Extra-Function of Ribosomal Protein L10a via Insulin Signaling Pathway

Abstract

Ribosomal protein L10a (RpL10a) is one of several ribosomal proteins which has functions besides protein synthesis in the ribosome. Many reports recently revealed the extra–ribosomal function of RpL10a, including a role in oogenesis, spermatogenesis, ovarian development, organogenesis, and embryogenesis. In Drosophila melanogaster, the overexpression of the shrimp RpL10a resulted in the loss of red pigment in the eye center. This finding suggests that RpL10a also plays a role in cell proliferation. In contrast, the lack of RpL10Ab (RpL10Ab-/-) in the germline of fruit flies showed the disappearance of the follicle cells surrounding the egg chamber, similar to the insulin receptor mutants (InR-/-), indicating that RpL10a plays a role in oogenesis. Normally, InR is the transmembrane receptor of the insulin signaling pathway that plays a key role in cell development, cell division, metabolism regulation especially the role in glucose homeostasis regulation. Therefore, RpL10a may be involved in the step of regulation of InR to regulate cell development and metabolism. To determine whether RpL10a is involved in regulating glucose homeostasis, carbohydrate content was observed in the eyes of normal and overexpressed flies by using an anthrone-sulfuric acid colorimetric assay. In this study, the function of shrimp RpL10a involved in insulin receptor (InR) and carbohydrate metabolism were investigated. In the eyes of RpL10a–overexpressed flies, the expression of InR was extensively increased in mRNA level and protein level, determined by qPCR and immunohistochemistry, respectively. Moreover, In the RpL10a–overexpressed eyes of the mutant fly showed the highly phosphorylated insulin signaling mediators such as Akt and dFOXO, whereas the mutant flies showed the reduction of the glycogen. This finding revealed that shrimp RpL10a affects cell proliferation and glycogenolysis through the insulin signaling pathway. Besides, The interaction between RpL10a and InR was performed both in silico and in vitro binding. The shrimp RpL10a protein and the human InR protein were obtained as a silico binding model. The ClusPro software indicated that RpL10a could interact with InR at the difference site on fibronectin type III (FnIII) domain of insulin bound to InR. Then, to reduce the culture time and to control the culture condition easily, the study of shrimp RpL10a function was performed in CHO-S cell line. The in vitro study results, including pull-down assay and immunofluorescence assay, confirmed the binding of the proteins. The binding of the two proteins could stimulate the insulin signaling pathway by binding at the FnIII domain of InR and increased the carbohydrate utilization while the lipid metabolism was unchanged. Also, the effect of RpL10a on glucose metabolism was investigated in insulin resistance conditions. The CHO-S cells were induced to insulin resistance under high glucose conditions before stimulating with RpL10a. After the healthy cells and insulin-resistant cells (IRCs) were incubated with RpL10a, RpL10a could induce glucose uptake by the IRCs better than healthy cells. Moreover, RpL10a could induce the expression of hexokinase in glycolysis and decreased the expression of glucose-6-phosphatase in gluconeogenesis. These findings suggested that RpL10a could alleviate the insulin resistance by inducing the glucose uptake into the cells, activating glycolysis, and suppressing glucose synthesis in IRCs induced under high glucose conditions. From this important function, RpL10a may have a function as an insulin-mimetic for insulin-resistant diabetes treatment.