Binding between insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 is not influenced by glucose or 2-deoxy-D-glucose

Abstract

A recent report (Zhong, D., Xiong, L., Liu, T., Liu, X., Liu, X., Chen, J., Sun, S. Y., Khuri, F. R., Zong, Y., Zhou, Q., and Zhou, W. (2009) J. Biol. Chem. 284, 23225-23233) details that 2-deoxy-D-glucose (2-DG), a well known inhibitor of glycolysis and a candidate antineoplastic agent, also induces insulin-like growth factor 1 receptor (IGF-1R) signaling through the inhibition of insulin-like growth factor 1-insulin-like growth factor-binding protein 3 (IGF-1-IGFBP-3) complex formation. Zhong et al. hypothesized that disrupted IGF-1/IGFBP-3 binding by 2-DG led to increased free IGF-1 concentrations and, consequently, activation of IGF-1R downstream pathways. Because their report suggests unprecedented off-target effects of 2-DG, this has profound implications for the fields of metabolism and oncology. Using ELISA, surface plasmon resonance, and novel "intensity-fading" mass spectrometry, we now provide a detailed characterization of complex formation between IGF-1 and IGFBP-3. All three of these independent methods demonstrated that there was no effect of glucose or 2-DG on the interaction between IGF-1 and IGFBP-3. Furthermore, we show examples of 2-DG exposure associated with reduced rather than increased IGF-1R and AKT activation, providing further evidence against a 2-DG increase in IGF-1R activation by IGF-1-IGFBP-3 complex disruption.

FIGURE 1.

ELISA to monitor binding of IGFBP3 (0–1000 nm in HBS-EP) to immobilized IGF-1 in the absence (open squares) or presence of 100 mm 2-DG (open triangles), 100 mm glucose (open circles), and 500 nm IGF-1 (closed squares). Error bars (standard deviation of triplicate measurements) are only depicted for the “control” and “500 nm IGF-1” series for simplicity.

FIGURE 2.

Reverse ELISA to monitor binding of IGF-1 (0–5000 nm in HBS-EP) to immobilized IGFBP-3 in the absence (open squares) or presence of 100 mm 2-DG (open triangles), 100 mm glucose (open circles), and 500 nm IGFBP-3 (closed squares). Error bars (standard deviation of triplicate measurements) are only depicted for the control and 500 nm IGFBP-3 series for simplicity.

FIGURE 3.

SPR analysis to monitor specificity of IGF-1 (25 nm in HBS-EP) binding to IGFBP-3 (1000 RU amine-coupled) in the absence (upper solid line) or presence of 100 mm 2-DG (dashed line) and 100 mm glucose (dotted line) at 75 μl/min. As a negative binding control, no significant response was observed with 25 nm BSA (bottom solid line). Double-referenced data are representative of duplicate injections acquired from two independent trials.

FIGURE 4.

SPR analysis to monitor real time kinetics of IGF-1 (0–25 nm in HBS-EP; 2-fold dilution series) binding to IGFBP-3 (1000 RU amine-coupled) in the absence (solid lines) or presence of 100 mm 2-DG (dashed lines) at 75 μl/min (3. 5 min association + 10 min dissociation). Double-referenced data are representative of duplicate injections acquired from two independent trials.

FIGURE 5.

MS analysis to monitor binding between IGF-1 (∼7600 Da) and IGFBP-3 (∼29,000 Da) in the absence or presence of 100 mm 2-DG (and internal EGF nonbinding control, ∼6400 Da). A, upper boxes represent 13 nm IGF-1 alone; middle boxes represent 13 nm IGF-1 with 13 nm IGFBP-3, and lower boxes represent 13 nm IGF-1 with 26 nm IGFBP-3. B, identical intensity fading experiments performed in the presence of 100 mm 2-DG. The data presented are representative of three independent experiments. y axes are representative of signal response (microampere (uA)), and x axes are representative of molecular mass (Da).

FIGURE 6.

Western blot assay of IGF-1R signaling. MCF-7, T47D, and HeLa cell lines were plated at 10⁶ cells/well in 6-well plates in DMEM containing 10% FBS. The following day, cells were treated with 10% FBS DMEM containing 2-DG (25 mm), IGF-1 (130 nm), or both. Four hours later, cells were harvested, and levels of signaling proteins were assayed by Western blot.