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Leucine-973 is a crucial residue differentiating insulin and IGF-1 receptor signaling
Hirofumi Nagao, … , Matthias Mann, C. Ronald Kahn
Hirofumi Nagao, … , Matthias Mann, C. Ronald Kahn
Published December 22, 2022
Citation Information: J Clin Invest. 2023;133(4):e161472. https://doi.org/10.1172/JCI161472.
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Research Article Endocrinology Metabolism

Leucine-973 is a crucial residue differentiating insulin and IGF-1 receptor signaling

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Abstract

Insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly conserved in IRs, to phenylalanine, the highly conserved homologous residue in IGF1Rs, resulted in decreased IRS-1/PI3K/Akt/mTORC1 signaling and increased Shc/Gab1/MAPK cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth, and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this led to decreased insulin sensitivity, a modest increase in growth, and decreased weight gain when mice were challenged with a high-fat diet. Thus, leucine-973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.

Authors

Hirofumi Nagao, Weikang Cai, Bruna B. Brandão, Nicolai J. Wewer Albrechtsen, Martin Steger, Arijeet K. Gattu, Hui Pan, Jonathan M. Dreyfuss, F. Thomas Wunderlich, Matthias Mann, C. Ronald Kahn

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Figure 2

L973F-IR mutation in regulation of metabolism and growth.

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L973F-IR mutation in regulation of metabolism and growth.
(A) Glucose up...
(A) Glucose uptake in WT-IR and L973F-IR preadipocytes stimulated with 0, 1, 10, or 100 nM insulin. Data are represented as mean ± SEM (n = 6). ***P < 0.001, WT-IR versus L973F-IR, 2-way ANOVA. (B) Glycolysis induced by 100 nM insulin for 6 hours. WT-IR and L973F-IR preadipocytes were FBS starved overnight before insulin stimulation. ECAR values were measured using a Seahorse X96 Bioanalyzer. Fold change of glycolysis rates in response to insulin stimulation were calculated by comparing ECARs on insulin stimulation after glucose injection. Data are represented as mean ± SEM (n = 8). *P < 0.05; ***P < 0.001, basal versus insulin. #P < 0.05, WT-IR versus L973F-IR, 2-way ANOVA. (C) Glycolytic rate (maximal glycolytic capacity) induced by 100 nM insulin for 6 hours. Fold change of the maximal glycolytic capacity in response to insulin stimulation as measured by ECAR on insulin stimulation after oligomycin injection was calculated. Data are represented as mean ± SEM (n = 8). ***P < 0.001, basal versus insulin. ###P < 0.001, WT-IR versus L973F-IR, 2-way ANOVA. (D) Triglyceride accumulation in WT-IR and L973F-IR adipocytes assessed by oil red O staining on day 7 after induction of differentiation (n = 3 per group). Data are represented as mean ± SEM. (E) Glucose uptake in WT-IR and L973F-IR differentiated adipocytes stimulated with 10 nM insulin. Data are represented as mean ± SEM (n = 6). *P < 0.05; ***P < 0.001, basal versus insulin. ###P < 0.001, WT-IR versus L973F-IR, 2-way ANOVA. (F) Proliferation rates of WT-IR and L973F-IR preadipocytes (n = 3) per day are shown as mean ± SEM. *P < 0.05, unpaired Student’s t test. (G) Immunoblotting of IRβ in whole cell lysates and biotin-labeled cell surface fraction at 0, 30, and 120 minutes after 100 nM insulin stimulation. Biotinylated receptors were pulled down by streptavidin agarose. (H) Quantification of relative surface receptors at 0, 30, and 120 minutes after 100 nM insulin stimulation. Data are represented as mean ± SEM. *P < 0.05; **P < 0.001, WT-IR versus L973F-IR, 2-way ANOVA.

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