Sequential cleavage of insulin receptor by calpain 2 and γ-secretase impairs insulin signalling.
Soluble insulin receptor (sIR), the ectodomain of the insulin receptor (IR), has been detected in human plasma and its concentration paralleled that of blood glucose. We have previously developed an in vitro model using HepG2 liver-derived cells, which mimics changes in sIR levels in plasma from diabetic patients and shows that calcium-dependent proteases cleave IR extracellularly (a process known as shedding). The present study aimed to reveal the mechanisms of IR cleavage.



Using the in vitro model, we investigated the molecular mechanisms of IR cleavage, which is accelerated by high-glucose treatment. We also analysed the relationship between IR cleavage and cellular insulin resistance, and the correlation between plasma sIR levels and insulin sensitivity, which was assessed by the euglycaemic-hyperinsulinaemic clamp technique.



Here, we determined that calpain 2, which is secreted into the extracellular space associated with exosomes, directly cleaved the ectodomain of the IRβ subunit (IRβ), which in turn promoted intramembrane cleavage of IRβ by γ-secretase. IR cleavage impaired insulin signalling and the inhibition of IR cleavage (by knockdown of calpain 2 and γ-secretase), restored IR substrate-1 and Akt, independent of IR. Furthermore, the glucose-lowering drug, metformin, prevented IR cleavage accompanied by inhibition of calpain 2 release in exosomes, and re-established insulin signalling. In patients with type 2 diabetes, plasma sIR levels inversely correlated with insulin sensitivity.



Sequential cleavage of IR by calpain 2 and γ-secretase may contribute to insulin signalling in cells and its inhibition may be partly responsible for the glucose-lowering effects of metformin. Thus, IR cleavage may offer a new mechanism for the aetiology of insulin resistance.
Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, Tokushima, Japan. yuasa.tomoyuki@tokushima-u.ac.jp.|Department of Community Medicine for Diabetes and Metabolic Disorders, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima-shi, Tokushima, 770-8503, Japan. yuasa.tomoyuki@tokushima-u.ac.jp.
Tomoyuki
T
Yuasa
Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, Tokushima, Japan.|Department of Diabetes Research, Yamaguchi University School of Medicine, Yamaguchi, Japan.
Kikuko
K
Amo-Shiinoki
Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, Tokushima, Japan.|Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
Shuhei
S
Ishikura
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.|Department of Diabetes Care Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Mitsuyoshi
M
Takahara
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Takaaki
T
Matsuoka
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.|Department of Diabetes, Endocrinology and Metabolism, Kawasaki Medical School, Okayama, Japan.
Hideaki
H
Kaneto
Diabetes Clinical Research and Development Center, Tokushima University, Tokushima, Japan.|Diabetes Therapeutics and Research Center, Institute for Advanced Medical Sciences, Tokushima University, Tokushima, Japan.
Akio
A
Kuroda
Diabetes Clinical Research and Development Center, Tokushima University, Tokushima, Japan.|Diabetes Therapeutics and Research Center, Institute for Advanced Medical Sciences, Tokushima University, Tokushima, Japan.
Munehide
M
Matsuhisa
Human Life Science, Tokushima Bunri University, Tokushima, Japan.
Seiichi
S
Hashida
D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D055354:Exosomes / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D047468:Immunoprecipitation; D007333:Insulin Resistance / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*
D034741:RNA, Small Interfering; D011972:Receptor, Insulin; D053829:Amyloid Precursor Protein Secretases; D002154:Calpain; C569870:CAPN2 protein, human
Calpain 2; Exosome; Insulin resistance; Metformin; Shedding; Soluble insulin receptor; Type 2 diabetes
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