The Supraphysiological Insulin Levels Amplify Pro-Inflammatory Response and Induce Apoptosis in Macrophages: An In Vitro Cell-Material Interaction Model




Continuous subcutaneous insulin infusion is a pivotal method of managing Type 1 diabetes, relying on the effectiveness of insulin infusion sets (IIS) to ensure the reliable delivery of insulin to a subcutaneous depot [1]. However, IIS face challenges marked by short wear time and high failure rates [2]. Emerging evidence idicates that the local inflammatory response to the IIS cannula may play a role in both the limited wear time and unreliable insulin adsorption [3-5]. However, a comprehensive understanding of the underlying mechanisms remains elusive. This study integrates a selected supraphysiological insulin concentration range with the “tissue damage” protein adsorption model. The primary objective is to investigate key parameters related to macrophage response, including nuclear factor-κB (NF-κB) and activating protein 1 (AP-1) activity, ROS accumulation, as well as assessments of cell density, viability, and apoptosis. The findings aim to advance our knowledge in the field and potentially inform strategies for enhancing the reliability of IIS in the management Type 1 diabetes.


3T3 fibroblast lysate were pre-adsorbed on tissue culture plates (TCPs) to model the damage-associated molecular patterns (DAMPs) released from tissue injury. A relevant supraphysiological insulin concentration range (0.1~10 U/ml) at the infusion site was chosen. Humulin-N (Eli Lilly), an insulin analog, was introduced to adherent RAW-Blue reporter macrophages on lysate-adsorbed surfaces to examine the impact on macrophage inflammatory response. NF-B/AP-1 dependent macrophage pro-inflammatory activity was assessed using the Quanti-Blue alkaline phosphatase activity assay (InvivoGen), normalized to cell density via QuantiFlour dsDNA assay (Promega). Intracellular ROS was measured with ROS-ID Total Detection Kit (Enzo Life Science) and normalized to adherent cell density. Statistical analysis employed a two-way ANOVA with Dunnett’s post hoc (n= 6-8). Real-time apoptotic cell measurement used the IncuCyte® Zoom imaging system (Essen BioScience) with multiplex staining of IncuCyte® Caspase-3/7 green dye. Data were collected via IncuCyte® Live-Cell Analysis System and plotted in GraphPad Prism software.


Supraphysiological insulin concentration (0.5~10 U/ml) enhanced the NF-kB/AP-1 activity and enhanced ROS accumulation at insulin concentration of 5 U/ml in macrophages on lysate-adsorbed surfaces. Notably, insulin had no significant impact on NF-B/AP-1 nor ROS in the absence of the inflammatory stimulus. Moreover, supraphysiological insulin (5 U/ml) induced macrophages apoptosis both with and without lysate adsorption.

In summary, the study demonstrates that insulin concentration ( 10 U/ml) alone had no significant effect, but concentration ( 0.5 U/ml) enhanced the activity of pro-inflammatory transcription factors NF-kB and AP-1, and showed significant increased intracellular ROS and increased macrophage apoptosis at 5 U/ml when combined with an inflammatory stimulus. This study contributes to the emerging evidence that infused insulin contributes to an enhanced inflammatory response to IIS.

Figure 1. The effect of supraphysiological insulin concentrations on the NF-κB/AP-1-dependent SEAP activity per cell. Data are displayed as mean ± SD (n = 9). * p < 0.05 compared to control of each group, # p < 0.05 for lysate control compared to media control.


Funding for this work was provided from the Canadian Institute of Health Research Project Grant and the Queen’s University Special Research Fund.


  1. Heinemann, L., et al., J. Diabetes Sci. Technol, 2012.
  2. Flores, M., et al., BMJ Open Diabetes Res. Care, 2020.
  3. Hauzenberger, J. R. et al., Diabetes Technol. Ther.2017.
  4. Eisler, G. et al., BMJ Open Diabetes Res. Care, 2019.
  5. Regittnig, W. et al., Diabetes Obes. Metab. 2022.




How to Cite

Y. Zhang and L. . Fitzpatrick, “The Supraphysiological Insulin Levels Amplify Pro-Inflammatory Response and Induce Apoptosis in Macrophages: An In Vitro Cell-Material Interaction Model”, CMBES Proc., vol. 46, Jun. 2024.