Selenium-Binding Protein 1 (SELENBP1) Supports Hydrogen Sulfide Biosynthesis and Adipogenesis

Abstract

Hydrogen sulfide (H₂S), a mammalian gasotransmitter, is involved in the regulation of a variety of fundamental processes including intracellular signaling, cellular bioenergetics, cell proliferation, and cell differentiation. Cystathionine g-lyase (CSE), cystathionine b-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) are currently considered the three principal mammalian H₂S-generating enzymes. However, recently, a fourth H₂S-producing enzyme, selenium-binding-protein 1 (SELENBP1), has also been identified. The cellular regulatory role(s) of SELENBP1 are incompletely understood. The current study investigated whether SELENBP1 plays a role in the regulation of adipocyte differentiation in vitro. 3T3-L1 preadipocytes with or without SELENBP1 knock-down were subjected to differentiation-inducing conditions, and H₂S production, cellular lipid accumulation, cell proliferation, and mitochondrial activity were quantified. Adipocyte differentiation was associated with an upregulation of H₂S biosynthesis. SELENBP1 silencing decreased cellular H₂S levels, suppressed the expression of the three "classical" H₂S-producing enzymes (CBS, CSE, and 3-MST) and significantly suppressed adipocyte differentiation. Treatment of SELENBP1 knock-down cells with the H₂S donor GYY4137 partially restored lipid accumulation, increased cellular H₂S levels, and exerted a bell-shaped effect on cellular bioenergetics (enhancement at 1 and 3 mM, and inhibition at 6 mM). We conclude that SELENBP1 in adipocytes (1) contributes to H₂S biosynthesis and (2) acts as an endogenous stimulator of adipocyte differentiation.

Keywords: fat; gasotransmitters, mitochondria, differentiation, metabolism; obesity.

Figure 1

Assessment of the adipogenesis process in wild-type 3T3-L1 and shSELENBP1 cells. (A) Schematic overview of the differentiation process in 3T3-L1 cells. (B) Representative immunoblot showing the expression of SELENBP1 and the adipocyte marker Adiponectin in two independent passages of wild-type cells before and after differentiation. (C) Representative immunoblot of SELENBP1 and adiponectin in stably transfected cells (shCtr and two independent shSELENBP1s, named sh85 and sh86), and corresponding densitometry analysis. β-Actin was used as loading control. D0: Day 0 (non-differentiated cells, i.e., pre-adipocytes); D7: Day 7 (mature (differentiated) adipocytes). DM: Differentiation Medium; MM: Maintenance Medium. ** p < 0.01 shows significant inhibition of SELENBP1 expression compared to control.

Figure 2

Effect of SELENBP1 knock-down on differentiation-associated lipid accumulation in 3T3-L1 cells. (A) Representative pictures of Oil Red O staining in shCtr and shSELENBP1 (sh85 and sh86 clones) cells before (Day 0) and after (Day 7) differentiation (scale bar: 200 µm). (B): numerical quantification of the findings. ** p < 0.01 indicates a significant difference between D7 and corresponding D0 values; ## p < 0.01 indicates a significant difference between shSELENBP1 (D7) and shCtr (D7).

Figure 3

Increases in cellular H₂S content during adipogenesis: role of SELENBP1. AzMC-assessed quantification of cellular H₂S levels before and after differentiation in shCtr and shSELENBP1 cells (sh85, sh86 clones). * p < 0.05 shows a significant difference between D7 and the corresponding D0; # p < 0.05 shows a significant difference between shSELENBP1 (D7) and shCtr (D7).

Figure 4

Effect of SELENBP1 silencing on adipocyte proliferation. Quantification of cell proliferation in shCtr and shSELENBP1 cells before and after differentiation. * p < 0.05 and ** p < 0.01 show significant differences between D7 and corresponding D0; # p < 0.05 shows a significant difference between shSELENBP1(D7) and shCtr(D7): ^§ p < 0.05 and ^§§ p < 0.01 show significant differences between shCtr (D0) and shSELENBP1(D0).

Figure 5

Effect of SELENBP1 silencing on the XTT-converting activity of adipocytes during differentiation. XTT conversion was quantified in control and SELENBP1 knock-down cells before and after differentiation. * p < 0.05 shows significant difference between D7 and corresponding D0; # p < 0.05 shows significant difference between shSELENBP1 (D7) and shCtr (D7); ^§ p < 0.05 shows significant difference between shCtr (D0) and shSELENBP1 (D0).

Figure 6

Effect of SELENBP1 silencing on mitochondrial respiration of mature adipocytes. (A) Oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) was measured in control and SELENBP1 knock-down cells after injection of oligomycin (Olig.), trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP,) and Rotenone/antimycin A (AMA). (C) Analysis of different bioenergetics parameters obtained from OCR values. * p < 0.05 and ** p < 0.01 show significant differences between shSELENBP1 and shCtr.

Figure 7

Effect of GYY4137 on H₂S-producing enzymes. (A) Schematic representation of GYY4137 treatment during the differentiation process. (B) Representative immunoblots of SELENBP1, CBS, CSE, and 3-MST in shCtr and ShSELENBP1 cells (clones sh85 and sh86), differentiated in the presence of the H₂S donor GYY4137. (C) Quantification of the densitometric data. * p < 0.05 and ** p < 0.01 show significant differences between GYY4137-treated cells and the corresponding control.

Figure 8

Effect of the H₂S donor GYY4137 on differentiation-associated lipid accumulation in shSELENBP1 knock-down cells. (A) quantification of Oil Red O staining in shCtr and shSELENBP1 (sh85, sh86 clones) treated with 1, 3, and 6 mM GYY4137. * p < 0.05 indicates a significant difference between GYY4137-treated and corresponding untreated values; # p < 0.05 indicates a significant difference between shSELENBP1 (untreated) and shCtr (untreated).

Figure 9

Increased cellular H₂S content in SELENBP1 knock-down cells treated with GYY4137. AzMC-assessed quantification of cellular H₂S levels in shCtr and shSELENBP1 cells (sh85, sh86 clones) treated with GYY4137 during the differentiation process. * p < 0.05 shows a significant difference between GYY4137-treated cells and the group control; ## p < 0.01 indicates a significant difference between shSELENBP1 (untreated) and shCtr (untreated).

Figure 10

Effect of GYY4137 on bioenergetics parameters in SELENBP1 knock-down cells. (A) Basal respiration, (B) maximal respiration, (C) proton leak, (D) ATP production, (E) non-mitochondrial oxygen consumption, and (F) spare respiratory capacity were calculated after extracellular flux analysis on GYY4137-treated shSELENBP1 cells. ** p < 0.01 shows a significant difference between GYY4137-treated cells and the group control; # p < 0.05 and ## p < 0.01 indicate significant differences between shSELENBP1 (untreated) and shCtr (untreated).