Cucurbitacin B-, E-, and I-Induced Browning of White Adipocytes Is Promoted by the Inhibition of Phospholipase D2

Abstract

The mechanism of white adipose tissue browning is not well understood; however, naturally occurring compounds are known to play a positive role. The effects of cucurbitacins B, E, and I on the browning of mature white adipocytes were investigated. First, the cell viability exhibited by cucurbitacins B, E, and I in pre- and mature adipocytes was verified. Cucurbitacins B, E, and I had no effect on cell viability in pre- and mature adipocytes at concentrations up to 300 nM. To investigate the characteristics of representative beige adipocytes, the formation and morphology of cucurbitacin B, E, and I lipid droplets were verified. The total lipid droplet surface area, maximum Feret diameter, and total Nile red staining intensity of cucurbitacin B-, E-, and I-treated adipocytes were lower than those of mature white adipocytes. Furthermore, treatment of white mature adipocytes with cucurbitacin B, E, and I led to the formation of several small lipid droplets that are readily available for energy expenditure. We evaluated the effect of cucurbitacins B, E, and I on the expression of representative browning markers UCP1, PGC1a, and PRDM16, which participate in the browning of white adipose tissue. Cucurbitacins B, E, and I increased the mRNA and protein expression levels of UCP1, PGC1a, and PRDM16 in a concentration-dependent manner. To promote energy consumption by beige adipocytes, active mitochondrial biogenesis is essential. Next, we investigated the effects of cucurbitacin B, E, and I on mitochondrial biogenesis in mature adipocytes. Mitochondrial mass increased when mature adipocytes were treated with cucurbitacin B, E, and I. The degree of cucurbitacin B-, E- and I-induced transformation of white adipocytes into beige adipocytes was in the order of Cu E > Cu B > Cu I. To verify the effect of phospholipase D2 on the browning of white adipocytes, CAY10594—a PLD2 pharmacological inhibitor, and a knockdown system were used. PLD2 inhibition and knockdown improved the expression levels of UCP1, PGC1a, and PRDM16. In addition, PLD2 inhibition and knockdown in mature white adipocytes promoted mitochondrial biosynthesis. The effect of PLD2 inhibition and knockdown on promoting browning of white adipocytes significantly increased when Cu B, Cu E, and Cu I were co-treated. These data indicate that mature white adipocytes’ beige properties were induced by cucurbitacins B, E, and I. These effects became more potent by the inhibition of PLD2. These findings provide a model for determining anti-obesity agents that induce browning and increase energy expenditure in mature white adipocytes.

Induction of Browning in White Adipocytes: Fucoidan Characterization and Gold Nanoparticle Synthesis from Undaria pinnatifida Sporophyll Extract

“…The principal distinction between these two types of adipose tissues lies in the presence or absence of UCP-1 (uncoupling protein-1) activity within the inner mitochondrial membrane of brown adipocytes. UCP1 plays a crucial role in adaptive thermogenesis by uncoupling ATP production via the catabolic pathways of lipids and carbohydrates [10,11,43]. The resulting energy is released in the form of heat, which is distributed throughout the body owing to the rich vascularization of BAT.…”

“…Images of the stained lipid droplets were acquired using a confocal microscope (LSM 800; Carl Zeiss, Jena, Germany), and the droplets were quantified using a flow cytometer (Fit NxT Flow Cytometer; Thermo Fisher Scientific). We used quantitative real-time polymerase chain reaction (qRT-PCR) to determine how the UPS extract, UPS-AuNPs, and fucoidan affected the UCP-1, PRDM16, and PGC1a mRNA transcripts [43]. Cellular RNA was isolated using the PureLink RNA Mini Kit and converted into complementary DNA (cDNA) using a high-dose cDNA reverse kit according to the manufacturer's instructions.…”

“…With 30-50 g of protein sample loaded, the conventional Western blot technique was used. UCP1, PRDM16, and PGC1a were used as primary antibodies [43], and -actin was used as the internal control antibodies. ImageQuant TL (version 8.1; Amersham, UK) was used to quantify the grayscale values.…”