22 research outputs found
Regulation of Insulin Receptor Substrate 1 (IRS-1)/AKT Kinase-mediated Insulin Signaling by O-Linked β-N-Acetylglucosamine in 3T3-L1 Adipocytes*
Increased O-linked β-N-acetylglucosamine (O-GlcNAc) is associated with insulin resistance in muscle and adipocytes. Upon insulin treatment of insulin-responsive adipocytes, O-GlcNAcylation of several proteins is increased. Key insulin signaling proteins, including IRS-1, IRS-2, and PDK1, are substrates for OGT, suggesting potential O-GlcNAc control points within the pathway. To elucidate the roles of O-GlcNAc in dampening insulin signaling (Vosseller, K., Wells, L., Lane, M. D., and Hart, G. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5313–5318), we focused on the pathway upstream of AKT. Increasing O-GlcNAc in 3T3-L1 adipocytes decreases phosphoinositide 3-kinase (PI3K) interactions with both IRS-1 and IRS-2. Elevated O-GlcNAc also reduces phosphorylation of the PI3K p85 binding motifs (YXXM) of IRS-1 and results in a concomitant reduction in tyrosine phosphorylation of Y608XXM in IRS-1, one of the two main PI3K p85 binding motifs. Additionally, insulin signaling stimulates the interaction of OGT with PDK1. We conclude that one of the steps at which O-GlcNAc contributes to insulin resistance is by inhibiting phosphorylation at the Y608XXM PI3K p85 binding motif in IRS-1 and possibly at PDK1 as well
Supplementary Figure S3 from XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies
Supplemental Figure S3: Repeated mXTX301 dosing was well tolerated and resulted in dose dependent TGI in mice bearing large (~360mm3) MC38 tumors (a) C57BL/6J mice were implanted subcutaneously with MC38 tumor cells and received a single intravenous injection of mXTX301, unmasked control, or vehicle (PBS) at indicated dose levels (N=12 per group). Tumor measurements were taken two/three times a week. Animals were euthanized due to health issues or body weight loss. The data represent individual survival curves. CR: complete regression, EU: euthanized. (b) C57BL/6J mice were implanted subcutaneously with MC38 tumor cells and received a total of 3 doses marked by red arrows of mXTX301. Body weight was monitored over time (c) Survival prolongation was assessed by Gehan-Breslow-Wilcoxon test comparison at all study time points (until Day 27). ***p = 0.0001; ****p < 0.0001.</p
Supplementary Figure S6 from XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies
Supplemental Figure S6: XTX301 induced cell signature and pathway analysis: single-sample Gene Set Enrichment Analysis (ssGSEA) was performed for all samples and for selected gene sets of interest (see figure 5e). Each ssGSEA score summarizes the overall expression level of genes involved in a certain biological process of interest in a biological sample. For each gene set, ssGSEA scores were analyzed with respect to treatment groups and significant differences compared to the vehicle group were determined by Wilcoxon rank sum test. Boxplots showing ssGSEA scores (y-axis) of all samples for gene sets of interest. Each point is a sample. Samples were grouped by treatment group (x-axis). Colors indicate different treatment groups. * (Wilcoxon test, p< 0.05) ** (Wilcoxon test, p<0.01).</p
Supplementary Figure S4 from XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies
Supplemental Figure S4: Gating strategy for multi-color flow cytometry analysis of tumor-infiltrating and splenic immune cell populations.</p
Supplementary Table S2 from XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies
Supplementary Table S2: pSTAT4 EC50 in HEK-Blue IL-12 RGA and primary CD8 T cells
IL-12 HEK-Blue reporter gene cells were incubated with either rh IL-12, unmasked control, or XTX301 at varying doses, and the reporter activity was measured. The data from three independent experiments, the average of replicates from each experiment are listed in the table. Pre-activated primary human PBMCs were incubated with either rhIL-12, unmasked control or XTX301 at varying doses, and STAT4 phosphorylation was assessed in CD8+ T cells via flow cytometry. The EC50 was calculated as described in the statistical analysis section. The data represents two independent experiments, each conducted with two different PBMC donors.</p
Supplementary Figure S5 from XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies
Supplemental Figure S5: IFN- production in plasma: MC38 tumor-bearing mice were treated with the indicated doses of mXT301 or unmasked control. Plasma IFN- concentration was measured by MSD assay at indicated time points. The data represent mean ± SEM, N = 3 to 5 mice per group, and IFN- level was assessed by a two-way ANOVA with Bonferroni’s post-hoc pairwise comparison test compared to vehicle (PBS) treated animals (*p < 0.05, **p < 0.005, ****p < 0.0001)</p
Supplementary Table S3 from XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies
Supplementary Table S3: IFN-γ production EC50 in mouse, human, and cynomolgus primary cells
The ability of mXTX301 to induce IFN-γ production in mouse splenocytes and XTX301 to induce IFN-γ production in human and cynomolgus monkey PBMCs was assessed via ELISA. The EC50 was calculated as described in the statistical analysis section.</p
Supplementary Figure S3 from XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies
Supplemental Figure S3: Repeated mXTX301 dosing was well tolerated and resulted in dose dependent TGI in mice bearing large (~360mm3) MC38 tumors (a) C57BL/6J mice were implanted subcutaneously with MC38 tumor cells and received a single intravenous injection of mXTX301, unmasked control, or vehicle (PBS) at indicated dose levels (N=12 per group). Tumor measurements were taken two/three times a week. Animals were euthanized due to health issues or body weight loss. The data represent individual survival curves. CR: complete regression, EU: euthanized. (b) C57BL/6J mice were implanted subcutaneously with MC38 tumor cells and received a total of 3 doses marked by red arrows of mXTX301. Body weight was monitored over time (c) Survival prolongation was assessed by Gehan-Breslow-Wilcoxon test comparison at all study time points (until Day 27). ***p = 0.0001; ****p < 0.0001.</p
Supplementary Figure S4 from XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies
Supplemental Figure S4: Gating strategy for multi-color flow cytometry analysis of tumor-infiltrating and splenic immune cell populations.</p