Flazin as a Lipid Droplet Regulator against Lipid Disorders

Abstract

Lipid disorders are closely related to numerous metabolic diseases, and lipid droplets (LDs) have been considered as a new target for regulating lipid metabolism. Dietary intervention and nutraceuticals provide safe and long-term beneficial effects for treating metabolic diseases. Flazin is a diet-derived bioactive constituent mainly existing in fermented foods, of which the lipid metabolism improvement function has not been studied. In this study, the effect of flazin on lipid regulation at both cell level and organelle level was investigated. Lipidomic profiling showed that flazin significantly decreased cellular triglyceride (TG) by 12.0-22.4% compared with modeling groups and improved the TG and free fatty acid profile. LD staining revealed that flazin efficiently reduced both cellular neutral lipid content by 17.4-53.9% and LD size by 10.0-35.3%. Furthermore, nanoelectrospray ionization mass spectrometry analysis proved that flazin exhibited a preferential suppression of LD TG and regulated LD morphology, including a size decrease and surface property improvement. An evaluation of related gene expression suggested the mechanism to be lipolysis promotion and lipogenesis inhibition. These findings indicated that flazin might be an LD regulator for reversing lipid metabolism disturbance. Moreover, the strategy proposed in this study may contribute to developing other nutraceuticals for treating lipid disorder-related metabolic diseases.

Keywords: diabetic nephropathy; functional foods; lipid metabolism; lipid-storage disorders; lipidomics; mass spectrometry; metabolic diseases; nutraceuticals; triglyceride.

Figure 1

Chemical structure of flazin.

Figure 2

Viability of cells treated with flazin (A), palmitic acid (PA) (B), or oleic acid (OA) (C). ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control (n = 3).

Figure 3

Effect of flazin on cellular lipids. (A) Cellular triglyceride (TG) content. (B) Cellular free fatty acid (FFA) content. PA, 200 μM palmitic acid; PA-F40, 200 μM palmitic acid + 40 μM flazin; PA-F80, 200 μM palmitic acid + 80 μM flazin; OA, 200 μM oleic acid; OA-F40, 200 μM oleic acid + 40 μM flazin; OA-F80, 200 μM oleic acid + 80 μM flazin. *** p < 0.001, **** p < 0.0001 vs. control, ^# p < 0.05 vs. PA group, ^$ p < 0.05, ^$$ p < 0.01 vs. OA group (n = 3).

Figure 4

(A,B) Heatmap of fatty acyl composition of TG in the cells treated with PA (A) or OA (B). (C,D) FFA ratio of 16:1 to 16:0 and 18:1 to 18:0 in the groups treated with PA (C) or OA (D). PA, 200 μM palmitic acid; PA-F40, 200 μM palmitic acid + 40 μM flazin; PA-F80, 200 μM palmitic acid + 80 μM flazin; OA, 200 μM oleic acid; OA-F40, 200 μM oleic acid + 40 μM flazin; OA-F80, 200 μM oleic acid + 80 μM flazin. ** p < 0.01 vs. control, ^$ p < 0.05, ^$$ p < 0.01 vs. OA group (n = 3).

Figure 5

Effect of flazin on lipid droplet (LD) accumulation. (A) Representative photographs of oil red O staining. (B) Neutral lipid content. PA, PA-F40 and PA-F80 groups are presented as relative amount to PA group; OA, OA-F40 and OA-F80 groups are presented as relative amount to OA group. (C) Average LD size. (D) LD number. PA, 200 μM palmitic acid; PA-F40, 200 μM palmitic acid + 40 μM flazin; PA-F80, 200 μM palmitic acid + 80 μM flazin; OA, 200 μM oleic acid; OA-F40, 200 μM oleic acid + 40 μM flazin; OA-F80, 200 μM oleic acid + 80 μM flazin. ^# p < 0.05, ^### p < 0.001 vs. PA group, ^$$ p < 0.01, ^$$$ p < 0.001 vs. OA group (n = 3).

Figure 6

Effect of flazin on LD TG content. (A) Representative photographs of LDs before and after aspiration. (B) Microscopic inspection on the tip of nanoESI emitter. (C,D) LD TG content in the cells treated with PA (C) or OA (D). PA, 200 μM palmitic acid; PA-F40, 200 μM palmitic acid + 40 μM flazin; PA-F80, 200 μM palmitic acid + 80 μM flazin; OA, 200 μM oleic acid; OA-F40, 200 μM oleic acid + 40 μM flazin; OA-F80, 200 μM oleic acid + 80 μM flazin. ^# p < 0.05, ^## p < 0.01 vs. PA group, ^$ p < 0.05, ^$$ p < 0.01 vs. OA group (n = 3).

Figure 7

Effect of flazin on LD surface properties. (A) Relative phosphatidylcholine–triglyceride (PC–TG) ratio of LD in the cells treated with PA or OA. (B) Relative phosphatidylcholine–phosphatidylethanolamine (PC–PE) ratio of LD in the cells treated with PA or OA. PA, 200 μM palmitic acid; PA-F40, 200 μM palmitic acid + 40 μM flazin; PA-F80, 200 μM palmitic acid + 80 μM flazin; OA, 200 μM oleic acid; OA-F40, 200 μM oleic acid + 40 μM flazin; OA-F80, 200 μM oleic acid + 80 μM flazin. ^# p < 0.05, ^## p < 0.01 vs. PA group (n = 3).

Figure 8

Effect of flazin on mRNA expression of adipose triglyceride lipase (ATGL) (A), acetyl-CoA carboxylase (ACC) (B), fatty acid synthase (FAS) (C), and stearoyl-CoA desaturase-1 (SCD-1) (D). PA, 200 μM palmitic acid; PA-F40, 200 μM palmitic acid + 40 μM flazin; PA-F80, 200 μM palmitic acid + 80 μM flazin; OA, 200 μM oleic acid; OA-F40, 200 μM oleic acid + 40 μM flazin; OA-F80, 200 μM oleic acid + 80 μM flazin. * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. control, ^#### p < 0.0001 vs. PA group, ^$ p < 0.05, ^$$$$ p < 0.0001 vs. OA group (n = 3).

Figure 9

Schematic illustration showing the effect of flazin on cellular lipid and lipid droplets (LDs). FA, fatty acid; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; TG, triglyceride; DG, diglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; ATGL, adipose triglyceride lipase; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase-1.