Selenoprotein P expression in glioblastoma as a regulator of ferroptosis sensitivity: preservation of GPX4 via the cycling-selenium storage.

Glioblastoma (GBM) is one of the most aggressive and deadly brain tumors; however, its current therapeutic strategies are limited. Selenoprotein P (SeP; SELENOP, encoded by the SELENOP gene) is a unique selenium-containing protein that exhibits high expression levels in astroglia. SeP is thought to be associated with ferroptosis sensitivity through the induction of glutathione peroxidase 4 (GPX4) via selenium supplementation. In this study, to elucidate the role of SeP in GBM, we analyzed its expression in GBM patients and found that SeP expression levels were significantly higher when compared to healthy subjects. Knock down of SeP in cultured GBM cells resulted in a decrease in GPX1 and GPX4 protein levels. Under the same conditions, cell death caused by RSL3, a ferroptosis inducer, was enhanced, however this enhancement was canceled by supplementation of selenite. These results indicate that SeP expression contributes to preserving GPX and selenium levels in an autocrine/paracrine manner, i.e., SeP regulates a dynamic cycling-selenium storage system in GBM. We also confirmed the role of SeP expression in ferroptosis sensitivity using patient-derived primary GBM cells. These findings indicate that expression of SeP in GBM can be a significant therapeutic target to overcome anticancer drug resistance.

Sulforaphane decreases serum selenoprotein P levels through enhancement of lysosomal degradation independent of Nrf2.

Selenoprotein P (SeP) is a major selenoprotein in serum predominantly produced in the liver. Excess SeP impairs insulin secretion from the pancreas and insulin sensitivity in skeletal muscle, thus inhibition of SeP could be a therapeutic strategy for type 2 diabetes. In this study, we examine the effect of sulforaphane (SFN), a phytochemical of broccoli sprouts and an Nrf2 activator, on SeP expression in vitro and in vivo. Treatment of HepG2 cells with SFN decreases inter- and intra-cellular SeP levels. SFN enhances lysosomal acidification and expression of V-ATPase, and inhibition of this process cancels the decrease of SeP by SFN. SFN activates Nrf2 in the cells, while Nrf2 siRNA does not affect the decrease of SeP by SFN or lysosomal acidification. These results indicate that SFN decreases SeP by enhancing lysosomal degradation, independent of Nrf2. Injection of SFN to mice results in induction of cathepsin and a decrease of SeP in serum. The findings from this study are expected to contribute to developing SeP inhibitors in the future, thereby contributing to treating and preventing diseases related to increased SeP.

An efficient selenium transport pathway of selenoprotein P utilizing a high-affinity ApoER2 receptor variant and being independent of selenocysteine lyase.

Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor. However, diverse variants of ApoER2 have been reported, and the details of its tissue specificity and the molecular mechanism of its efficiency remain unclear. In the present study, we found that human T lymphoma Jurkat cells have a high ability to utilize selenium via SeP, while this ability was low in human rhabdomyosarcoma cells. We identified an ApoER2 variant with a high affinity for SeP in Jurkat cells. This variant had a dissociation constant value of 0.67 nM and a highly glycosylated O-linked sugar domain. Moreover, the acidification of intracellular vesicles was necessary for selenium transport via SeP in both cell types. In rhabdomyosarcoma cells, SeP underwent proteolytic degradation in lysosomes and transported selenium in a Sec lyase-dependent manner. However, in Jurkat cells, SeP transported selenium in Sec lyase-independent manner. These findings indicate a preferential selenium transport pathway involving SeP and high-affinity ApoER2 in a Sec lyase-independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP.

Piceatannol Prevents Obesity and Fat Accumulation Caused by Estrogen Deficiency in Female Mice by Promoting Lipolysis.

Postmenopausal women have a higher susceptibility to obesity and chronic disease. Piceatannol (PIC), a natural analog of resveratrol, was reported to inhibit adipogenesis and to have an antiobesity effect. In this study, PIC's effect on postmenopausal obesity and the mechanism of its action were investigated. C57BL/6J female mice were divided into four groups and half of them were ovariectomized (OVX). Both OVX and sham-operated mice were fed a high-fat diet (HFD) with and without the addition of 0.25% of PIC for 12 weeks. The abdominal visceral fat volume was higher in the OVX mice than the sham-operated mice, and PIC significantly decreased the fat volume only in the OVX mice. Unexpectedly, expression levels of adipogenesis-related proteins in white adipose tissue (WAT) were suppressed in the OVX mice, and PIC did not affect lipogenesis in either the OVX or sham-operated mice. Regarding the expression of proteins associated with lipolysis, PIC activated the phosphorylation of hormone-sensitive lipase much more in the OVX mice, but it did not affect the expression of adipose triglyceride lipase. PIC also tended to induce the expression of uncoupled protein 1 in brown adipose tissue (BAT). These results suggest that by promoting lipolysis in WAT and deconjugation in BAT, PIC is a potential agent to inhibit fat accumulation caused by menopause.

GPER/PKA-Dependent Enhancement of Hormone-Sensitive Lipase Phosphorylation in 3T3-L1 Adipocytes by Piceatannol.

We previously reported that piceatannol (PIC) had an anti-obesity effect only in ovariectomized (OVX) postmenopausal obesity mice. PIC was found to induce the phosphorylation of hormone-sensitive lipase (pHSL) in OVX mice. To elucidate the mechanism by which PIC activates HSL, we investigated the effect of PIC using 3T3-L1 adipocytes. PIC induced HSL phosphorylation at Ser563 in 3T3-L1 cells, as in vivo experiments showed. pHSL (Ser563) is believed to be activated through the ß-adrenergic receptor (ß-AR) and protein kinase A (PKA) pathways; however, the addition of a selective inhibitor of ß-AR did not inhibit the effect of PIC. The addition of a PKA inhibitor with PIC blocked pHSL (Ser563), suggesting that the effects are mediated by PKA in a different pathway than ß-AR. The addition of G15, a selective inhibitor of the G protein-coupled estrogen receptor (GPER), reduced the activation of HSL by PIC. Furthermore, PIC inhibited insulin signaling and did not induce pHSL (Ser565), which represents its inactive form. These results suggest that PIC acts as a phytoestrogen and phosphorylates HSL through a novel pathway that activates GPER and its downstream PKA, which may be one of the inhibitory actions of PIC on fat accumulation in estrogen deficiency.

Role of selenoprotein P expression in the function of pancreatic ß cells: Prevention of ferroptosis-like cell death and stress-induced nascent granule degradation.

Selenoprotein P (SELENOP) is a major selenium (Se)-containing protein (selenoprotein) in human plasma that is mainly synthesized in the liver. SELENOP transports Se to the cells, while SELENOP synthesized in peripheral tissues is incorporated in a paracrine/autocrine manner to maintain the levels of cellular selenoproteins, called the SELENOP cycle. Pancreatic ß cells, responsible for the synthesis and secretion of insulin, are known to express SELENOP. Here, using MIN6 cells as a mouse model for pancreatic ß cells and Selenop small interfering (si)RNA, we found that Selenop gene knockdown (KD) resulted in decreased cell viability, cellular pro/insulin levels, insulin secretion, and levels of several cellular selenoproteins, including glutathione peroxidase 4 (Gpx4) and selenoprotein K (Selenok). These dysfunctions induced by Selenop siRNA were recovered by the addition of Se. Ferroptosis-like cell death, regulated by Gpx4, was involved in the decrease of cell viability by Selenop KD, while stress-induced nascent granule degradation (SINGD), regulated by Selenok, was responsible for the decrease in proinsulin. SINGD was also observed in the pancreatic ß cells of Selenop knockout mice. These findings indicate a significant role of SELENOP expression for the function of pancreatic ß cells by maintaining the levels of cellular selenoproteins such as GPX4 and SELENOK.

Saturated fatty acid in the phospholipid monolayer contributes to the formation of large lipid droplets.

The degree of saturation of fatty acid chains in the bilayer membrane structure is known to control membrane fluidity and packing density. However, the significance of fatty acid composition in the monolayers of lipid droplets (LDs) has not been elucidated. In this study, we noted a relationship between the size of LDs and the fatty acid composition of the monolayer. To obtain large LDs, we generated NIH3T3 cells overexpressing fat-specific protein 27 (FSP27). This induced the fusion of LDs, resulting in larger LDs in FSP27-overexpressing cells compared with LDs in control cells. Moreover, the lipid extracts of LDs from FSP27-overexpressing cells reconstituted large-droplet emulsions in vitro, implying that the lipid properties of LDs might affect the size of LDs. FSP27-overexpressing cells had more saturated fatty acids in the phospholipid monolayer of the LDs compared with control cells. To further investigate the effects of the degree of phospholipid unsaturation on the size of LDs, we synthesized artificial emulsions of a lipid mixed with distearoylphosphatidylcholine (DSPC, diC18:0-PC) and with dioleoylphosphatidylcholine (DOPC, diC18:1n-9-PC) and compared the sizes of the resulting LDs. The emulsions prepared from saturated PC had larger droplets than those prepared from unsaturated PC. Our results suggest that saturated fatty acid chains in phospholipid monolayers might establish the form and/or stability of large LDs.

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n-9), an indicator of essential fatty acid deficiency.

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n-9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1-6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n-9, 20:1n-9 and 20:2n-9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n-9→(Fads2)→18:2n-9→(Elovl5)→20:2n-9→(Fads1)→20:3n-9 and pathway 2) 18:1n-9→(Elovl5)→20:1n-9→(Fads2)→20:2n-9→(Fads1)→20:3n-9.

Changes in the phospholipid fatty acid composition of the lipid droplet during the differentiation of 3T3-L1 adipocytes.

Lipid droplets (LDs) are independent organelles in adipocytes that are composed of a lipid ester core surrounded by a phospholipid monolayer. The fatty acid composition of the phospholipid monolayer should determine the metabolism and dynamics of LDs. In this study, we examined the fatty acid composition of phospholipid monolayer in LDs during the differentiation of 3T3-L1 adipocytes. The levels of saturated fatty acids (SFAs), such as 16:0 and 18:0, in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of LDs decreased during differentiation. In contrast, the levels of monounsaturated fatty acids (MUFAs) such as 16:1n-7 and 18:1n-9 in PC and PE of LDs and the level of polyunsaturated fatty acids (PUFAs) such as 20:3n-6, 20:4n-6 and 22:6n-3 in PE of LDs increased during differentiation. These results suggest that the phospholipid monolayer in mature LDs is more fluid than that in nascent LDs. The fatty acid compositions of the LD monolayer were different from those of the microsome bilayer in the early stage of differentiation, but similar to those of the microsome bilayer in the late stage of differentiation. These data provide evidence that biophysical properties of the phospholipid monolayer in LDs change during adipocyte differentiation.