Autoregulation of insulin receptor signaling through MFGE8 and the αvβ5 integrin

Abstract

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvβ5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/β5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvβ5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/β5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvβ5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.

Keywords: MFGE8; insulin receptor; insulin sensitivity; insulin signaling; integrins.

Fig. 1.

MFGE8 regulates insulin-induced skeletal-muscle glucose uptake in vivo. (A) GTT in 7 to 8 wk old male WT and Mfge8 ^−/− mice after IP injection of β5 blocking (β5 block) or isotype control antibody (Con ab). n = 3 to 4 for WT mice and n = 3 for Mfge8 ^−/− mice per group per experiment and merged data from two to four independent experiments are presented. Statistical analysis compares the β5 blocking and control antibody groups in WT mice. (B) Serum insulin levels during GTT in WT mice. n = 3 to 4 mice per group per experiment, and merged data from two independent experiments are presented. (C) GTT in male WT mice after IP injection of MFGE8 blocking (MFGE8 block) or isotype control antibody (Con ab). n = 3 mice in each group per experiment, and merged data from two independent experiments are presented. (D) GTT in WT male mice after IP injection of recombinant MFGE8 (rMFGE8) or RGE control construct. n = 2 to 3 mice per group per experiment, and merged data from three independent experiments are presented. (E) ITT in WT male mice treated with β5 blocking or control antibody. n = 3 to 4 mice per group per experiment, and the merged data from four independent experiments are presented. (F) ITT after IP administration of rMFGE8 or RGE control in WT male mice. n = 2 to 3 mice per group per experiment, and merged data from three independent experiments are presented. (G and I) PET/CT scan images showing radioactive 18FDG deposition (red arrowheads) in 7 to 8 wk old WT male mice treated with β5 blocking, (G) MFGE8 blocking, (I) or control antibody in presence of insulin (1 U/kg) and 18FDG. n = 4 independent experiments. (H and J) Quantification of 18FDG deposition in vastus and gastrocnemius skeletal-muscle compartments in the setting of β5 (H) or MFGE8 (J) blockade. Data are expressed as % injected dose of 18FDG per cubic centimeter (cc) tissue (%ID cc⁻¹). All data expressed as mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001. Data in A through F were analyzed by two-way repeated measures ANOVA followed by Bonferroni’s posttest. Data in H and J were analyzed by Mann–Whitney U test.

Fig. 2.

MFGE8 regulates insulin-induced glucose uptake in vitro. (A–C) 2NBDG uptake assay in mouse primary WT and Mfge8^−/− myotubes (A, n = 3 independent experiments), primary HsKM (B, n = 3 to 6 independent experiments), and differentiated C2C12 myotubes (C, n = 3 to 6 independent experiments) in the presence or absence of rMFGE8 or RGE control protein, β5 blocking, MFGE8 blocking, or isotype control antibody and insulin (100 nM). Data are expressed as relative fold changes compared to the untreated cells (NT). (D and E) Dose response (D, n = 4 to 6 independent experiments) and time course of insulin action (E, n = 6 independent experiments) on 2NBDG uptake in C2C12 myotubes in the presence of either β5 blocking or control antibody. Data are expressed as relative fold changes compared to control antibody-treated cells in the absence of insulin. (F) 2NBDG uptake assay in β5 blocking antibody–treated C2C12 cells in the presence and absence of insulin and wortmannin. Data are expressed as relative fold changes compared to the untreated cells (NT). n = 4 to 6 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Data analyzed by one-way ANOVA followed by Bonferroni’s posttest (A, D, and F). Data in E were analyzed by two-way repeated measures ANOVA followed by Bonferroni’s posttest. (G) Proposed model of how the MFGE8/β5 pathway dampens insulin-stimulated glucose uptake.

Fig. 3.

Persistent activation of insulin receptor signaling with disruption of the MFGE8/β5 pathway in skeletal muscle. (A–C) Coimmunoprecipitation experiments demonstrating the effect of β5 blockade and rMFGE8 treatment on IRβ and IRS-1 tyrosine phosphorylation in presence or absence of insulin (INS). (A) C2C12 myotubes were treated with insulin (100 nM) for 5 min (INS 5′) or 30 min (INS 30′) in presence of either β5 (A) blocking or isotype control antibody or (C) rMFGE8 or RGE protein coadministered with insulin. Western blots are representative of three independent experiments. (B) Dose response of insulin action on IRβ and IRS-1 tyrosine phosphorylation in presence of β5 blocking or isotype control antibody. Western blots are representative of four independent experiments. (D) Coimmunoprecipitation studies showing tyrosine phosphorylation of IRβ and IRS-1 in skeletal-muscle lysates from WT mice treated with IP insulin (1 U/kg) for 15 min (INS 15′) or 60 min (INS 60′) in presence of β5 blocking or control antibody. Western blots are representative of three independent experiments. WT male mice were used for all in vivo experiments.

Fig. 4.

Enhanced hepatic insulin sensitivity after β5 blockade. (A) PTT in 6 wk old male WT mice after IP injection of β5 blocking (β5 block) or isotype control antibody (Con ab). n = 3 to 4 in each group per experiment; data merged from two independent experiments are presented. (B) Coimmunoprecipitation experiments demonstrating the effect of β5 blockade on IRβ and IRS-1 tyrosine phosphorylation in the presence or absence of insulin (INS,100 nM for 30 min) in HepG2 cells after treatment with β5 blocking or isotype control antibody. Western blots are representative of three independent experiments. (C and D) Densitometric analysis of the Western blots (including B) of IRβ and IRS-1 tyrosine phosphorylation in HepG2 cells. (E) Coimmunoprecipitation experiments demonstrating the effect of β5 blockade on IRβ and IRS-1 tyrosine phosphorylation in the presence or absence of insulin (INS,100 nM for 30 min) in murine primary hepatocytes in the presence of β5 blocking or isotype control antibody. Western blots are representative of three independent experiments. Both male and female mice were used for primary hepatocyte isolation. (F) Coimmunoprecipitation studies showing IRβ and IRS-1 tyrosine phosphorylation from liver lysates of WT male mice treated with IP insulin (1 U/kg) for 15 min (INS 15′) or 60 min (INS 60′) in the presence of β5 blocking or control antibody. Western blots are representative of three independent experiments. (G and H) Densitometric analysis of Western blots (including F) showing fold changes in IRβ and IRS-1 tyrosine phosphorylation relative to NT. *P < 0.05 and **P < 0.01. Data in A were analyzed by two-way repeated measures ANOVA followed by Bonferroni’s posttest. Data in C, D, G, and H were analyzed by one-way ANOVA followed by Bonferroni’s posttest.

Fig. 5.

Interaction between IRβ and the β5 integrin. (A) PLA showing proximity between IRβ and β5 in C2C12 myotubes and primary HskM treated with β5 blocking or isotype control antibody and with or without 30 min of insulin (INS, 100 nM) treatment. n = 3 for C2C12, and n = 2 for HsKM myotubes. (Scale bar, 5 μm.) Magnification of 60× for C2C12 myotubes; 40× magnification for HsKM. (B–E) Coimmunoprecipitation experiments and respective densitometric analyses demonstrating the effect of β5 blockade (B and C) and rMFGE8 treatment (D and E) on the interaction between IRβ and β5 in the presence or absence of insulin (INS, 100 nM) using an antibody recognizing the cytosolic domain of β5 antibody for pulldown and subsequent Western blot for IRβ. Western blots are representative of three independent experiments. Densitometric data represented as fold changes relative to no treatment group (NT). (F) Coimmunoprecipitation studies of skeletal-muscle lysates from WT mice treated with IP insulin (1 U/kg) for 15 min (INS 15′) and 60 min (INS 60′) in presence of β5 blocking or control antibody. Western blots are representative of three independent experiments (three mice total per condition). (G) Densitometric analysis of Western blots (including F) showing fold changes in IRβ–β5 interaction relative to no treatment (NT). (H) Immunostaining showing colocalization of IRβ and β5 integrin in the hind-leg skeletal muscles of mice 60 min after insulin treatment (INS 60′) in presence of β5 blocking or control antibody. For all in vivo experiments, 7 to 8 wk old WT male mice were used. Data analyzed by ANOVA followed by Bonferroni’s posttest. *P < 0.05, **P < 0.01, and ***P < 0.001. (I) Proposed model showing how enhanced interaction between IRβ and β5 impedes insulin signaling.

Fig. 6.

Insulin induces cell-surface enrichment of MFGE8. (A–C) Cell fractionation of insulin-treated (1U/kg IP) skeletal-muscle tissue samples followed by Western blotting showing (A) MFGE8 expression in the cytoplasmic and cell-surface membrane fraction. Western blotting for HSP90 and CAVEOLIN-1 (CAV-1) confirmed cytoplasmic and membrane fractions, respectively. Western blots are representative of five independent experiments. (B) Densitometric analysis of Western blots (including A) showing relative fold changes in membrane MFGE8 in insulin-treated groups compared to NT. (C) Western blot showing surface membrane (CAV-1), ER (BIP), Golgi (STX) and cytoplasmic (HSP90) marker expression in different fractions from ultracentrifugation. Fraction 1 represents the cell-surface membrane used in A. WT male mice were used for all in vivo experiments. (D–F) Western blot showing cell-surface (streptavidin-bound) (D) and intracellular (streptavidin-unbound) (E) MFGE8 protein levels before and after 30 min of insulin (100 nM) treatment (INS) in C2C12 myotubes pretreated with either GCA (20 μM) or DMSO. Western blots are representative of four independent experiments. Western blotting for ABCA-1 confirmed successful inhibition of GBF-1 by GCA. (F) Densitometric analysis of Western blots (including D) showing fold changes in streptavidin-bound MFGE8 relative to NT. (G and H) Western blot showing cell-surface (streptavidin-bound, G) and intracellular (streptavidin-unbound, H) MFGE8 protein levels before and after 30 min of insulin (100 nM) treatment (INS) in C2C12 myotubes pretreated with either AICAR (10 mM) or DMSO. Data represents three independent experiments. (I) Densitometric analysis of Western blots (including G) showing fold changes in streptavidin-bound MFGE8 relative to NT. For all biotinylation experiments (D, E, G, and H), streptavidin-bound and unbound fractions were probed for HSP90 as a control for the intracellular (streptavidin-unbound) and Na,K-ATPase for the membrane-bound (streptavidin-bound) fractions. Total cell lysates not exposed to biotin reagent served a negative control (No bio). Densitometric data (B, F, and I) were analyzed by one-way ANOVA followed by Bonferroni’s posttest. *P < 0.05, **P < 0.01, and ***P < 0.001. (J) Proposed model showing how cell-surface enrichment of MFGE8 impedes insulin receptor signaling via ligation of β5 integrin.

Fig. 7.

Metabolic regulation of serum MFGE8 levels. (A–C) Blood glucose (A), serum insulin (B), and serum MFGE8 (C) levels in WT mice subjected to fasting for 5 and 16 h before a refeeding period of 1 h. n = 7 to 10 mice in A and C, n = 5 mice for B. Both male and female mice were used for these experiments. Data are from two to three independent experiments. (D) Coimmunoprecipitation experiments using hind-leg skeletal muscle lysates showing an interaction between IRβ and β5 in 16 h fasted mice and mice refed for 1 h after a 16 h fast. Data represents two independent experiments (four mice total per condition). WT male mice were used for this experiment. (E) Densitometric analysis of Western blots (including D) showing fold changes in IRβ–β5 interaction in the refed state relative to the fasted condition. Densitometry data were analyzed by Student's t test. Data in A–C were analyzed by one-way ANOVA followed by Bonferroni’s posttest. *P < 0.05, **P < 0.01, and ***P < 0.001.