Interactions among insulin resistance, epigenetics, and donor sex in gene expression regulation of iPSC-derived myoblasts
Nida Haider, C. Ronald Kahn
Nida Haider, C. Ronald Kahn
Published November 30, 2023
Citation Information: J Clin Invest. 2024;134(2):e172333. https://doi.org/10.1172/JCI172333.
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Interactions among insulin resistance, epigenetics, and donor sex in gene expression regulation of iPSC-derived myoblasts

Abstract

About 25% of people in the general population are insulin resistant, increasing the risk for type 2 diabetes (T2D) and metabolic disease. Transcriptomic analysis of induced pluripotent stem cells differentiated into myoblasts (iMyos) from insulin-resistant (I-Res) versus insulin-sensitive (I-Sen) nondiabetic individuals revealed that 306 genes increased and 271 genes decreased in expression in iMyos from I-Res donors with differences of 2-fold or more. Over 30 of the genes changed in I-Res iMyos were associated with T2D by SNPs and were functionally linked to insulin action and control of metabolism. Interestingly, we also identified more than 1,500 differences in gene expression that were dependent on the sex of the cell donor, some of which modified the insulin resistance effects. Many of these sex differences were associated with increased DNA methylation in cells from female donors and were reversed by 5-azacytidine. By contrast, the insulin sensitivity differences were not reversed and thus appear to reflect genetic or methylation-independent epigenetic effects.

Authors

Nida Haider, C. Ronald Kahn

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Figure 5

DNA methylation contributes to sexual dimorphism but not insulin resistance.

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DNA methylation contributes to sexual dimorphism but not insulin resista...
(A) DNMT1 and DNMT3A protein levels comparing male versus female I-Sen and I-Res iMyos (blue squares, male; red circles, female; dark shade, I-Sen; light shade, I-Res) from LC-MS/MS-based proteomics. n = 10 males, n = 9 females. **P < 0.01, ****P < 0.0001 males vs. females. (B) ELISA measuring 5-methylcytosine (5-mC) percentage changes relative to total DNA amount in I-Sen and I-Res iMyos from postpubertal male and female donors and prepubertal male and female donors. n = 10 males, n = 8 females (postpuberty), and n = 4 males and females each (prepuberty). *P < 0.05 males vs. females. (C) Overlap of autosomal sex-biased iMyo genes (P < 0.05, n = 3,447) with autosomal sex-biased methylation positions on n = 15,724 genes from (from ref. 39) showing an overlap of 1,356 of 3,447 genes (39.3%). (D) mRNA levels of male-dominant genes, GLIPR1 and COL8A1, and female-dominant genes, USP11 and GALNT18, in I-Sen iMyos from male and female donors with and without 5-azacytidine (5-Az) treatment. n = 4 per group. *P < 0.05 males vs. females in controls, or #P < 0.05, ##P < 0.01, ###P < 0.001 controls vs. 5-Az in females. (E) mRNA levels of genes altered in I-Res and I-Sen iMyos from male and female donors with and without 5-Az treatment. n = 5–8 per group. *P < 0.05, **P < 0.01, ***P < 0.001 I-Sen vs. I-Res. (F) Western blot of I-Sen iMyos from male and female cell lysates with and without 5-Az treatment processed through the active RhoA pull-down (PD) experiment and total cell lysates (TCL). Quantification of PD Western blot showing the active form of RhoA over TCL showing total RhoA levels. n = 4 per group. ***P < 0.001 males vs. females, ####P < 0.0001 DMSO vs. 5-Az in males. (G) 2-NBDG glucose uptake assay in iMyos stimulated with 100 nM of insulin for 30 minutes. n = 9–10 per group. **P < 0.01, ***P < 0.001 basal vs. insulin. (A and B) unpaired t test; (D–F) 1-way ANOVA followed by correction for multiple comparison by controlling the FDR; (G) 2-way ANOVA followed by correction for multiple comparison by controlling the FDR. Data are shown as the mean ± SEM.