The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells.

In obesity, chronic membrane-localization of CD36 free fatty acid (FFA) translocase, but not other FFA transporters, enhances FFA uptake and intracellular lipid accumulation. This ectopic lipid accumulation promotes insulin resistance by inhibiting insulin-induced GLUT4 glucose transporter trafficking and glucose uptake. GLUT4 and CD36 cell surface delivery is triggered by insulin- and contraction-induced signaling, which share conserved downstream effectors. While we have gathered detailed knowledge on GLUT4 trafficking, the mechanisms regulating CD36 membrane delivery and subsequent FFA uptake in skeletal muscle are not fully understood. The exocyst trafficking complex facilitates the docking of membrane-bound vesicles, a process underlying the controlled surface delivery of fuel transporters. The exocyst regulates insulin-induced glucose uptake via GLUT4 membrane trafficking in adipocytes and skeletal muscle cells and plays a role in lipid uptake in adipocytes. Based on the high degree of conservation of the GLUT4 and CD36 trafficking mechanisms in adipose and skeletal muscle tissue, we hypothesized that the exocyst also contributes to lipid uptake in skeletal muscle and acts through the targeted plasma membrane delivery of CD36 in response to insulin and contraction. Here, we show that the exocyst complex is necessary for insulin- and contraction-induced CD36 membrane trafficking and FFA uptake in muscle cells.

Disrupted glucose homeostasis and skeletal-muscle-specific glucose uptake in an exocyst knockout mouse model.

Skeletal muscle is responsible for the majority of glucose disposal following meals, and this is achieved by insulin-mediated trafficking of glucose transporter type 4 (GLUT4) to the cell membrane. The eight-protein exocyst trafficking complex facilitates targeted docking of membrane-bound vesicles, a process underlying the regulated delivery of fuel transporters. We previously demonstrated the role of exocyst subunit EXOC5 in insulin-stimulated GLUT4 exocytosis and glucose uptake in cultured rat skeletal myoblasts. However, the in vivo role of EXOC5 in skeletal muscle remains unclear. Using mice with inducible, skeletal-muscle-specific knockout of exocyst subunit EXOC5 (Exoc5-SMKO), we examined how muscle-specific disruption of the exocyst would affect glucose homeostasis in vivo. We found that both male and female Exoc5-SMKO mice displayed elevated fasting glucose levels. Additionally, male Exoc5-SMKO mice had impaired glucose tolerance and lower serum insulin levels. Using indirect calorimetry, we observed that male Exoc5-SMKO mice have a reduced respiratory exchange ratio during the light period and lower energy expenditure. Using the hyperinsulinemic-euglycemic clamp method, we further showed that insulin-stimulated skeletal muscle glucose uptake is reduced in Exoc5-SMKO males compared with wild-type controls. Overall, our findings indicate that EXOC5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle and regulate glucose homeostasis in vivo.

Combined Omics Reveals That Disruption of the Selenocysteine Lyase Gene Affects Amino Acid Pathways in Mice.

Selenium is a nonmetal trace element that is critical for several redox reactions and utilized to produce the amino acid selenocysteine (Sec), which can be incorporated into selenoproteins. Selenocysteine lyase (SCL) is an enzyme which decomposes Sec into selenide and alanine, releasing the selenide to be further utilized to synthesize new selenoproteins. Disruption of the selenocysteine lyase gene (Scly) in mice (Scly-/- or Scly KO) led to obesity with dyslipidemia, hyperinsulinemia, glucose intolerance and lipid accumulation in the hepatocytes. As the liver is a central regulator of glucose and lipid homeostasis, as well as selenium metabolism, we aimed to pinpoint hepatic molecular pathways affected by the Scly gene disruption. Using RNA sequencing and metabolomics, we identified differentially expressed genes and metabolites in the livers of Scly KO mice. Integrated omics revealed that biological pathways related to amino acid metabolism, particularly alanine and glycine metabolism, were affected in the liver by disruption of Scly in mice with selenium adequacy. We further confirmed that hepatic glycine levels are elevated in male, but not in female, Scly KO mice. In conclusion, our results reveal that Scly participates in the modulation of hepatic amino acid metabolic pathways.

From Selenium Absorption to Selenoprotein Degradation.

Selenium is an essential dietary micronutrient. Ingested selenium is absorbed by the intestines and transported to the liver where it is mostly metabolized to selenocysteine (Sec). Sec is then incorporated into selenoproteins, including selenoprotein P (SELENOP), which is secreted into plasma and serves as a source of selenium to other tissues of the body. Herein, we provide an overview of the biology of selenium from its absorption and distribution to selenoprotein uptake and degradation, with a particular focus on the latter. Molecular mechanisms of selenoprotein degradation include the lysosome-mediated pathway for SELENOP and endoplasmic reticulum-mediated degradation of selenoproteins via ubiquitin-activated proteasomal pathways. Ubiquitin-activated pathways targeting full-length selenoproteins include the peroxisome proliferator-activated receptor gamma-dependent pathway and substrate-dependent ubiquitination. An alternate mechanism is utilized for truncated selenoproteins, in which cullin-RING E3 ubiquitin ligase 2 targets the defective proteins for ubiquitin-proteasomal degradation. Selenoproteins, particularly SELENOP, may have their Sec residues reutilized for new selenoprotein synthesis via Sec decomposition. This review will explore these aspects in selenium biology, providing insights to knowledge gaps that remain to be uncovered.

Relationship between selenoprotein P and selenocysteine lyase: Insights into selenium metabolism.

Selenoprotein P (SelenoP) functions as a plasma transporter of selenium (Se) from liver to other tissues via incorporation into multiple selenocysteine (Sec) residues. Selenocysteine lyase (Scly) is an intracellular enzyme that decomposes Sec into selenide, providing Se for the synthesis of new selenoproteins. Both SelenoP and Scly are mostly produced by the liver. Previous studies demonstrated that male mice lacking SelenoP (SelenoP KO) or Scly (Scly KO) had increased or decreased total hepatic Se, respectively. While SelenoP regulation by Se is well-studied, Scly regulation by Se has not been reported. We hypothesize that Scly is negatively regulated by Se levels, and that absence of SelenoP jeopardizes Scly-dependent Se recycling. Using in vitro and in vivo models, we unveiled a tissue-specific Se regulation of Scly gene expression. We also determined that SelenoP, a considered source of intracellular Se, affects Scly expression and activity in vitro but not in vivo, as in the absence of SelenoP, Scly levels and activity remain normal. We also showed that absence of SelenoP does not increase levels of transsulfuration pathway enzymes, which would result in available selenocompounds being decomposed by the actions of cystathionine γ-lyase (CGL or CTH) and cystathionine ß-synthase (CBS). Instead, it affects levels of thioredoxin reductase 1 (Txnrd1), an enzyme that can reduce selenite to selenide to be used in selenoprotein production. This study evaluates a potential interplay between SelenoP and Scly, providing further insights into the regulation of selenium metabolism.

Sexual Dimorphism in the Selenocysteine Lyase Knockout Mouse.

Selenium (Se) is an essential micronutrient known for its antioxidant properties and health benefits, attributed to its presence in selenoproteins as the amino acid, selenocysteine. Selenocysteine lyase (Scly) catalyzes hydrolysis of selenocysteine to selenide and alanine, facilitating re-utilization of Se for de novo selenoprotein synthesis. Previously, it was reported that male Scly-/- mice develop increased body weight and body fat composition, and altered lipid and carbohydrate metabolism, compared to wild type mice. Strikingly, females appeared to present with a less severe phenotype, suggesting the relationship between Scly and energy metabolism may be regulated in a sex-specific manner. Here, we report that while body weight and body fat gain occur in both male and female Scly-/- mice, strikingly, males are susceptible to developing glucose intolerance, whereas female Scly-/- mice are protected. Because Se is critical for male reproduction, we hypothesized that castration would attenuate the metabolic dysfunction observed in male Scly-/- mice by eliminating sequestration of Se in testes. We report that fasting serum insulin levels were significantly reduced in castrated males compared to controls, but islet area was unchanged between groups. Finally, both male and female Scly-/- mice exhibit reduced hypothalamic expression of selenoproteins S, M, and glutathione peroxidase 1.