Insulin oxidation and oxidative modifications alter glucose uptake, cell metabolism, and inflammatory secretion profiles

Abstract

Insulin participates in glucose homeostasis in the body and regulates glucose, protein, and lipid metabolism. Chronic hyperglycemia triggers oxidative stress and the generation of reactive oxygen species (ROS), leading to oxidized insulin variants. Oxidative protein modifications can cause functional changes or altered immunogenicity as known from the context of autoimmune disorders. However, studies on the biological function of native and oxidized insulin on glucose homeostasis and cellular function are lacking. Native insulin showed heterogenous effects on metabolic activity, proliferation, glucose carrier transporter (GLUT) 4, and insulin receptor (INSR) expression, as well as glucose uptake in cell lines of five different human tissues. Diverse ROS compositions produced by different gas plasma approaches enabled the investigations of variously modified insulin (oxIns) with individual oxidative post-translational modification (oxPTM) patterns as identified using high-resolution mass spectrometric analysis. Specific oxIns variants promoted cellular metabolism and proliferation in several cell lines investigated, and nitrogen plasma emission lines could be linked to insulin nitration and elevated glucose uptake. In addition, insulin oxidation modified blood glucose levels in the chicken embryos (in ovo), underlining the importance of assessing protein oxidation and function in health and disease.

Keywords: CAP; Cold physical plasma; Gas plasma technology; Mass spectrometry; OxPTM; ROS; Reactive oxygen species; kINPen.

Fig. 1

Native insulin promotes metabolic activity in five different cell lines and affects proliferation and surface marker expression. Expression of surface markers (a) insulin receptor (INSR) and (b) glucose transporter (GLUT4) was determined in cells of different origin by flow cytometry; five cell lines were selected for further studies and incubated with different insulin concentrations before (c, d) metabolic activity and (e,f) proliferation were measured; (g) glucose uptake was determined by adding a fluorescent glucose probe (2-NBDG). Data are shown as mean ± SEM from three to five independent experiments with three to six technical replicates for each measurement. Statistical analysis was performed by the one-way analysis of variance (ANOVA), comparing insulin-treated cells vs. vehicle-treated cells (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, n.s. = not significant); (h) representative histogram of GLUT4 signal and (j, l) quantified signals; (i,k,m) INSR signal in different cell lines after adding native insulin. d, f, j-m are mean values that include data from three to five independent experiments with three to six technical replicates for each measurement. Statistical analysis was performed by the Mann-Whitney test, comparing insulin-treated cells vs. vehicle-treated cells (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, n.s. = not significant). j, k are mean values ± SEM from three biological replicates, including three technical replicates for each measurement. Statistical analysis was performed by the two-way analysis of variance (ANOVA), comparing treated samples versus untreated proteins for each time point (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, n.s. = not significant).

Fig. 2

Cold physical plasma generated reactive oxygen species that affect insulin's structure. (a) Reactive species generated by cold physical plasma varied when using different feed gases to ignite the plasma; (b) scheme of insulin exposed to argon gas as control or five different plasma conditions; effects on insulin were determined by dynamic light scattering, where (c, d) correlation coefficient, (e) polydispersity index, and (f, g) size were determined. d, e, g are mean values ± SEM from six independent experiments with three technical replicates each. Statistical analysis was performed by the Mann-Whitney test, comparing treated samples versus untreated proteins (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, n.s. = not significant).

Fig. 3

Identification of oxidative post-translational modifications in oxidized insulin. Insulin was exposed to different plasmas, and oxPTMs were identified by mass spectrometry analysis. (a) overview of summarized oxPTM distribution of the A and B chain sequences (the B chain sequence GFFYTPKT with high PSM numbers marked in yellow); (b) A and B chain's amino acid sequence and PSM count of unmodified GFFYTPKT sequence; (c) total number of oxPTMs detected in native insulin and plasma-treated insulin variants; (d) percentage of individual oxPTMs in oxidized insulin variants; (e) individual oxPTMs on specific amino acids in the detected (GFFYTPKT) sequence; (f) accumulated oxPTMs on amino acids of the GFFYTPKT sequence. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Oxidized insulin promotes metabolic activity, proliferation, and INSR and GLUT4 expression. (a) workflow of the study in which different cell lines were seeded and incubated with native or oxidized insulin for various durations before different aspects were determined; (b) metabolic activity was determined 8h after adding insulin or oxidized insulin by resazurin assay; (c) proliferation was determined by high-content imaging and flow cytometry; (d) metabolic activity and viability in pancreatic beta cells after incubation with native or oxidized insulin; after the cells were incubated with Ins or oxIns variants, surface marker expression of (e) INSR and (f) GLUT4 was determined by flow cytometry. b-f are mean values ± SEM from three to six independent experiments with three technical replicates each. Statistical analysis was performed by the Mann-Whitney test, comparing treated samples versus untreated proteins (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, n.s. = not significant). e,f are mean values of three biological replicates with three technical replicates each. Statistical analysis was performed by the two-way analysis of variance (ANOVA), comparing treated samples versus untreated proteins for each time point (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, n.s. = not significant).

Fig. 5

Glucose uptake is increased after incubation with oxidized insulin in vitro but not in ovo. (a) glucose uptake was determined by adding a fluorescent glucose probe (2-NBDG) together with native or oxidized insulin and measuring fluorescent intensity after 1 h. Data are shown as mean ± SEM from three independent experiments with three or 12 technical replicates each; (b) transgenic, human INSR expressing CHO cells were incubated with Ins or oxIns and 2-NBDG to determine glucose uptake. Data are shown as mean ± SEM from three independent experiments with three or 12 technical replicates each; (c) workflow of in ovo experiment; (d) photo of a blood vessel separated from the chicken egg to take blood for blood glucose testing; (e) Ins was applied on the CAM following determination of blood sugar level after one, three and 5 h; (f) blood glucose level in eggs 3 h after adding native or oxidized insulin. All individual data points are shown Data are shown as mean ± SEM from five to eight independent experiments. a-c,e-f Statistical analysis was performed by the Mann-Whitney test, comparing Ins vs. oxIns (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, n.s. = not significant).