IntroductionSkeletal muscle is one of the largest organs in the body and is a primary site for insulin action to control blood glucose.1,2 In skeletal muscle, insulin facilitates glucose uptake, promotes glycogen and protein synthesis, and inhibits protein degradation. Dysfunction of skeletal muscle metabolism in type 2 diabetes (T2D) is a major contributor to whole body insulin resistance and precedes and predicts T2D in offspring of diabetic parents.1,3,4 Our group recently demonstrated that induced pluripotent stem (iPS) cells from patients with T2D or insulin resistance without T2D differentiated into myoblasts in vitro (iMyos) exhibit multiple defects found in skeletal muscle in vivo, including altered insulin signaling, decreased insulin-stimulated glucose uptake, and reduced mitochondrial respiration.5,6 Thus, the insulin resistance observed in T2D iMyos in vitro mirrors many features of muscle insulin resistance in vivo but in the absence of potentially confounding circulating factors.Cell-intrinsic drivers of insulin resistance can include both genetic and epigenetic factors, such as changes in DNA sequence, DNA methylation, histone modification, and altered levels of noncoding RNAs, including miRNAs.6,7,8,9,10 Indeed, miRNAs are major regulators of mRNA and protein levels and have been implicated in regulating various aspects of T2D, including insulin resistance, glucose metabolism, and diabetic complications.11,12 Thus, studies have shown that miR-133, miR-206, and miR-1, which are capable of regulating myoblast differentiation and insulin sensitivity, are all decreased in the muscle of T2D patients.13,14 Likewise, a recent meta-analysis identified miR-144, miR-29a, and miR-133 as regulators of insulin signaling, glucose metabolism, and insulin resistance in muscle, thus contributing to the pathogenesis of T2D.15In addition, previous reports have shown that circulating miRNAs, such as miR-122, miR-192, miR-194, and miR-193, are increased in the serum of T2D patients.16,17,18,19 miR-122 can target the regulatory subunit of AMP-activated protein kinase (PRKAB1), interleukin 1 receptor type 1, the REL proto-oncogene, and the NF-kB subunit, potentially contributing to the pathogenesis of insulin resistance.20 Likewise, miR-192 is known to target the transcription factor RFX6, which regulates islet formation and insulin production, whereas miR-194 can target RUNX1, FOXA1, AKT, and GSK3, all of which regulate metabolic genes and phosphorylation in muscle. Similarly, elevated circulating miR-193b levels have been shown to negatively correlate with muscle mass in T2D patients, suggesting its involvement in muscle dysfunction in T2D. This is thought to occur via effects of miR-193b to suppress Pdk1 expression, leading to the deactivation of the Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase (S6K) signaling pathway, which in turn reduces protein synthesis.19In the present study, we sought to determine whether changes in miRNA abundance and secretion are part of a cell-intrinsic program using a disease-in-a-dish approach with iPS cells from control and T2D patients differentiated into myoblasts (iMyos) in vitro and performed a sensitive nuclease protection assay to assess the expression profile of cellular and secreted miRNAs. We found that 22 miRNAs were significantly upregulated and 49 were downregulated in iMyos in T2D. Overlapping the predicted targets of these miRNAs with gene and protein expression data indicated that these miRNAs may contribute to altered expression of over 200 proteins. Interestingly, this occurs in most cases with little to no change in mRNA levels. This can be reproduced in vitro using miRNA mimics. We also show cell-intrinsic alterations in miRNA secretion in sEVs in T2D iMyos, most of which occur independent of changes in cellular miRNA content. Together, these data demonstrate that miRNAs may serve as important epigenetic regulators of protein expression in skeletal muscle in T2D and may mediate additional systemic effects through independent changes in secreted miRNAs.ResultsCell-intrinsic alterations in expression of cellular miRNAs in T2DTo identify cell-intrinsic drivers of skeletal muscle insulin resistance in T2D without the interference of systemic factors, we derived iPS cell lines from people with T2D or healthy controls and differentiated these cells into myoblasts (iMyos) in vitro (Fig. 1a). Patient characteristics of donors are provided in Supplementary Fig. 1a. Cellular and secreted miRNAs were then assessed using the HTG EdgeSeq nuclease protection assay, which detects over 2,000 known human miRNAs. In total, 71 miRNAs were significantly altered by T2D, with 22 miRNAs being increased and 49 being decreased in T2D iMyos (Fig. 1b). In general, there were not only more downregulated than upregulated miRNAs, but the magnitude of change was also greater. The upregulated miRNAs in T2D iMyos included miR-7161-3p, miR-5004-3p, and miR-1272, which were increased by 1.8-3.0-fold, while the downregulated miRNAs, including miR-222-5p, miR-155-5p, and miR-142-5p, were decreased by 1.9-2.3-fold (Fig. 1c, d). In T2D iMyos, upregulation was observed for both mature miRNAs and their precursors (Fig. 1e), indicating that upregulation was likely due to enhanced transcription rather than changes in half-life. In contrast, for the miRNAs that were decreased in T2D iMyos, including miR-222-5p, miR-155-5p, and miR-142-5p, there was no change in precursor levels, suggesting that these decreases reflect altered processing, increased degradation or secretion of these miRNAs from the cell, rather than changes at the transcriptional level (Fig. 1f). Interestingly, of the 22 miRNAs increased in T2D iMyos, 2 miRNAs were significantly increased only in males (Supplementary Fig. 1b). Likewise, of the 49 miRNAs decreased in T2D iMyos, 24 were significantly decreased in males only, while 1 was decreased in females only, indicating some level of sexual dimorphism. Representative examples of miRNAs that were decreased only in males with T2D (miR-335-5p), only in females with T2D (miR-23a-5p), or in both males and females (miR-142-5p, miR-193a-3p, and miR-193a-5p) are shown in Supplementary Fig. 1c, d. Importantly, two of the most abundant miRNAs downregulated in T2D iMyos, miR-206 and miR-133 (Supplementary Fig. 2a), have also been found to be decreased in the muscle of patients with T2D.13 Similarly, miR-194-5p was decreased in T2D iMyos (Supplementary Fig. 2b) and has also been found to be decreased in the muscle of patients with T2D.18 Likewise, we observed increased expression of miR-135a-5p in T2D iMyos (Supplementary Fig. 2b), consistent with previous studies of skeletal muscle biopsies of humans with T2D and db/db mice.21,22 As summarized in Table 1, several of the downregulated miRNAs have been previously linked to insulin resistance, impaired glucose metabolism, and inflammatory signaling in metabolic tissues.13,23,24,25,26,27,28,29,30,31,32,33,34Fig. 1Full size imageCell-intrinsic alterations in expression of miRNAs in T2D iMyos. a Schematic overview of the experimental design. Eight healthy (controls) and eight type 2 diabetic (T2D) subjects were included in the study cohort. Muscle-derived cells were reprogrammed into induced pluripotent stem (iPS) cells using a non-integrating approach (e.g., Sendai virus-based reprogramming). iPS cells were differentiated into iPS-derived myoblasts (iMyoblasts). Harvested cells underwent integrative analysis through miRNA profiling, global proteomics, and RNA sequencing to identify cell-intrinsic drivers. b Volcano plot of changes in miRNA levels in T2D iMyos cells, determined by using the HTG EdgeSeq nuclease protection assay, compared with controls. Red dots are miRNAs that significantly increased or decreased in T2D (both defined as fold change from baseline >1 and p