The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.
Acanthamoeba castellanii/*enzymology/genetics/growth & development ; Amebiasis/*parasitology ; Cell Wall/*metabolism ; Cellulose/*biosynthesis/genetics ; Glucosyltransferases/genetics/metabolism ; Glycogen Phosphorylase/genetics/metabolism ; Protozoan Proteins/genetics/*metabolism ; UTP-Glucose-1-Phosphate Uridylyltransferase/genetics/metabolism

AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid (MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a (GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β (IRβ), protein kinase B (also known as Akt), and glycogen synthase kinase-3β (GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet (HFD). RESULTS: The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. CONCLUSION The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase.
Animals ; Diabetes Mellitus ; drug therapy ; enzymology ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Enzyme Inhibitors ; administration & dosage ; Glycogen ; metabolism ; Glycogen Phosphorylase ; antagonists & inhibitors ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Insulin ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Signal Transduction ; drug effects ; Triterpenes ; administration & dosage