdh was used as reference gene. (A) mRNA expression of gene markers specifically expressed in BAT cells. Ucp1 only expressed in differentiated BAT (day 8). Prdm16 and Zic1 were expressed in both undifferentiated (day 0) and differentiated BAT (day 8). WAT cells did not express Ucp1, Prdm16 and Zic1. (B) mRNA expression of gene markers detected in both WAT and BAT cells. Ucp2, Tle3, Tmem26, Shox2, Ppargc1a, Erap1 are all expressed in WAT and BAT cells. Ppargc1a and Erap1 are strongly expressed in differentiated BAT cells.

Figure 3. mRNA expression levels of Ppargc1a and Pdk4 tested by quantitative real time PCR (qRT-PCR)

Data was normalized using the geometric mean of Gapdh and β-actin, and presented as mean ± SD. * P<0.05; ** P<0.005; *** P<0.001. (A) Left: BAT Ppargc1a expression level; Right: WAT Ppargc1a expression level. Ppargc1a was expressed 15 fold higher in differentiated BAT cells (day 8) than undifferentiated cells (day 0); no differences were detected in WAT. (B) Left: BAT Pdk4 expression level; Right: WAT Pdk4 expression level. Compared to undifferentiated cells, differentiated BAT cells had significantly higher expression of Pdk4, whereas, differentiated WAT cells had significantly lower expression.

Figure 4. mRNA expression levels of Fads1 and Fads2 tested by quantitative real time PCR (qRT-PCR)

Data was normalized using the geometric mean of Gapdh and β-actin, and presented as mean ± SD. * P<0.05; ** P<0.005; *** P<0.001. (A) Left: BAT Fads1 expression level; Right: WAT Fads1 expression level. Fads1 was significantly downregulated in differentiated BAT and WAT cells (day 8) compared to undifferentiated BAT and WAT cells (day 0). (B) Left: BAT Fads2 expression level; Right: WAT Fads2 expression level. Fads2 expression in differentiated BAT cells was only forty percent of that in undifferentiated BAT cells. No difference in Fads2 expression was found in WAT.

Figure 5. De novo biosynthesis of omega-3 LCPUFA from d5-18:3n-3 in cultured BAT and WAT cells

Cells were treated with isotopically labeled d5-18:3n-3 and omega-3 LCPUFA biosynthesis monitored. The relative amount of de novo LCPUFA biosynthesis was shown as a ratio of d5-product/d5-n3C_20-22. (A) BAT cells LCPUFA biosynthesis. Undifferentiated BAT cells (day 0) mainly accumulated EPA (20:5n-3) and DPA (22:5n-3) whereas differentiated BAT cells (day 8) accumulated 20:4n-3, EPA and DPA. No labeled 20:4n-3 was detected in undifferentiated BAT. Differentiated BAT synthesize more DHA. (B) WAT cells LCPUFA biosynthesis. Undifferentiated WAT cells mainly accumulated EPA whereas differentiated WAT cells accumulated DPA. Very little DHA was synthesized in both undifferentiated and differentiated WAT cells.