Dietary Supplementation with Dunaliella Tertiolecta Prevents Whitening of Brown Fat and Controls Diet-Induced Obesity at Thermoneutrality in Mice

Abstract

We investigated the effect of evodiamine-containing microalga Dunaliella tertiolecta (DT) on the prevention of diet-induced obesity in a thermoneutral C57BL/6J male (30 °C). It attenuates the activity of brown adipose tissue (BAT), which accelerates diet-induced obesity. Nine-week-old mice were fed a high-fat diet supplemented with 10 g (Low group) or 25 g (High group) DT powder per kg food for 12 weeks. Compared to control mice without DT supplementation, body weight gain was significantly reduced in the High group with no difference in food intake. Tissue analyses indicated maintenance of multilocular morphology in BAT and reduced fat deposition in liver in DT-supplemented mice. Molecular analysis showed a significant decrease in mammalian target of rapamycin-ribosomal S6 protein kinase signaling pathway in white adipose tissue and upregulation in mRNA expression of brown fat-associated genes including fibroblast growth factor-21 (Fgf21) and uncoupling protein 1 (Ucp1) in BAT in the High group compared to the control. In the experiments using C3H10T1/2 adipocytes, DT extract upregulated mRNA expression of brown fat-associated genes in dose-dependent and time-dependent manners, accompanied by a significant increase in secreted FGF21 levels. Our data show the ability of DT as a nutraceutical to prevent brown fat attenuation and diet-induced obesity in vivo.

Keywords: brown fat; fibroblast growth factor-21; microalga; obesity; thermoneutrality; uncoupling protein 1.

Figure 1

Protocol of animal experiment. Nine-week-old mice were fed the high-fat diet (HFD) supplemented with or without Dunaliella tertiolecta (DT) powder for 12 weeks.

Figure 2

Effect of DT supplementation on diet-induced obesity at thermoneutrality in mice. Nine-week-old male C57BL/6J mice were fed the HFD supplemented with 0 g (Control), 10 g (Low) or 25 g (High) DT powder per kg food for 12 weeks. (A) Body weight gain. (B) Food intake. (C) Tissue weights. IBAT: interscapular BAT, IWAT: inguinal WAT, EWAT: epidydimal WAT, RWAT: retroperitoneal WAT. (D) Histology of IBAT, IWAT, EWAT, and liver were analyzed. Tissue sections were stained with hematoxylin and eosin. Representative images are shown. Scale bars, 50 μm for IBAT and liver, 200 μm for IWAT and EWAT. (E–G) Serum levels of fed glucose (E), insulin (F), triglyceride: TG, and total cholesterol: TC (G) were examined. (H,I) Hepatic levels of TG (H) and TC (I) were also analyzed. Data are expressed as the mean ± SE (Control: n = 5, Low: n = 6, High: n = 5). * p < 0.05, ** p < 0.01 vs. Control.

Figure 3

Effect of DT supplementation on mTOR-S6K signaling in WAT and liver of mice. Western blot analyses of mTOR (A,D), S6K (B,E), and rpS6 (C,F) were performed using tissue lysates of EWAT (A–C) and liver (D–F) from mice in Figure 2. Representative images are shown. Phosphorylation levels of mTOR Ser2448, S6K Thr389, and rpS6 Ser235/236 were normalized to total level of each protein. Data are expressed as mean ± SE (n = 5–6). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control. ^# p < 0.05, ^## p < 0.01 vs. Low.

Figure 4

DT supplementation stimulates expression of brown fat-associated genes in IBAT in HFD-fed mice at thermoneutrality. (A–C) mRNA levels of Ucp1, Cidea, Ppargc1a, Prdm16, and Fgf21 in IBAT (A), IWAT (B), and EWAT (C). (D) UCP1 and tubulin levels in IBAT. Representative images are shown. (E) Fgf21 mRNA level in liver. (F) Serum FGF21 level. Data are the means ± SE (n = 4–6). * p < 0.05, *** p < 0.001 vs. Control. ^# p < 0.05, ^## p < 0.01 vs. Low.

Figure 5

DT extract stimulates expression of brown fat-associated genes in C3H10T1/2 adipocytes. Cells were treated with the indicated concentration of DT extract for 18 h. (A) Cell morphology. (B–F) Dose dependent upregulation of Ucp1 (B), Cidea (C), Ppargc1a (D), Prdm16 (E), and Fgf21 (F) mRNAs. (n = 7, except n = 4 for 3%). Data are the means ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0%.

Figure 6

Effects of DT on Ucp1, Fgf21 mRNA expression, and FGF21 production in C3H10T1/2 adipocytes. Cells were stimulated by 0.3% DT extract for the indicated time (A–C). (A) Ucp1 mRNA level. (B) Fgf21 mRNA level (n = 2 for each point, representative of two independent experiments). (C) FGF21 level in the conditioned medium (CM) when treated with DT (n = 3–5). (D) FGF21 level in the CM when treated with DT and DS. Cells were stimulated by the indicated concentration of DT or DS extract for 18 h (n = 4). Data are the means ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 h, 4 h or 0% in the same extract. ^# p < 0.05, ^### p < 0.001 vs. DT+ (24 h) or DS extract at same concentration.