title: Development of in vitro model of insulin receptor cleavage induced by high glucose in HepG2 cells.
abstract: Soluble insulin receptor (sIR), the ectodomain of IR, has been detected in human plasma, and its concentration parallels that of blood glucose in patients with diabetes. IR has a pivotal role in glucose homeostasis and diabetes development; therefore, cleavage of IR promoted by hyperglycemia is involved in insulin resistance and glucose toxicity. To elucidate the physiology of sIR, we developed an in vitro model mimicking the changes in sIR levels in plasma from patients with diabetes. Among four human cell lines that expressed IR, spontaneous cleavage of IR occurred only in HepG2 cells. The molecular characteristics of sIR derived from HepG2 cells were similar to those of sIR detected in human plasma. The concentration of sIR in the medium did not differ between basal and high-glucose conditions in the initial 24-h period, but increasing the duration of pre-stimulation (>48 h) led to a significant increase in sIR levels in cells exposed to high glucose. Additionally, glucose-dependent increment of sIR was reversible in this model. These results are consistent with the observation of plasma sIR in patients with diabetes. Using this model, O-linked N-acetylglucosamine modification was determined to be involved in high-glucose-induced IR cleavage. A calcium-dependent protease was shown to cleave IR extracellularly. These findings show that this in vitro model could be useful for determining the molecular mechanism underlying IR cleavage.
authors: Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, 3-18-15 Kuramotocho, Tokushima 770-8503, Japan.
authors: Tomoyuki
authors: T
authors: Yuasa
authors: Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, 3-18-15 Kuramotocho, Tokushima 770-8503, Japan.
authors: Kikuko
authors: K
authors: Amo
authors: Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, 3-18-15 Kuramotocho, Tokushima 770-8503, Japan.
authors: Shuhei
authors: S
authors: Ishikura
authors: Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, 3-18-15 Kuramotocho, Tokushima 770-8503, Japan.
authors: Hisao
authors: H
authors: Nagaya
authors: Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, 3-18-15 Kuramotocho, Tokushima 770-8503, Japan.
authors: Keiji
authors: K
authors: Uchiyama
authors: Institute for Health Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
authors: Seiichi
authors: S
authors: Hashida
authors: Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, 3-18-15 Kuramotocho, Tokushima 770-8503, Japan. Electronic address: ebina@za3.so-net.ne.jp.
authors: Yousuke
authors: Y
authors: Ebina
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000215:Acylation / Q000187:drug effects; D001786:Blood Glucose / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D003920:Diabetes Mellitus / Q000097:blood; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000494:pharmacology*; D057809:HEK293 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D008954:Models, Biological; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects*; D034622:RNA Interference; D011972:Receptor, Insulin / Q000097:blood / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001786:Blood Glucose; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D011972:Receptor, Insulin; D010447:Peptide Hydrolases; D005947:Glucose; D002118:Calcium; D000117:Acetylglucosamine
keywords: Calcium-dependent protease; HepG2 cells; O-GlcNAcylation; Shedding; Soluble insulin receptor